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.