eli lilly presentation · lineage and pathway-specific markers can be introduced ... epilepsy,...
TRANSCRIPT
New Techniques for MEA-Based Interrogation of Human
Stem Cell Derived Cardiomyocytes to Support CiPA and
In-vitro Safety Pharmacology Assays
Blake Anson PhD Cellular Dynamics International
Jim Ross PhD Axion Biosystems
Oct 21 2014
Outline
Proarrhythmia Testing
- moving from single channel to holistic
assessments
iCell Cardiomyocytes
Population Diversity
Key manufacturing components
3
In-vitro detection of proarrhythmia
The road to in-vitro
proarrhythmia testinghellip
hellip started in a fly
4
Drug ndashinduced Electrophysiological Aberrations
not a new phenomenon
Quinidine Syncope and Delayed
Repolarization SyndromesReynolds E and Vander Ark C M
Modern Concepts of Cardiovascular Disease 45117-
122 1976
Davies et al BMJ 1989298
Wyosowski and Bacsanyi NEJM 1995335
Astemizole-induced Arrhythmmia
From Vorperian et al JACC 199615
hellipbut took on a new meaning when
caused by non-cardiac compounds
5
Fruit flies provided insight to arrhythmia
Leg shaking
EAG mutant
(ether-a-go-go)
Wild
type
EAG Gene Ether-sedated Drosophila (Fruit Flies)
B Ganetzky
6
hERG is a member of the EAG
superfamily of K+ channels
Library Screen(s)
Hippocampal mRNA
Hit
Analysis
Heterologous
Expression
hERG ndash human ether a
go-go related gene
The hERG gene is linked
to Long QT Syndrome
EAG ndashether a go-go
gene sequence
The hERG gene
encdes lKr
7
DIA
Arrhythmogenic drugs block hERG channels and
prolong the cardiac AP
Zhou and January 1997
8
hERG and In-vitro Safety Assessments
Highly sensitive with
questionable specificity
9
Comprehensive in-vitro Proarrhythmia Testing
The Future
Comprehensive in-vitro Proarrhythmia
Assessment (CiPA)1 Assess effects on multiple individual ion
channels
2 Model effects (if any) on the ventricular
action potential and proarrhythmia
3 Verify conclusions with cardiomyocyte
recordings
httpwwwilsiextraorghesisciencecardiaccipa
Sager et al American Heart Journal (2014)
10
iCell CardiomyocytesDevelopment Regulatory Guidance
Nature Reviews Drug Discovery (Aug Sept 2013)
Product launch regulatory evaluation in 3 years
iPS cell-derived cardiomyocytes are
being evaluated for use in arrhythmia
assessment amp as a replacement for
thorough QT studies
11
Company Overview
Cellular Dynamics International (CDI) is the worldrsquos largest producer of human
iPS cells and iPS cell-derived cell types
Headquartered in Madison WI
Currently employs ~138 total staff
~650 yrs human stem cell experience
gt800 patents (owned or licensed)
Core competencies
Creation and culture of human iPS cells
Normal and disease phenotypes
Genetic engineering of iPS cells
Lineage and pathway-specific markers can be introduced
Development of new differentiation protocols
Differentiated cells from all three germ layers
Manufacture of human iPS cell-derived cell types
Scalable production of highly purified cells
Partnership with iPS Academia Japan enables access
and support for CDIrsquos products in Japan
12
iCell Cardiomyocytes
Human Cardiomyocytes
gt95 pure cryopreserved ready to use
gt4x106 cardiomyocytes per unit
Normal human biology
Broad platform utility for life science research drug
discovery and toxicity testing
Improved workflow with greater predictivity
Full product solution unlimited quantities
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Outline
Proarrhythmia Testing
- moving from single channel to holistic
assessments
iCell Cardiomyocytes
Population Diversity
Key manufacturing components
3
In-vitro detection of proarrhythmia
The road to in-vitro
proarrhythmia testinghellip
hellip started in a fly
4
Drug ndashinduced Electrophysiological Aberrations
not a new phenomenon
Quinidine Syncope and Delayed
Repolarization SyndromesReynolds E and Vander Ark C M
Modern Concepts of Cardiovascular Disease 45117-
122 1976
Davies et al BMJ 1989298
Wyosowski and Bacsanyi NEJM 1995335
Astemizole-induced Arrhythmmia
From Vorperian et al JACC 199615
hellipbut took on a new meaning when
caused by non-cardiac compounds
5
Fruit flies provided insight to arrhythmia
Leg shaking
EAG mutant
(ether-a-go-go)
Wild
type
EAG Gene Ether-sedated Drosophila (Fruit Flies)
B Ganetzky
6
hERG is a member of the EAG
superfamily of K+ channels
Library Screen(s)
Hippocampal mRNA
Hit
Analysis
Heterologous
Expression
hERG ndash human ether a
go-go related gene
The hERG gene is linked
to Long QT Syndrome
EAG ndashether a go-go
gene sequence
The hERG gene
encdes lKr
7
DIA
Arrhythmogenic drugs block hERG channels and
prolong the cardiac AP
Zhou and January 1997
8
hERG and In-vitro Safety Assessments
Highly sensitive with
questionable specificity
9
Comprehensive in-vitro Proarrhythmia Testing
The Future
Comprehensive in-vitro Proarrhythmia
Assessment (CiPA)1 Assess effects on multiple individual ion
channels
2 Model effects (if any) on the ventricular
action potential and proarrhythmia
3 Verify conclusions with cardiomyocyte
recordings
httpwwwilsiextraorghesisciencecardiaccipa
Sager et al American Heart Journal (2014)
10
iCell CardiomyocytesDevelopment Regulatory Guidance
Nature Reviews Drug Discovery (Aug Sept 2013)
Product launch regulatory evaluation in 3 years
iPS cell-derived cardiomyocytes are
being evaluated for use in arrhythmia
assessment amp as a replacement for
thorough QT studies
11
Company Overview
