society of toxicology annual meeting 13 m h 2012 13 march 2012 · 13 march 2012 presentation...
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Human iPS Cell Technology in Predictive and Mechanism-based Drug Discovery and Toxicity T ti U i Ph t t i B d A
Society of Toxicology Annual Meeting13 M h 2012
Testing Using Photometric-Based Assays
13 March 2012
Presentation Outline
iPS Cell Technology & Company Overview
iC ll P d t iCell Products iCell® Cardiomyocytes
iCell® Hepatocytes
Assessing Mechanisms of Toxicity through g y gMultiparametric Analysis of iCell Cellular Test Systems
iCell Hepatocyte VignettesCe epatocyte g ettes
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iPS Cell TechnologyUnlimited Potential
iPS cells are uniquely useful stem cellsf Derived from adult tissue via non-invasive methods
Can be expanded indefinitely Can be differentiated into any cell type in the body Fully pluripotent
iPS cells offer distinct advantages to ES cellsCan be created ia streamlined & non in asi e methods Can be created via streamlined & non-invasive methods
Eliminates political/social issues regarding tissue source Enables diversity of genotype and phenotype
Reprogram iPS cells multiplyDraw 1 small Differentiate iPS cells into
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Reprogram sample tissue into iPS cells
iPS cells multiply and expand in culture indefinitely
Draw 1 small sample from 1 person
any cell type in the body (unlimited numbers)
CDI Overview
Cellular Dynamics International (CDI) is the world’s largest producer of human iPS cells d iPS ll d i d ll tand iPS cell-derived cell types
Currently employs ~107 people in Madison, WI
Core competencies
Creation and culture of human iPS cells
Genetic engineering of iPS cells
Manufacture of human iPS cell-derived cell types Manufacture of human iPS cell derived cell types
iCell® Cardiomyocytes – first commercial product
iCell® Endothelial Cells – launched Q3 2011
iCell® Neurons - launched Q4 2011
iCell® Hepatocytes – pre-commercial evaluation
MyCell Custom Services
iPS cell reprogramming
iPS cell genetic engineering
iPS cell differentiation The Wall Street Journal
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iPS cell differentiation The Wall Street JournalGold Winner: CDITechnology Innovation Awards 2011
CDI OverviewQuality, Quantity, Purity
Quality Exhibit key cellular characteristics Recapitulate normal human biology Reproducible Target Cell (non proliferating) Reproducible Known and relevant genotype
Purit
y
Target Cell (non proliferating)
Cel
l
Days in Culture
Non-Target Cell (proliferating)
Quantity Purity
ays Cu tu e
Sufficient to support HTP drug screening and safety testing
Currently 1Bn iCell Cardiomyocytes/day
>95% pure Maintained over time
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y y y y Currently up to 1.2 Bn iCell Hepatocytes/week
(shipped >5 Bn to external partners in Q4 2011)
CDI OverviewPeer-Reviewed Publications
Booth #1507Workshop, Tues. Mar 13, 6-8:30PM , Intercontinental Hotel
Presentation Outline
iPS Cell Technology & Company Overview
iC ll P d t iCell Products iCell Cardiomyocytes
iCell Hepatocytes
Assessing Mechanisms of Toxicity through g y gMultiparametric Analysis of iCell Cellular Test Systems
iCell Hepatocyte VignettesCe epatocyte g ettes
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iCell CardiomyocytesProtein Characterization
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iCell CardiomyocytesGenomic Characterization
Gene ExpressioniCell Cardiomyocytes d28 and d120
Stem cellGene Category
Transition
Primary Heart culturesNovartis GNF expression atlas
Heart expression ≥ 10X median tissue expression
GO analysis confirms Cardiac-specific enrichment
Cardiomyocyte“More human than human”
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Data courtesy of Uli Certa and Josh Babiarz
iCell CardiomyocytesElectrophysiology Characterization
I ISpontaneous Action PotentialsIonic Currents
INa ICa-L
Ito IKr
Ifunny IK1 ElicitedAction Potentials
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iCell HepatocytesMorphology
Adherent monolayer
Round nucleus
Distinct nucleoli Distinct nucleoli
High cyto/nuclear ratio
Bi-nucleation (circles)
Bile canaliculi (arrows)
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iCell HepatocytesMorphology
Bile Canaliculi – 4 days post-plating
2 hours
4 days Hoechst1 day
2 d
CDFDA O l
2 days
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CDFDA Overlay
3 days
iCell HepatocytesLong-term culture vs. PHH
Day 1 Day 21Day 1 Day 21
Thank You
iCell Hepatocytes
Thank You
Primary Human Hepatocytes
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Poster #519
iCell HepatocytesProtein Characterization
Alpha-1-antitrypsin HNF4a
Albumin Hoechst - Occludin
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iCell HepatocytesIntrinsic Function
PAS
Oil Red BODIPY
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Presentation Outline
iPS Cell Technology & Company Overview
iC ll P d t iCell Products iCell Cardiomyocytes
iCell Hepatocytes
Assessing Mechanisms of Toxicity through g y gMultiparametric Analysis of iCell Cellular Test Systems
iCell Hepatocyte VignettesCe epatocyte g ettes
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Cell ViabilityCellTiter-Glo®
CellTiter-Glo® Cell Viability Assay iCell Cardiomyocytes Luminescent, ATP-based assay Single reagent “add-mix-measure” format Amenable to automated HTS, cell
proliferation & cytotoxicity assaysproliferation & cytotoxicity assays
- 2- 4- 6- 8- 10- 12Log [Compound] M
iCell HepatocytesTroglitazone
TamoxifenTerfenadineTheophylline
iCell Hepatocytes
AcetaminophenChlorpromazine
17Log [Compound] M
Cell ViabilityCellTox-Green
iCell Hepatocytes
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Apoptosis/NecrosisApoTox-Glo
ApoTox-Glo Triplex Assay Viability, Cytotoxicity, Apoptosis
Step 1: Live cell measurement of viability & cytotoxicity
Step 2: Measurement of apoptosis
y, y y, p p
Utilizes two distinct fluorogenic substrates Cell permeable live-cell protease substrate Cell impermeable dead-cell protease substrate
Utilizes a luminogenic caspase-3/7 substrate
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Apoptosis/NecrosisApoTox-Glo
iCell Cardiomyocytes iCell HepatocytesiCell Cardiomyocytes iCell Hepatocytes
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Cell Type SpecificityProteasome Inhibitors
Bortezomib (PS-341; marketed as Velcade): first FDA-approved proteasome
iCell Cardiomyocytes*K562
inhibitor for use in the treatment of certain myeloma cancers
*Bortezomib (and other proteasome inhibitors) are known to partially
On-target potency(cytotoxicity by apoptosis)
Off-target safety
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( ) yinhibit the viability assay protease biomarker at concentrations >1μM; >50 fold the EC50 for K562 cells.
