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