Cellular Dynamics International (CDI) is the worldrsquos largest producer of human
iPS cells and iPS cell-derived cell types
Headquartered in Madison WI
Currently employs ~138 total staff
~650 yrs human stem cell experience
gt800 patents (owned or licensed)
Core competencies
Creation and culture of human iPS cells
Normal and disease phenotypes
Genetic engineering of iPS cells
Lineage and pathway-specific markers can be introduced
Development of new differentiation protocols
Differentiated cells from all three germ layers
Manufacture of human iPS cell-derived cell types
Scalable production of highly purified cells
Partnership with iPS Academia Japan enables access
and support for CDIrsquos products in Japan
12
iCell Cardiomyocytes
Human Cardiomyocytes
gt95 pure cryopreserved ready to use
gt4x106 cardiomyocytes per unit
Normal human biology
Broad platform utility for life science research drug
discovery and toxicity testing
Improved workflow with greater predictivity
Full product solution unlimited quantities
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
3
In-vitro detection of proarrhythmia
The road to in-vitro
proarrhythmia testinghellip
hellip started in a fly
4
Drug ndashinduced Electrophysiological Aberrations
not a new phenomenon
Quinidine Syncope and Delayed
Repolarization SyndromesReynolds E and Vander Ark C M
Modern Concepts of Cardiovascular Disease 45117-
122 1976
Davies et al BMJ 1989298
Wyosowski and Bacsanyi NEJM 1995335
Astemizole-induced Arrhythmmia
From Vorperian et al JACC 199615
hellipbut took on a new meaning when
caused by non-cardiac compounds
5
Fruit flies provided insight to arrhythmia
Leg shaking
EAG mutant
(ether-a-go-go)
Wild
type
EAG Gene Ether-sedated Drosophila (Fruit Flies)
B Ganetzky
6
hERG is a member of the EAG
superfamily of K+ channels
Library Screen(s)
Hippocampal mRNA
Hit
Analysis
Heterologous
Expression
hERG ndash human ether a
go-go related gene
The hERG gene is linked
to Long QT Syndrome
EAG ndashether a go-go
gene sequence
The hERG gene
encdes lKr
7
DIA
Arrhythmogenic drugs block hERG channels and
prolong the cardiac AP
Zhou and January 1997
8
hERG and In-vitro Safety Assessments
Highly sensitive with
questionable specificity
9
Comprehensive in-vitro Proarrhythmia Testing
The Future
Comprehensive in-vitro Proarrhythmia
Assessment (CiPA)1 Assess effects on multiple individual ion
channels
2 Model effects (if any) on the ventricular
action potential and proarrhythmia
3 Verify conclusions with cardiomyocyte
recordings
httpwwwilsiextraorghesisciencecardiaccipa
Sager et al American Heart Journal (2014)
10
iCell CardiomyocytesDevelopment Regulatory Guidance
Nature Reviews Drug Discovery (Aug Sept 2013)
Product launch regulatory evaluation in 3 years
iPS cell-derived cardiomyocytes are
being evaluated for use in arrhythmia
assessment amp as a replacement for
thorough QT studies
11
Company Overview
Cellular Dynamics International (CDI) is the worldrsquos largest producer of human
iPS cells and iPS cell-derived cell types
Headquartered in Madison WI
Currently employs ~138 total staff
~650 yrs human stem cell experience
gt800 patents (owned or licensed)
Core competencies
Creation and culture of human iPS cells
Normal and disease phenotypes
Genetic engineering of iPS cells
Lineage and pathway-specific markers can be introduced
Development of new differentiation protocols
Differentiated cells from all three germ layers
Manufacture of human iPS cell-derived cell types
Scalable production of highly purified cells
Partnership with iPS Academia Japan enables access
and support for CDIrsquos products in Japan
12
iCell Cardiomyocytes
Human Cardiomyocytes
gt95 pure cryopreserved ready to use
gt4x106 cardiomyocytes per unit
Normal human biology
Broad platform utility for life science research drug
discovery and toxicity testing
Improved workflow with greater predictivity
Full product solution unlimited quantities
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
4
Drug ndashinduced Electrophysiological Aberrations
not a new phenomenon
Quinidine Syncope and Delayed
Repolarization SyndromesReynolds E and Vander Ark C M
Modern Concepts of Cardiovascular Disease 45117-
122 1976
Davies et al BMJ 1989298
Wyosowski and Bacsanyi NEJM 1995335
Astemizole-induced Arrhythmmia
From Vorperian et al JACC 199615
hellipbut took on a new meaning when
caused by non-cardiac compounds
5
Fruit flies provided insight to arrhythmia
Leg shaking
EAG mutant
(ether-a-go-go)
Wild
type
EAG Gene Ether-sedated Drosophila (Fruit Flies)
B Ganetzky
6
hERG is a member of the EAG
superfamily of K+ channels
Library Screen(s)
Hippocampal mRNA
Hit
Analysis
Heterologous
Expression
hERG ndash human ether a
go-go related gene
The hERG gene is linked
to Long QT Syndrome
EAG ndashether a go-go
gene sequence
The hERG gene
encdes lKr
7
DIA
Arrhythmogenic drugs block hERG channels and
prolong the cardiac AP
Zhou and January 1997
8
hERG and In-vitro Safety Assessments
Highly sensitive with
questionable specificity
9
Comprehensive in-vitro Proarrhythmia Testing
The Future
Comprehensive in-vitro Proarrhythmia
Assessment (CiPA)1 Assess effects on multiple individual ion
channels
2 Model effects (if any) on the ventricular
action potential and proarrhythmia
3 Verify conclusions with cardiomyocyte
recordings
httpwwwilsiextraorghesisciencecardiaccipa
Sager et al American Heart Journal (2014)