Cell Type SpecificityHDAC Inhibitors
U937 iCell Cardiomyocytes
Log [Apicidin] M Log [Apicidin] M
K562 iCell Cardiomyocytes
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On-target potency(cytotoxicity by apoptosis)
Off-target safety
Mitochondrial ToxicityMitochondrial ToxGlo Assay
Mitochondrial Tox-Glo AssayStep 1: iCell CardiomyocytesStep 1: Live cell measurement of cytotoxicity Utilizes a cell-impermeable dead-cell protease
fluorogenic substrateSt 2Step 2: Utilizes a luminogenic measure of ATP
K562
No MitoTox
MitoTox
• Oligomycin: phosphorylation inhibitor• Antimycin: respiratory chain inhibitor• Na Azide: electron transport chain inhibitor
CCCP: uncoupling agent (respiratory & phosphorylation)
MitoTox
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• CCCP: uncoupling agent (respiratory & phosphorylation)
Presentation Outline
iPS Cell Technology & Company Overview
iC ll P d t iCell Products iCell Cardiomyocytes
iCell Hepatocytes
Assessing Mechanisms of Toxicity through g y gMultiparametric Analysis of iCell Cellular Test Systems
iCell Hepatocyte VignettesCe epatocyte g ettes
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Hepatotoxicity: AflatoxinHepatotoxicity: Aflatoxin
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iCell HepatocytesHepatotoxicity: Aflatoxin
Aflatoxins: • Naturally occurring mycotoxins produced by the fungus Aspergillus
120150
• Naturally occurring mycotoxins produced by the fungus Aspergillus• Metabolized by the liver to a reactive intermediate
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80
100
120
+ Keto + ANF- Keto + ANF
100
150HepG2Hum HepiPS Hep
lity
(RLU
)
HepG2PHHiCell Hepatocytes
00
20
40
10 20 30 40 50 60 70 80 90 1000 10 20 30 40 500
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% V
iabi
0 10 20 30 40 50 60 70 80 90 1000 10 20 30 40 50 Aflatoxin-B1 (M)
Conclusions:Conclusions: iCell Hepatoyctes exhibit a hepatotoxic response to Aflatoxin-B1,
similar to primary human hepatoyctes
Aflatoxin-B induced hepatotoxicity is inhibited by ketoconazole
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Aflatoxin-B1 induced hepatotoxicity is inhibited by ketoconazoleand alpha-naphthoflavone, inhibitors of P450 enzymes
iCell HepatocytesHepatotoxicity: Aflatoxin
ApoTox-GloCellTiter-Glo®
GSH/GSSG-Glo Luminescent Glutathione Assay
SH/G
SSG
SH/G
SSG
Glutathione Assay
PHHiCell Hepatocytes
Rat
io G
S
Rat
io G
S
PHHiCell Hepatocytes
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Aflatoxin (μM) Menadione (μM)
Hepatotoxicity: Rheumatoid ArthritisHepatotoxicity: Rheumatoid Arthritis
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iCell HepatocytesHepatotoxicity: Rheumatoid Arthritis
Conclusions:Conclusions: No apoptosis or cytotoxicity is observed in RO-1 treated cells
Both RO-2 and RO-3 cause rapid increase in caspase 3/7 within 5 h resulting in cell death within 24 h
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resulting in cell death within 24 hPoster #519
iCell HepatocytesHepatotoxicity: Rheumatoid Arthritis
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Poster #519
Summary
• iPS cell technology provides unprecedented access to human biology.
• CDI has developed a novel commercial platform for reprogramming and differentiating iPS cells and products.
• Promega has developed an extensive portfolio of simple, predictive assays for determining mechanisms of toxicityassays for determining mechanisms of toxicity.
• When used together, CDI’s iCell products & Promega’s photometric-based assays provide a relevant & accessible platform for assessing mechanisms of toxicity.
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Thank YouThank You
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Proteasome-Glo Cell-Based Assays Luminescent, real-time assays to measure:
Chymotrypsin-like proteasome activity Trypsin-like proteasome activity Caspase-like proteasome activity
“Add-mix-measure” format amenable to automated HTS
“The proteasome: a suitable antineoplastic target”Julian Adams (2004) Nature Reviews Cancer. 4:349-360( )
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