©2013 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

High-content Assays for Hepatotoxicity Using Induced Pluripotent Stem Cell (iPSC)-Derived Cells

Oksana Sirenko1, Jayne Hesley1, Ivan Rusyn2, and Evan F. Cromwell1

1Molecular Devices, LLC, 1311 Orleans Drive, Sunnyvale, CA 94089, 2 University of North Carolina, Chapel Hill, NC 27599

Introduction

Materials & Methods

Summary • We developed and validated assays for measuring impact of pharmacological compounds on human iPSC-derived hepatocytes as a predictor of Drug Induced Liver Toxicity. • The live-cell assays used the automated ImageXpress Micro XL High Content Imaging System with human iCell Hepatocytes and measured hepatocyte viability and intrinsic hepatocyte functions. • We demonstrate utility of these in vitro assay models for toxicity screening and understanding potential hepatotoxic effects early in the drug development process.

Hepatotoxicity High Content Assay A multi-parametric imaging based assay was used to monitor changes in cell viability (Calcein AM), nuclear shape (Hoechst), and mitochondria integrity (MitoTracker Orange) associated with different types of toxicity. The assay was used to asses toxicity of various compounds in the ScreenWell™ Hepatotoxicity Library (ENZO) (72 hr, 5 concentrations). In addition, mitochondria potential/oxidative stress assay (JC-10) 60min was also used to increase overall predictivity.

Output parameters: – Number of Calcein pos. cells

– Number of MitoTracker pos.

– Total cell number

– Calcein AM intensity

– Calcein AM cell area

– MitoTracker intensity

– MitoTracker cell area

– Nuclear area

– Nuclear average intensity

– JC-10 Granules/Cell

– JC-10 Total Granules

Calcein AM, MitoTracker Orange, Hoechst

3mM 10mM control

High Content Imaging •Images were acquired with an ImageXpress® Micro XL System using 20x, 10x, or 4x objectives. •The following filters were used

• Calcein AM, Cyto-ID, Neutral Lipids: FITC Filter Cube • MitoTracker Orange, Phospholipids: TRITC Filter Cube • Hoechst: DAPI Filter Cube.

Cell Preparation • iPSC-derived hepatocytes (iCell® Hepatocytes) from Cellular Dynamics International (CDI) were plated according to their recommended protocol. • Cells were plated at a density of 60K/well (96-well plate) or 15K/well (384-well plate) on collagen coated plates and incubated for 2-3 days. Then cells were treated with compounds for 72 hr.

Development of predictive in vitro assays for early toxicity evaluation is extremely important for improving the drug development process and reducing drug attrition rates during clinical development. High-content imaging based in vitro toxicity assays are emerging as efficient tools for safety and efficacy testing to improve drug development efficiency. In this report we have used an iPSC-derived hepatocyte cell model having a primary tissue-like phenotype, unlimited availability, and the potential to compare cells from different individuals. We examined a number of assays and phenotypic markers and developed automated screening methods for assessing multi-parameter readouts of general and mechanism-specific hepatotoxicity. Endpoints assessed were: cell viability, nuclear shape, average and integrated cell area, mitochondrial membrane potential, phospholipid accumulation, cytoskeleton integrity, and apoptosis. We assayed compounds with known mechanisms of toxicity and also evaluated a diverse hepatotoxicity library of 240 compounds. We conclude that high-content automated screening assays using iPSC-derived hepatocytes are feasible, provide information about mechanisms of toxicity, and can facilitate the safety assessment of drugs and chemicals.

Figure 1. Left: Images of iCell Hepatocytes treated with indicated concentrations of amitryptiline for 72 hr. Images were taken with ImageXpress Micro XL system using a 10x objective. Bottom: Response for various compounds of known mechanism of action using analysis for viable cells.

Compound Library Screen Results

JC - 10 Granules

Calcein AM Nuclear Positive cell count

JC - 10

Ln (

fold

change v

s D

MS

O)

-16

-14

-12

-10

-8

-6

-4

-2

0

2

1 2 3 4 5 6 7 8 9

Lamivudine

Miconazole

Simvastatin

Aflatoxin B1

Leflunomide

Nimesulide

Flecainide

Amiodarone

Daunorubicin

Mitomycin C

Chlorprothixene

Prochloperazine

No/c

ell

Tota

l

Tota

l Are

a

Ave A

rea

Inte

nsity

Ave A

rea

Ave Inte

nsity

Calc

ein

AM

MitoT

racker

Tota

l cells

No/c

ell

Tota

l

Figure 2. Image processing by Multi-Wavelength Cell Scoring application module. Masks shown define FITC staining (green), MitoTracker staining (orange), and Hoechst 33258 staining (grey).