10
iCell CardiomyocytesDevelopment Regulatory Guidance
Nature Reviews Drug Discovery (Aug Sept 2013)
Product launch regulatory evaluation in 3 years
iPS cell-derived cardiomyocytes are
being evaluated for use in arrhythmia
assessment amp as a replacement for
thorough QT studies
11
Company Overview
Cellular Dynamics International (CDI) is the worldrsquos largest producer of human
iPS cells and iPS cell-derived cell types
Headquartered in Madison WI
Currently employs ~138 total staff
~650 yrs human stem cell experience
gt800 patents (owned or licensed)
Core competencies
Creation and culture of human iPS cells
Normal and disease phenotypes
Genetic engineering of iPS cells
Lineage and pathway-specific markers can be introduced
Development of new differentiation protocols
Differentiated cells from all three germ layers
Manufacture of human iPS cell-derived cell types
Scalable production of highly purified cells
Partnership with iPS Academia Japan enables access
and support for CDIrsquos products in Japan
12
iCell Cardiomyocytes
Human Cardiomyocytes
gt95 pure cryopreserved ready to use
gt4x106 cardiomyocytes per unit
Normal human biology
Broad platform utility for life science research drug
discovery and toxicity testing
Improved workflow with greater predictivity
Full product solution unlimited quantities
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
5
Fruit flies provided insight to arrhythmia
Leg shaking
EAG mutant
(ether-a-go-go)
Wild
type
EAG Gene Ether-sedated Drosophila (Fruit Flies)
B Ganetzky
6
hERG is a member of the EAG
superfamily of K+ channels
Library Screen(s)
Hippocampal mRNA
Hit
Analysis
Heterologous
Expression
hERG ndash human ether a
go-go related gene
The hERG gene is linked
to Long QT Syndrome
EAG ndashether a go-go
gene sequence
The hERG gene
encdes lKr
7
DIA
Arrhythmogenic drugs block hERG channels and
prolong the cardiac AP
Zhou and January 1997
8
hERG and In-vitro Safety Assessments
Highly sensitive with
questionable specificity
9
Comprehensive in-vitro Proarrhythmia Testing
The Future
Comprehensive in-vitro Proarrhythmia
Assessment (CiPA)1 Assess effects on multiple individual ion
channels
2 Model effects (if any) on the ventricular
action potential and proarrhythmia
3 Verify conclusions with cardiomyocyte
recordings
httpwwwilsiextraorghesisciencecardiaccipa
Sager et al American Heart Journal (2014)
10
iCell CardiomyocytesDevelopment Regulatory Guidance
Nature Reviews Drug Discovery (Aug Sept 2013)
Product launch regulatory evaluation in 3 years
iPS cell-derived cardiomyocytes are
being evaluated for use in arrhythmia
assessment amp as a replacement for
thorough QT studies
11
Company Overview
Cellular Dynamics International (CDI) is the worldrsquos largest producer of human
iPS cells and iPS cell-derived cell types
Headquartered in Madison WI
Currently employs ~138 total staff
~650 yrs human stem cell experience
gt800 patents (owned or licensed)
Core competencies
Creation and culture of human iPS cells
Normal and disease phenotypes
Genetic engineering of iPS cells
Lineage and pathway-specific markers can be introduced
Development of new differentiation protocols
Differentiated cells from all three germ layers
Manufacture of human iPS cell-derived cell types
Scalable production of highly purified cells
Partnership with iPS Academia Japan enables access
and support for CDIrsquos products in Japan
12
iCell Cardiomyocytes
Human Cardiomyocytes
gt95 pure cryopreserved ready to use
gt4x106 cardiomyocytes per unit
Normal human biology
Broad platform utility for life science research drug
discovery and toxicity testing
Improved workflow with greater predictivity
Full product solution unlimited quantities
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
6
hERG is a member of the EAG
superfamily of K+ channels
Library Screen(s)
Hippocampal mRNA
Hit
Analysis
Heterologous
Expression
hERG ndash human ether a
go-go related gene
The hERG gene is linked
to Long QT Syndrome
EAG ndashether a go-go
gene sequence
The hERG gene
encdes lKr
7
DIA
Arrhythmogenic drugs block hERG channels and
prolong the cardiac AP
Zhou and January 1997
8
hERG and In-vitro Safety Assessments
Highly sensitive with
questionable specificity
9
Comprehensive in-vitro Proarrhythmia Testing
The Future
Comprehensive in-vitro Proarrhythmia
Assessment (CiPA)1 Assess effects on multiple individual ion
channels
2 Model effects (if any) on the ventricular
action potential and proarrhythmia
3 Verify conclusions with cardiomyocyte
recordings
httpwwwilsiextraorghesisciencecardiaccipa
Sager et al American Heart Journal (2014)
10
iCell CardiomyocytesDevelopment Regulatory Guidance
Nature Reviews Drug Discovery (Aug Sept 2013)
Product launch regulatory evaluation in 3 years
iPS cell-derived cardiomyocytes are
being evaluated for use in arrhythmia
assessment amp as a replacement for
thorough QT studies
11
Company Overview
Cellular Dynamics International (CDI) is the worldrsquos largest producer of human
iPS cells and iPS cell-derived cell types
Headquartered in Madison WI
Currently employs ~138 total staff
~650 yrs human stem cell experience
gt800 patents (owned or licensed)
Core competencies
Creation and culture of human iPS cells
Normal and disease phenotypes
Genetic engineering of iPS cells
Lineage and pathway-specific markers can be introduced
Development of new differentiation protocols
