cellular dynamics international, inc., madison, wi usa · 2019-08-26 · similar to adrenaline...
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Application of Human iPS Cell-Derived Models for
Highly Predictive Toxicity Screening
Target
Identification
Target
Validation
Compound
Screening
Lead
Optimization
Preclinical
Trials
Clinical
Trials
Coby Carlson, Giorgia Salvagiotto, Shannon Einhorn, Blake Anson,
Eugenia Jones, Susan DeLaura, David Mann, and Vanessa Ott Cellular Dynamics International, Inc., Madison, WI USA
www.cellulardynamics.com Madison, WI USA +1 (608) 310-5100
Problem: The most widely-accepted method for
measuring the potency of botulinum neurotoxin
(BoNT) is a mouse bioassay (MBA); however, it
takes four days to complete, has a large error
rate, is not standardized between labs, and
requires a large number of animals (~50 per
assay).
Current approach: Many cell-based model
systems do exist, but none examine toxin
function with species-specific relevance while
exhibiting high sensitivity. The most sensitive
cell type for BoNT detection are from primary rat
spinal cord (RSC) cells, which still require the
use of animals and skilled technical expertise
for culture preparation.
iCell-based solution: iCell Neurons were
shown to provide an ideal and highly sensitive
platform for BoNT potency determination,
neutralizing antibody detection, and for
mechanistic studies. This novel application of
human iPS cell-derived neurons offers a reliable
and scalable method that does not rely on the
use of animals for testing.
iCell Neurons have an intact system for
BoNT intoxication. iCell Neurons were
analyzed by Western Blot for expression of
the receptors and enzymatic targets
necessary for BoNT cell entry and catalytic
activity. These data indicate that the cells
express primarily SV2A, SYT1, and VAMP2
isoforms of these proteins (consistent with a
forebrain-like phenotype).
Highly sensitive in vitro assays. (Left) iCell Neurons were compared
directly to RSC cells and were found to be equally (if not more) sensitive
to BoNT/A1 exposure (concentration range of 0.014–56 U) as detected
by cleaved SNAP-25 Western blot analysis. (Right) iCell Neurons were
protected from SNAP-25 cleavage due to BoNT/A1 treatment (1.5 U of
toxin for 24 h) in the presence of a dose-response of a BoNT/A1-specific
neutralizing antibody.
Reference: Whitemarsh et al. Toxicol. Sci. (2012) 126(2), 426–435.
Cell-based Alternative to Animal Testing
iCell Neurons
RSC cells
0 0
Uncleaved
Cleaved
Toxin Detection Toxin Neutralization
A major challenge in disease research and drug
development is access to clinically relevant cell
models. Induced pluripotent stem (iPS) cell
technology offers the potential to generate such
model systems. Over the past 5 years, a rapidly
growing body of literature has demonstrated the
use of iPS cells to derive tissue-specific cell types
that are proving to be more predictive of the
human condition than immortalized cell lines or
primary rodent cultures. Here we present the
development of an industrial-scale manufacturing
platform for the production of terminally-
differentiated, human iPS cell-derived tissue types
(e.g. neurons, cardiomyocytes, and hepatocytes)
that are highly pure (>95%) and exhibit normal
genotypic, phenotypic, and functional
characteristics of native cells. It is the quantity,
quality, and purity of these cells that has been the
driving force for rapid adoption within the scientific
community. Example application data of their
functional utility will be presented, illustrating how
these cellular models have been used for various
toxicity studies and are now creating new
opportunities for therapeutic decision-making.
Abstract
Any cell type
(relevant biology)
Any individual
(population
diversity)
iPS Cell
Cardiomyocytes
Hepatocytes
Neurons
Endothelial cells
Hematopoietic cells
Other…
Somatic cells from adult tissue are used to generate human iPS cells. Cells are first
reprogrammed to a pluripotent state, and then banked, characterized, and expanded
in culture indefinitely. From the iPS cell bank, terminal cell types representing
mesoderm, endoderm, and ectoderm can be derived. By combining the power of
iPS cell technology with CDI’s refined large scale manufacturing capabilities,
commercial quantities of terminal cells can be produced from the same source
material, making them an ideal model system for studying biology in human cells.