Figure 6. Multi-Parametric Hepatotoxicity Analysis. ToxPi scores were used to create a relative ranking of 240 screened compounds using multi-parametric data (total cells, viable cells, cells with intact mitochondria, area covered by live cells, mean area of live cells (cell spreading), mean intensity of Calcein AM, nuclear mean area, nuclear stain average intensity). Yellow circles – non-toxic drugs, clear diamonds – hepatotoxic drugs. Representative ToxPi-es for three compounds are shown to visualize the relative contribution of each parameter (represented in colors of each slice) to the overall score. Note: Non-toxic compounds are expected to align with a higher Relative Activity Index value. Automated Image Analysis

•Images were analyzed using MetaXpress Software with the following application modules. • Multitoxicity Assay: Multi-Wavelength Cell Scoring • JC-10 Assay: Granularity • Nuclear Condensation: Custom Module Editor

Hepatotoxicity Phenotypes

Colchicine Atorvastatin

Imipramine

Control

Tolcapone Naproxen

Sulindac Tolmetin Papaverin

While a decrease in cell number is a very strong indicator for cytotoxicity, more subtle changes in cell appearance and morphology may be good early indicators of cell stress. We used phenotypic characterization of hepatotoxicity by quantitation of the average and total stained cell areas. Certain compounds

Figure 3. Images of iCell Hepatocytes treated with 100 mM of indicated compounds for 72 hr, then stained with Calcein AM and Hoechst 33258. Examples presented show impact of different compounds on total positive cell area and/or average positive cell area. Images were acquired with a 10X objective

Mitochondrial depolarization is an early signal for hypoxic damage or oxidative stress. Mitochondria membrane potential was monitored with the mitochondria active dye JC-10. Data was analyzed using the MetaXpress 5 software Granularity module. This assay can be used either as an end-point or live-cell real time assay.

Mitochondria Potential Assay

Figure 5. iCell Hepatocytes treated with compounds for 72h. Cells stained with Hoechst (nuclei), and JC-10 (mitochondria integrity). Images taken with 10x objective and analyzed with MetaXpress software Granularity module. Analysis results are shown in the bottom images. Concentration response curves and IC50 values are shown on right.

Antimycin A 5

CCCP 47

Valinomycin 1

Tolcapone 25.7

Nefazodone 33.6

Leflunomide 34.3

Ketoconazole 71.4

Isoniazid -

Fusariotoxin 132

Flutamide 22.2

Concentration, uM

1e-7 1e-6 1e-5 1e-4 0.001 0.01 0.1 1 10 100 1000

0

10

20

30

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2

antA (AntA: Concentration vs MeanValue) 23 3.22 0.00522 1.14 0.958

Plot#9 (Tolcapone: Concentration vs MeanValue) 19.1 3.64 25.7 0.331 0.993

Plot#8 (Valinomycin: Concentration vs MeanValue) 22.2 2.53 0.000841 0.372 0.978

Plot#7 (Nefazodone: Concentration vs MeanValue) 19.3 28.8 33.6 3.25 0.985

Plot#6 (Leflunomide: Concentration vs MeanValue) 19 27.3 34.3 1.52 0.988

Plot#5 (Ketoconazole: Concentration vs MeanValue) 18.4 2.23 71.4 0.795 0.997

Plot#4 (Isoniazid: Concentration vs MeanValue) 19.9 0.153 6.96e+36 -5.31e+05 0.547

Plot#3 (Fusariotoxin: Concentration vs MeanValue) 18.4 5.23 132 11.4 0.963

Plot#2 (Flutamide: Concentration vs MeanValue) 21.4 3.61 22.2 0.399 0.998

CCCP (CCCP: Concentration vs MeanValue) 21.8 13.2 0.047 0.8 0.972__________

Weighting: Fixed

Concentration, uM

1e-7 1e-6 1e-5 1e-4 0.001 0.01 0.1 1 10 100 1000

0

10

20

30

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2

antA (AntA: Concentration vs MeanValue) 23 3.22 0.00522 1.14 0.958

Plot#9 (Tolcapone: Concentration vs MeanValue) 19.1 3.64 25.7 0.331 0.993

Plot#8 (Valinomycin: Concentration vs MeanValue) 22.2 2.53 0.000841 0.372 0.978

Plot#7 (Nefazodone: Concentration vs MeanValue) 19.3 28.8 33.6 3.25 0.985

Plot#6 (Leflunomide: Concentration vs MeanValue) 19 27.3 34.3 1.52 0.988

Plot#5 (Ketoconazole: Concentration vs MeanValue) 18.4 2.23 71.4 0.795 0.997

Plot#4 (Isoniazid: Concentration vs MeanValue) 19.9 0.153 6.96e+36 -5.31e+05 0.547