Differentiated cells from all three germ layers
Manufacture of human iPS cell-derived cell types
Scalable production of highly purified cells
Partnership with iPS Academia Japan enables access
and support for CDIrsquos products in Japan
12
iCell Cardiomyocytes
Human Cardiomyocytes
gt95 pure cryopreserved ready to use
gt4x106 cardiomyocytes per unit
Normal human biology
Broad platform utility for life science research drug
discovery and toxicity testing
Improved workflow with greater predictivity
Full product solution unlimited quantities
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
7
DIA
Arrhythmogenic drugs block hERG channels and
prolong the cardiac AP
Zhou and January 1997
8
hERG and In-vitro Safety Assessments
Highly sensitive with
questionable specificity
9
Comprehensive in-vitro Proarrhythmia Testing
The Future
Comprehensive in-vitro Proarrhythmia
Assessment (CiPA)1 Assess effects on multiple individual ion
channels
2 Model effects (if any) on the ventricular
action potential and proarrhythmia
3 Verify conclusions with cardiomyocyte
recordings
httpwwwilsiextraorghesisciencecardiaccipa
Sager et al American Heart Journal (2014)
10
iCell CardiomyocytesDevelopment Regulatory Guidance
Nature Reviews Drug Discovery (Aug Sept 2013)
Product launch regulatory evaluation in 3 years
iPS cell-derived cardiomyocytes are
being evaluated for use in arrhythmia
assessment amp as a replacement for
thorough QT studies
11
Company Overview
Cellular Dynamics International (CDI) is the worldrsquos largest producer of human
iPS cells and iPS cell-derived cell types
Headquartered in Madison WI
Currently employs ~138 total staff
~650 yrs human stem cell experience
gt800 patents (owned or licensed)
Core competencies
Creation and culture of human iPS cells
Normal and disease phenotypes
Genetic engineering of iPS cells
Lineage and pathway-specific markers can be introduced
Development of new differentiation protocols
Differentiated cells from all three germ layers
Manufacture of human iPS cell-derived cell types
Scalable production of highly purified cells
Partnership with iPS Academia Japan enables access
and support for CDIrsquos products in Japan
12
iCell Cardiomyocytes
Human Cardiomyocytes
gt95 pure cryopreserved ready to use
gt4x106 cardiomyocytes per unit
Normal human biology
Broad platform utility for life science research drug
discovery and toxicity testing
Improved workflow with greater predictivity
Full product solution unlimited quantities
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
8
hERG and In-vitro Safety Assessments
Highly sensitive with
questionable specificity
9
Comprehensive in-vitro Proarrhythmia Testing
The Future
Comprehensive in-vitro Proarrhythmia
Assessment (CiPA)1 Assess effects on multiple individual ion
channels
2 Model effects (if any) on the ventricular
action potential and proarrhythmia
3 Verify conclusions with cardiomyocyte
recordings
httpwwwilsiextraorghesisciencecardiaccipa
Sager et al American Heart Journal (2014)
10
iCell CardiomyocytesDevelopment Regulatory Guidance
Nature Reviews Drug Discovery (Aug Sept 2013)
Product launch regulatory evaluation in 3 years
iPS cell-derived cardiomyocytes are
being evaluated for use in arrhythmia
assessment amp as a replacement for
thorough QT studies
11
Company Overview
Cellular Dynamics International (CDI) is the worldrsquos largest producer of human
iPS cells and iPS cell-derived cell types
Headquartered in Madison WI
Currently employs ~138 total staff
~650 yrs human stem cell experience
gt800 patents (owned or licensed)
Core competencies
Creation and culture of human iPS cells
Normal and disease phenotypes
Genetic engineering of iPS cells
Lineage and pathway-specific markers can be introduced
Development of new differentiation protocols
Differentiated cells from all three germ layers
Manufacture of human iPS cell-derived cell types
Scalable production of highly purified cells
Partnership with iPS Academia Japan enables access
and support for CDIrsquos products in Japan
12
iCell Cardiomyocytes
Human Cardiomyocytes
gt95 pure cryopreserved ready to use
gt4x106 cardiomyocytes per unit
Normal human biology
Broad platform utility for life science research drug
discovery and toxicity testing
Improved workflow with greater predictivity
Full product solution unlimited quantities
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
9
Comprehensive in-vitro Proarrhythmia Testing
The Future
Comprehensive in-vitro Proarrhythmia
Assessment (CiPA)1 Assess effects on multiple individual ion
channels
2 Model effects (if any) on the ventricular
action potential and proarrhythmia
3 Verify conclusions with cardiomyocyte
recordings
httpwwwilsiextraorghesisciencecardiaccipa
Sager et al American Heart Journal (2014)
10
iCell CardiomyocytesDevelopment Regulatory Guidance
Nature Reviews Drug Discovery (Aug Sept 2013)
Product launch regulatory evaluation in 3 years
iPS cell-derived cardiomyocytes are
being evaluated for use in arrhythmia
assessment amp as a replacement for
thorough QT studies
11
Company Overview
Cellular Dynamics International (CDI) is the worldrsquos largest producer of human
iPS cells and iPS cell-derived cell types
Headquartered in Madison WI
Currently employs ~138 total staff
~650 yrs human stem cell experience
gt800 patents (owned or licensed)
Core competencies