Power of iPS Cell Technology Mechanism-Based Toxicity
Adverse effect profiles of Amiodarone. (Top) Treatment of iCell
Hepatocytes with a dose-response of drug leads to an induction of
phospholipidosis (red curve) and cytotoxicity at higher concentrations
(black curve). (Bottom) Fluorescent images of cells either untreated or
dosed with 33 M amiodarone illustrate how the increased accumulation
of phospholipids can be detected by high content imaging and specific
fluorescent probes.
Problem: Liver toxicity is a major
problem as drug candidates (and their
metabolites) can lead to undesired
effects. Relevant and reliable cell models
are lacking.
Current approach: HepG2 cells and
primary hepatocytes are cellular models
commonly used to represent toxic effects
on the liver. However, the applicability
and reproducibility of these systems are
not totally sufficient.
iCell-based solution: iCell Hepatocytes
provide a consistent source of human
cells that are compatible with many HTS-
methods, making them ideal for routine
toxicity screening of early-phase or
advanced-stage compounds.
Drug-Induced Phospholipidosis
Untreated + Amiodarone
Problem: Drug-induced adverse cardiovascular
events are the number one cause of drug with-
drawal or drug development termination. There
is a need for a higher throughput approach that
goes beyond hERG-mediated QT prolongation.
Irregular cardiac beating patterns
revealed. Known arrhythmogenic
compounds were profiled on the
xCELLigence instrument. All traces
are 20 seconds in duration.
Treatment xCELLigence MEA Notes
Baseline control
Uncouples excitation
and contraction
Na+ channel blocker
Pace-making (funny)
channel blocker
Structurally / functionally
similar to adrenaline
Platform comparison. Impedance (xCELLigence) measures
the effects of functional cardiac channels and detects physical
beating rather than electrical changes (MEA). Results
between the two platforms are quantitatively similar.
Relaxed Cardiomyocyte Contracted Cardiomyocyte
Predicted proarrhythmic score (PPS). This metric developed
by Roche establishes a threshold value for compounds tested on
iCell Cardiomyocytes using xCELLigence to separate safe drugs
from high risk molecules. It can correctly identify compounds that
inhibit hERG in vitro but have normal ECG in vivo, and is a very
valuable tool to prioritize drug candidates in early safety.
Quantitative System for Predicting Cardiac Arrhythmias
Reference:
Guo et al. Toxicol. Sci. (2011) 123(1), 281–289.
xCELLigence RTCA system from Acea offers a
reproducible and HTS-compatible solution.
Scientists at Roche pioneered the analysis of
iCell Cardiomyocytes on this platform.
Current approach: Multielectrode array (MEA) measures
electrical field potential and is a dependable technology that can
identify drug-induced liabilities in cardiac cells. Typical cell
models are unsuitable, however, because immortalized cell lines
express only one channel at high levels (and out of context),
and primary cells will eventually stop beating in culture.
iCell-based solution: The xCELLigence platform can monitor
the cellular behavior of iCell Cardiomyocytes through real-time
impedance measurements in 96-well format. Rhythmic beating
of the cardiomyocytes is ideally matched with this technology to
provide an indirect but sensitive readout of contraction.
Summary CDI is the leading producer of human iPS
cell-derived tissue types. Differentiation of
terminal cells into each of the 3 different
germ layers is now routinely possible. These
data presented here highlight the increasing
impact that the commercial availability of
human cells is having on assay development
and toxicity testing. Moreover, these
published case studies underscore the
meaning of “predictivity” that these cells can
offer other scientific researchers in the field.
CDI is constantly developing new cell types,
while at the same time working to develop
new applications for existing products to push
the limits for how iPS cell-derived products
(eg. iCell Products) can be utilized like this in
the future for safety and toxicology, drug
discovery, and cellular therapeutics.
CDI would like to acknowledge Prof. Eric
Johnson and his lab @ the University of
Wisconsin-Madison and Dr. Kyle Kolaja,
formerly @ Roche, for the use of their
published data in this poster.
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