Plot#3 (Fusariotoxin: Concentration vs MeanValue) 18.4 5.23 132 11.4 0.963

Plot#2 (Flutamide: Concentration vs MeanValue) 21.4 3.61 22.2 0.399 0.998

CCCP (CCCP: Concentration vs MeanValue) 21.8 13.2 0.047 0.8 0.972__________

Weighting: Fixed

Gra

nule

s/c

ell

Concentration, mM

IC50, mM

IC50, nM

Antimycin A

Control

Control cells Staurosporine

Nuclear Characterization

Concentration, uM

0.01 0.1 1 10 100 1000

0

1000

2000

3000

4000

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2

Plot#1 (Afla1: Concentration vs MeanValue) 3.73e+03 3.48 5.45 79.5 0.993

Plot#6 (Ida: Concentration vs MeanValue) 3.71e+03 3.25 7.44 246 0.999

Plot#5 (RetA: Concentration vs MeanValue) 3.79e+03 1.39 482 -684 0.991

Plot#4 (Stauro: Concentration vs MeanValue) 3.83e+03 3.97 1.96 436 0.994

Plot#3 (MitoC: Concentration vs MeanValue) 3.69e+03 28 18.8 232 0.99

Plot#2 (Afla4: Concentration vs MeanValue) 3.63e+03 4.34 1.93 100 0.998__________

Weighting: Fixed

Aflatoxin 1 10.7

Idarubicin 8.9

Retinoic acid 351

Staurosporine 3.1

Mitomycin C 19.8

Aflatoxin 4 3.4

Concentration, uM

0.1 1 10 100 100038

48

58

68

78

88

98

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2

Plot#1 (Afla1: Concentration vs MeanValue) 77.6 27.3 10.7 46.6 0.994

Plot#6 (Ida: Concentration vs MeanValue) 74.1 2.91 8.94 37.7 0.992

Plot#5 (RetA: Concentration vs MeanValue) 79.8 25.3 351 55.7 0.94

Plot#4 (Stauro: Concentration vs MeanValue) 80.8 28 3.12 41.7 0.976

Plot#3 (MitoC: Concentration vs MeanValue) 76.7 28.7 19.8 55.1 0.96

Plot#2 (Afla4: Concentration vs MeanValue) 77.6 28.8 3.4 46.5 0.996__________

Weighting: Fixed

Concentration, mM

Nucle

ar A

rea

IC50 (mM)

Nuclear condensation is characterized by: “decreased nuclear area” + “increased average intensity”. This can provide additional sensitivity to toxicity. Small, bright nuclei can also be used as an indicator of apoptosis.

Figure 4. Top: Images of iCell Hepatocytes untreated and treated with 10 mM of staurosporine, stained with Hoechst 33258. Right: Concentration-dependent responses for several compounds using average nuclear area as a read-out.

Screen-Well Hepatotoxicity Library contains 240 compounds including anti-cancer , anti-inflammatory, neuroleptic, antibiotics, and other classes. Compounds represent different mechanisms of hepatotoxicity: ALT elevation, steatosis, phospholipidosis, mitochondria damage, etc. A multi-parameter hepatotoxicity assay (Calcein AM, MitoTracker, Hoechst) 72 hr, 5 concentrations was used to asses toxicity of compounds in the library. In addition, mitochondria potential/oxidative stress assay (JC-10) 60min was also used to increase overall predictivity.

(e.g. atorvastatin) caused a significant decrease in cell area as a result of rounding or loss of cell-cell contact. In other examples (e.g., tomeltin or suldinac), an increase in average cell size was observed as a result of vacuolization, or swelling. In the case of papaverin treatment, a decrease in average cell size was observed as a result of reduced cell spreading. Such phenotypes usually appear once the cell has committed, but still prior, to cell death.

Amiodarone 7.7

Pimozide 33

Idarubicin 1.2

Mitomycin C 11

Tamoxifen 30

IC50 (mM)

Calc

ein

AM

Pos C

ells

Concentration, mM