Creation and culture of human iPS cells
Normal and disease phenotypes
Genetic engineering of iPS cells
Lineage and pathway-specific markers can be introduced
Development of new differentiation protocols
Differentiated cells from all three germ layers
Manufacture of human iPS cell-derived cell types
Scalable production of highly purified cells
Partnership with iPS Academia Japan enables access
and support for CDIrsquos products in Japan
12
iCell Cardiomyocytes
Human Cardiomyocytes
gt95 pure cryopreserved ready to use
gt4x106 cardiomyocytes per unit
Normal human biology
Broad platform utility for life science research drug
discovery and toxicity testing
Improved workflow with greater predictivity
Full product solution unlimited quantities
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
10
iCell CardiomyocytesDevelopment Regulatory Guidance
Nature Reviews Drug Discovery (Aug Sept 2013)
Product launch regulatory evaluation in 3 years
iPS cell-derived cardiomyocytes are
being evaluated for use in arrhythmia
assessment amp as a replacement for
thorough QT studies
11
Company Overview
Cellular Dynamics International (CDI) is the worldrsquos largest producer of human
iPS cells and iPS cell-derived cell types
Headquartered in Madison WI
Currently employs ~138 total staff
~650 yrs human stem cell experience
gt800 patents (owned or licensed)
Core competencies
Creation and culture of human iPS cells
Normal and disease phenotypes
Genetic engineering of iPS cells
Lineage and pathway-specific markers can be introduced
Development of new differentiation protocols
Differentiated cells from all three germ layers
Manufacture of human iPS cell-derived cell types
Scalable production of highly purified cells
Partnership with iPS Academia Japan enables access
and support for CDIrsquos products in Japan
12
iCell Cardiomyocytes
Human Cardiomyocytes
gt95 pure cryopreserved ready to use
gt4x106 cardiomyocytes per unit
Normal human biology
Broad platform utility for life science research drug
discovery and toxicity testing
Improved workflow with greater predictivity
Full product solution unlimited quantities
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
11
Company Overview
Cellular Dynamics International (CDI) is the worldrsquos largest producer of human
iPS cells and iPS cell-derived cell types
Headquartered in Madison WI
Currently employs ~138 total staff
~650 yrs human stem cell experience
gt800 patents (owned or licensed)
Core competencies
Creation and culture of human iPS cells
Normal and disease phenotypes
Genetic engineering of iPS cells
Lineage and pathway-specific markers can be introduced
Development of new differentiation protocols
Differentiated cells from all three germ layers
Manufacture of human iPS cell-derived cell types
Scalable production of highly purified cells
Partnership with iPS Academia Japan enables access
and support for CDIrsquos products in Japan
12
iCell Cardiomyocytes
Human Cardiomyocytes
gt95 pure cryopreserved ready to use
gt4x106 cardiomyocytes per unit
Normal human biology
Broad platform utility for life science research drug
discovery and toxicity testing
Improved workflow with greater predictivity
Full product solution unlimited quantities
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
12
iCell Cardiomyocytes
Human Cardiomyocytes
gt95 pure cryopreserved ready to use
gt4x106 cardiomyocytes per unit
Normal human biology
Broad platform utility for life science research drug
discovery and toxicity testing
Improved workflow with greater predictivity
Full product solution unlimited quantities
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
13
iCell CardiomyocytesCharacterization
Electrophysiology E-C Coupling Contractility
Enables mechanistic toxicity testing
Whole-Genome Gene Expression
Relevant amp stable over time in culture
------ Adult myocardium____ d28 iCell CM____ d120 iCell CM
____ PromoCell____ Celprogen
-------Myocardium
Babiarz et al 2012 Kattman et al 2011 Rana et al 2012 Ma et al 2011
(For a complete list of iCell Cardiomyocytes publications go to
wwwcellulardynamicscom)
Metabolism
Recapitulates normal human
cardiac function
Protein Expression
Appropriate for interrogating
mitochondrial toxicity
iCell Cardiomyocytes native human biology enables
Mechanistic interrogation of cardiac function
Toxicity testing disruption of normal processes
Disease modeling corruption of normal processes
Well represented in the peer reviewed literature
~40 iCell Cardiomyocytes pubs to-date
More than all other commercial iPSC-CMs combined
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
14
CDI CommitmentQuality Quantity Purity
Quality
Quantity Purity
Exhibit key cellular characteristics
Recapitulate normal human biology
Reproducible
Known and relevant genotype
Sufficient to support HTP drug screening
and safety testing
Currently 1Bn iCell Cardiomyocytesday
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
15
StandardizationManufacturing Benchmarks
NHLBI Next Generation Genetic
Association Studies(RFA-HL-11-066)
250 patient samples - HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes from
all 250 individuals
Induce hypertrophy phenotype perform
molecular analyses
Correlate GWAS findings with in vitro
phenotype
Scale-Out
Manufacturing
bull 1000rsquos of individuals
bull Billions of cells
Scale-Up
Manufacturing
bull Quality
bull Quantity
