application of human ips cell-derived models for highly ... · similar to adrenaline platform...
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Application of Human iPS Cell-Derived Models for Highly
Predictive Toxicity Screening Dominic Hussey, Giorgia Salvagiotto, Shannon Einhorn, Coby Carlson,
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. Rat spinal cord
(RSC) cells exhibit high sensitivity to BoTN
but 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 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 assess 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
withdrawal 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
predictivity that these cells can offer the
scientific community for toxicity screening.
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 (e.g.. 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 at the University of
Wisconsin-Madison and Dr. Kyle Kolaja,
formerly at Roche, for the use of their
published data in this poster.
A
Industrial-Scale Production of
iPS Cell-Derived Tissue Cells
Cardiomyocytes Neurons
Hepatocytes
iPS Cells
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 Cardiomyocytes/day
Ce
ll P
uri
ty
Days in Culture
Target Cell (non proliferating)
Non-Target Cell (proliferating)
MyCell® Products
iPS Cell Reprogramming
iPS Cell Genetic Engineering
iPS Cell Differentiation
Endothelial Cells
iCell® Products
Astrocytes
CD34+ HPC
Additional cell types in development
Quality
Target
Identification Target
Validation
Compound
Screening
Lead
Optimization Preclinical
Trials
Clinical
Trials