bull Purity
CDI Manufacturing Benchmarks (cells per day gt95 purity)
2 billion iPS cells
1 billion cardiomyocytes
1 billion neurons
05 billion endothelial cells
04 billion hepatocytes
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
16
NHLBI Next Generation Genetic
Association Studies (RFA-HL-11-066)
250 patient samples ndash HyperGEN cohort
GWAS ndash Left Ventricular Hypertrophy (LVH)
Derive iPS cells and cardiomyocytes
Induce hypertrophy perform molecular analyses
Correlate GWAS findings with in vitro phenotype
Progress as of July 2014
250 donors reprogrammed
Differentiation protocol optimized to work robustly across all lines
128 iPS cell lines (1 per donor) are differentiated or in progress
Cardiomyocytes from 89 donors cryopreserved amp all pass QC
20 batches of cardiomyocytes are in currently being tested in
hypertrophy assays
Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)
Progress ReportPopulation genomics and left ventricular hypertrophy
CDIrsquos iPSC technology is enabling population studies
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
17
CIRM AwardiPS Cell Manufacture amp Banking
California Institute for Regenerative Medicine (CIRM)
Human iPS Cell Initiative ndash 3 Awards
Sample Collection iPSC Derivation (CDI) iPS Cell Banking
iPS Cell Derivation (CDI)
3000 donors (healthy amp disease phenotypes)
3 iPS cell clones per donor
Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos
disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)
pulmonary fibrosis
Derived from peripheral blood (preferred) or skin fibroblasts
Episomal ldquofootprint-freerdquo method
CDI ndash Coriell Partnership
Extensive collaboration to bring together expertise in electronic record-keeping
sample tracking iPS cell derivation amp characterization cell banking amp distribution
Joint facility located within the Buck Institute Novato CA
Demonstrated success in generating high quality iPSC lsquopopulationsrsquo
Well poised to enable population diversity in toxicity and safety assessments
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
QMS Framework Overview
Key Systems Objectives
QAQC Compliance and product consistency
Standard Operating Procedures Consistent procedures
CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use
Change Management Changes are documented assessed for risk and tested
CAPA Report correct and prevent product quality issues
Supplier Qual amp Mgmt Quality and reliability of raw materials
Materials Management Control trace and monitor stock inventory
Training Education and proficiency
Complaint Handling Customer satisfaction and continuous improvement
New Product Introduction Improve likelihood that product meets market need
An ISO GMP hybrid QMS system ensures customer safety and satisfaction
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
20
1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for
drug-induced repolarization delay and arrhythmias in an iPS cell-
derived cardiomyocyte sheet multi-site validation study J
Pharmacol Sci 124(4)494-501
2 Eldridge S Guo L et al (2014) Examining the Protective Role of
ErbB2 Modulation in Human Induced Pluripotent Stem Cell-
Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150
[Epub ahead of print]
3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their
use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-
61
4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to
induce cardiac hypertrophy and electrical remodeling in highly
purified human iPS cell-derived cardiomyocytes J Pharmacol
Toxicol Methods 69(2)177-88
5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-
derived HLA-A1-presented peptide as a cross-reactive target for
engineered MAGE A3-directed T cells Sci Transl Med
5(197)197ra103
6 Carlson C Koonce C et al (2013) Phenotypic screening with
human iPS cell-derived cardiomyocytes HTS-compatible assays
for interrogating cardiac hypertrophy J Biomol Screen
18(10)1203-11
7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity
testing of crizotinib sunitinib erlotinib and nilotinib in human
cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55
8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-
derived Cardiomyocytes for Studies of Cardiac Ion Transporters
Am J Physiol Cell Physiol 305(5)C481-91
9 Guo L Coyle l et al (2013) Refining the Human iPSC-
Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci
136(2)581-94
10 Harris K Aylott M et al (2013) Comparison of
Electrophysiological Data from Human Induced Pluripotent Stem
Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-
clinical Safety Assays Toxicol Sci 134(2)412-26
11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced
pluripotent stem cell-derived cardiomyocytes exhibit temporal
changes in phenotype Am J Physiol Heart Circ Physiol
305(6)H913-22
12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced
Long QT Ayndrome Acute Inhibition of Recombinant hERG K+
Channels and Action Potential Prolongation in Human
Cardiomyocytes Derived from Induced Pluripotent Stem Cells
British J Pharm 1681215-29
13 Puppala D Collis LP et al (2013) Comparative Gene Expression
Profiling in Human Induced Pluripotent Stem Cell Derived
Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol
Sci 131292-301
14 Rao C Prodromakis T et al (2013) The effect of microgrooved
culture substrates on calcium cycling of cardiac myocytes
derived from human induced pluripotent stem cells Biomaterials
34(10)2399-411
15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on
Human Cardiomyocyte Function and Cardiac Ion Channel Activity
Toxicol in Vitro 27745-51
16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro
Assessment of Compound Effects on Cardiomyocyte Physiology
Using iPS Cells J Biomol Screening 1839-53
17 Sirenko O Cromwell EF et al (2013) Assessment of beating
parameters in human induced pluripotent stem cells enables
quantitative in vitro screening for cardiotoxicity Toxicol Appl
Pharmacol 273(3)500-07
18 Babiarz JE Ravon M et al (2012) Determination of the Human
Cardiomyocyte mRNA and miRNA Differentiation Network by
Fine-scale Profiling Stem Cells Dev 211956-65
19 Cerignoli R Charlot D et al (2012) High Throughput Measurement
of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-
derived Cardiomyocytes by Kinetic Image Cytometry
J Pharmacol Toxicol Methods 66246-256
20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium
Mapping of Genetically Purified Human Induced Pluripotent Stem
Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63
21 Mioulane M Foldes G et al (2012) Development of High Content
Imaging Methods for Cell Death Detection in Human Pluripotent
Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res
5593-604
22 Rana P Anson BD et al (2012) Characterization of Human-
induced Pluripotent Stem Cell-derived Cardiomyocytes
Bioenergetics and Utilization in Safety Screening Toxicol Sci
130117-31
23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal
Doxorubicin Displays Enhanced Anti-tumorigenic Effects without
Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10
24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses
Outward Rectifier Potassium Currents in Human Pluripotent Stem
Cell-Derived Cardiomyocytes Plos One 7(11)e50641
25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an
iPSC-derived Cardiomyocyte Model Provides a Powerful Platform
for Gene Discovery in Left Ventricular Hypertrophy Frontiers in
Genetics 392
26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-
derived Cardiomyocytes to Examine Sunitinib Mediated
Cardiotoxicity and Electrophysiological Alterations Toxicol Appl
Pharmacol 25774-83
27 Guo L Qian JY et al (2011) The Electrophysiological Effects of
Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and
in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462
28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-
induced Proarrhythmia Using Human Induced Pluripotent Stem
Cell-derived Cardiomyocytes Toxicol Sci 123281-289
29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection
of Beating Rhythm and Proarrhythmic Effects of Compounds on
Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-
11
30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent
Stem Cell-derived Cardiomyocytes Electrophysiological
Properties of Action Potentials and Ionic Currents Am J Physiol
Heart Circ Physiol 301H2006-H2017
iCell CardiomyocytesMarket Validation (82014)
~40 Peer-reviewed
Publications (102014)
bull Characterization
bull Toxicity testing
bull Disease modeling
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
21
Measuring Cardiomyocyte FunctionElectrical and Mechanical Activity
iCell Cardiomyocytes provide a human system for
measuring all aspects of EC functionality
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
22
Predictivity ScreensElectrical Measurements
iPSC within frac12 log of wedge in some cases more sensitive
ldquoMEA assays using iPSC-CMs offer a reliable
cost effective surrogate to preclinical in vitro
testing in addition to the 3Rs (refine reduce
and replace animals in research) benefitrdquoHarris et al Toxicol Sci 2013
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
23
Summary
Proarrhythmia Testing
- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes
iCell Cardiomyocytes
- Human biology validated in the peer-reviewed literature
Population Diversity
- Well poised to enable population testing in toxicity and safety assessments
Key manufacturing components
- Quality is king
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
New techniques for MEA-based assays to support CiPA and in-vitro
safety pharmacology assays
October 21 2014
Jim Ross PhD CTO Axion Biosystems
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
1 Introduc+on13 to13 Axion13 2 Brief13 MEA13 Technology13 Review13 3 Role13 of13 MEAs13 in13 CiPA13 4 Arrhythmia13 assessment13 5 Cardiomyocyte13 Pacing13 6 New13 developments13
Agenda
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Analysis13
Quality13 Control13
Experiments13
Questions
Answers
Axionrsquos Objective Enable Great Science Complicated Network Electrophysiology Made Accessible
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Engineering
Applications
Hardw
areDesign
App
lica1
onsDevelop
men
t
SensorMicrofab
SowareD
esign
Applica1ons Engineering
Electronics
Sensors
Software
Applications Driven Innovation High Performance Scalable Technologies that are Simple to Use
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Proarrhythmia Score
Mechanism Based Continuous Scale
Rank Ordered Contextual Data
CiPA Objective
Clinical Assessment Human ECG
Ion Channel Panel
In Silico Simulations
Integrated Human Cellular Studies
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Integrated Human Cellular Studies
6
Ion Channel Panel
In Silico Simulations
Clinical Assessment Human ECG
1 Repolarization13
2 Arrhythmia13
3 ScalabilityReproducibility13
4 Risk Assessment13
Myocyte Objective
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
7
CM-MEA bridges the gap between AP and ECG
Na13 Ca13 K13
Clinical13 ECG13
Ac8on13 Poten8al13
CM-shy‐MEA13 Field13
Poten8al13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
813
Criteria13 for13 monitoring13 cardiac13 networks13
(1) Real-shy‐+me13 measurement13 of13 the13 phenotypic13 signal13 voltage13 13
(2) Sufficient13 resolu+on13 to13 capture13 field13 poten+als13
(3) Mul+ple13 recording13 sites13 to13 improve13 reliability13 and13 assess13 field13 poten+al13 propaga+on13 13
(4) Label-shy‐free13 non-shy‐invasive13 opera+on13 to13 observe13 natural13 cell13 func+on13 (5) Preserva+on13 of13 cellular13 interconnec+vity13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
A13 grid13 of13 microelectrodes13 interfaces13 with13 electro-shy‐ac+ve13 +ssue13 modeling13 complex13
human13 systems13 in13 a13 dish13
CM-shy‐MEA13 Assay13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Measures13 13 Field13 poten+al13 dura+on13 (ldquoQTrdquo)13 Conduc+on13 velocity13 Beat13 rate13 Field13 poten+al13 metrics13 (Amplitude13 Slope13 etc)13
Applica+ons13 13 Cardiac13 Safety13 Screening13 Drug13 Discovery13 Func+onal13 Disease13 Models13 Stem13 Cells13 Pa+ent-shy‐specific13 Therapies13 13 13
Propagation
100micro
V
1 sec15 2 25 3 35 4 45 5 55
minus1
0
1
2
3
x 10minus4
Depolarization
ms
0
1
2
3
4 Propagation Delay
Repolarization
CM-shy‐MEA13 Assay13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
MEAs in CiPA Current Status Characterizing cross-site reliability of CM Assays
1113
13 MEA13 Pilot13 Studies13 beginning13 in13 October13 201413 13
1213 Pilot13 Sites13 including13 Cyprotex13 Bristol-shy‐Myers13 Squibb13 Chantest13 NCI13 Janssen13 (JNJ)13 Merck13 NMI13 Sanofi13 and13 others13
1113 of13 1213 test13 sites13 are13 independent13 from13 the13 MEA13 supplier13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Tracking Repolarization Direct Measures of Depolarization amp Repolarization
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Tracking Repolarization Assessing stable beating
Con+nuous13 monitoring13 enables13 evalua+on13 of13 stable13 spontaneous13 bea+ng13 Red13 data13 points13 highlight13 the13 most13 stable13 region13 of13 spontaneous13 bea+ng13 between13 1513 ndash13 2013 minutes13 post13 dose13 avoiding13 a13 dri^13 in13 beat13 rate13 at13 1813 minutes13 (red13 square)13 13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Dofe+lide13
Quinidine13
Detecting Arrhythmia Beat Irregularity
Beat13 irregulari+es13 can13 emerge13 and13 disappear13 over13 +me13 13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Beat13 Irregularity13 NotchEAD13 Alternans13 amp13 Triggered13 Ac+vity13
Quinidine 1microM
Nakamura13 201413
Navarrete13 201313
Time13 (sec)13
BP13 (sec)13
Harris13 201313
Detecting Arrhythmia Arrhythmic Event Classification
MEA13 classifies13 aberrant13 events13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
DMSO13 113 microM13 di-shy‐4-shy‐ANEPPS13 613 microM13 di-shy‐4-shy‐ANEPPS13
113 minute13 dye-shy‐load13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Detecting Arrhythmia Advantages for Label-Free Assessment
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
N=413 DMSO13
113 microM13 di-shy‐4-shy‐ANEPPS13
Time13 (min)13 Time13 (min)13
Detecting Arrhythmia Advantages for Label-Free Assessment
Persistent13 dye13 Serum-shy‐containing13 media13 Label-shy‐free13 assessment13 minimizes13 perturba+ons13 to13 cardiomyocytes13 13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Evoked13 field13 potenals13 in13 a13 high13 throughput13 instrument13 13
Pacing with AxIS 20
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Pacing Entraining Cultures
Cardiomyocytes13 rapidly13 entrain13 to13 electrical13 s+muli13 13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
FPD13 shortens13 as13 pacing13 rate13 increases13
Pacing Specifying Beat Period
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Changes13 in13 FPD13 occur13 over13 the13 +mescale13 of13 minuteshellip13
Pacing Practical Considerations
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
hellipbut13 are13 reliable13 across13 beats13 and13 across13 wells13
Pacing Practical Considerations
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Pacing13 enables13 accurate13 beat13 rate13 correc+on13
Control13 of13 beat13 rate13 reduces13 well-shy‐to-shy‐well13 variability13
Pacing Improving Accuracy
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Pacing13 uncovers13 reverse13 use-shy‐dependence13 of13 candidate13 compounds13
Reverse Use-Dependence
Pacing Adding Depth
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
hellip
Beat13 113 Beat13 213 Beat13 313
Beat13 413 Beat13 513 Beat13 613 Beat13 10413
Spon
tane
ous13
Pacing13
Pacing Controlling Propagation
Pacing13 establishes13 consistent13 propaga+on13 paherns13 13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13
Conduc+on13 Velocity13 (mms)13
13 of13 Trials13
Paced13
Spontaneous13
Pacing Controlling Conduction
Pacing13 establishes13 consistent13 conduc+on13 measures13
2813
Questions
The13 Maestro13