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Predictive High-Content/High-Throughput Assays for Hepatotoxicity Using Induced Pluripotent Stem Cell (iPSC)-Derived Hepatocytes

Oksana Sirenko, Jayne Hesley, Emile Nuwaysir,1 Ivan Rusyn,2 and Evan F Cromwell Molecular Devices, LLC, 1311 Orleans Drive, Sunnyvale, CA 94089, 1Cellular Dynamics International, 525 Science Drive, Madison WI 53711, 2Univeristy of North Carolina, Chapel Hill NC 27599

Human iPSC-derived hepatocytes have been developed as a replacement for primary cells and show promise with respect to liver-like phenotype, unlimited availability, and a potential to establish cells from individuals who are prone/resistant to adverse drug reactions. Accordingly, there is great interest in using iPSC-derived hepatocytes as tools for screening in drug development. While unlimited supply of such cells from multiple donors addresses one common bottleneck (i.e., availability of cells), it is yet to be shown that iPSC-derived hepatocytes are amenable to high-throughput and high-content screening analyses. In this project we tested several automated screening approaches for assessing general and mechanism-specific hepatotoxicity using iPSC-derived hepatocytes. We found that multi-parametric automated image analysis greatly increases assay sensitivity while also providing important information about possible toxicity mechanisms. Specifically, we found for testing a library of 240 compounds an assay sensitivity of 60% with a specificity of 91% and predictivity of 75%. This was superior to evaluation of cell viability endpoint only. We conclude that the high-throughput and high-content automated screening assays using iPSC-derived hepatocytes is feasible and can facilitate safety assessment or drugs and chemicals.

References 1. O'Brien PJ, Irwin W, Diaz D, Howard-Cofield E, Krejsa CM, Slaughter MR, Gao B, Kaludercic N, Angeline A, Bernardi P, Brain P, Hougham C. High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening. Arch Toxicol. 2006 Sep;80(9):580-604. 2. Anderson N, Borlak J (2006). "Drug-induced phospholipidosis". FEBS Lett 580 (23): 5533–540.

Summary

• Live-cell assays using the ImageXpress Micro XL High Content Imaging System with human iCell Hepatocytes can measure the impact of pharmacological compounds on hepatocyte viability and intrinsic hepatocyte functions.

• Multi-parametric read-outs allow simultaneous assessment of viability, membrane permeability, lipid accumulation, cytoskeleton integrity, and mitochondrial depolarization in live cells and increased assay sensitivity.

• We demonstrate utility of these in vitro assay models for toxicity screening and understanding potential hepatotoxic effects early in the drug development process.

High Content Imaging •Images were acquired using the 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.

Multi-Parameter Cytotoxicity Assay Multi-parametric Image Analysis can be used to monitor changes in cell viability (Calcein AM), nuclear shape (Hoechst), and mitochondria integrity (MitoTracker Orange) associated with different types of toxicity. The ImageXpress Micro system allows automatic analysis on a cell-by-cell basis using the MetaXpress 5 software Multi-Wavelength Cell Scoring (MWSC) module.

Figure 2. Top: iCell Hepatocytes treated with Amitriptyline for 48 hours. Images taken with 10x

objective and analyzed with MWSC module. Response for various compounds of known

mechanism of action using analysis for viable cells. All IC50 values given in mM.

Automated Image Analysis • Image analysis was done using MetaXpress® 5 Software and image processing modules, including Multi-Wavelength Cell Scoring, Live-Dead and Granularity.

• New Custom Module Editor (CME) capabilities derived from industry leading

MetaXpress 5 software have been developed to allow users to expand their abilities to characterize phenotypic changes.

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.

Compound Library Screening Screen-Well™ Hepatotoxicity Library (ENZO) 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.

Calcein AM, MitoTracker Orange, Hoechst

High Content Imaging Characterization

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)

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

Top: Albumin secretion was shown to be similar to primary human hepatocytes. Cells characterized by glycogen storage using periodic acid-Schiff (PAS) staining (Top Right) and lipid production using Oil Red and BODIPY staining (Bottom)

Methods 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 appropriate compounds for 72 hr.

PAS

Oil

Red BODIPY

Autophagy & Phospholipidosis Selective degradation of intracellular targets, such as misfolded proteins and damaged organelles is an important homeostatic function of the cell. In disease, autophagy may function as a survival mechanism by removing damaged organelles and toxic metabolites to maintain viability during periods of stress. Autophagic machinery can be manipulated to treat human diseases. Phospholipidosis is a lysosomal storage disorder characterized by the excess accumulation of phospholipids in tissues. Many cationic amphiphilic drugs, including anti-depressants, antianginal, antimalarial, and cholesterol-lowering agents, are reported to cause drug-induced phospholipidosis (DIPL) in humans.

Figure 7. A: Autophagy Assay. Images of iCell Hepatocytes treated with Rapamycin for 24 hr and stained with Cyto-ID Autophagy detection kit. Analysis with Granularity module is shown on the right. B: Phospholipidosis, lipid accumulation assay. Images of iCell Hepatocytes treated with propranolol for 48 hr. Phospholipidosis and steatosis detected using LipidTOX reagent showing phospholipids and neutral lipids as indicated. C: Concentration dependent responses using Total Granular Area output and corresponding IC50 values for phospholipidosis.

Specific Toxicity Assays

3mM 10mM control

Nuclear condensation is characterized by: “decreased nuclear area” + “increased average intensity”. This can provide additional sensitivity to toxicity.

Output parameters:

– Number of Calcein positive cells

– Number of MitoTracker positive

– 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

Toxicity Cell Count Other

Parameters

Sensitivity 40% 60%

Specificity 97% 91%

Predictive value 69% 75%

Assay Sensitivity by Compound Class

• High predictivity was found for many classes of compounds, e.g. neuroleptic, anti-cancer, cardiac drugs, toxins. • Lower predictivity was observed for anti-inflammatory, antibiotics, and anti-viral drugs.

Hepatotoxicity Phenotypes

•Assay Sensitivity “hits” based on output parameters deviating more than ±3s from DMSO controls.

•Number of compounds in group shown in parentheses

AC

E inhib

itors

(7)

Nucle

osid

e a

nalo

g R

TIs

(10)

Non

-he

pa

toto

xic

(3

2)

Oth

er

cla

sses (

30)

Ora

l hypogly

cem

ics (

4)

Anti-I

nflam

mato

ry (

23)

Antibio

tics (

32)

Sta

tins (

chole

ste

rol) (

11)

Neuro

lep

tics (

27)

Horm

onal (7

)

Anti-C

ancer

(14)

Vascula

r (7

)

Anti-f

ungal (8

)

Card

iac d

rugs (

11)

Know

n toxin

s (

13)

Anti-H

ista

min

es (

7)

0

10

20

30

40

50

60

70

80

90

100

Assay s

ensitiv

ity (

%)

JC - 10 Granules

Calcein AM Nuclear Positive cell count

JC - 10

Ln (

fold

ch

an

ge

vs 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

Colchicine Atorvastatin

Imipramine

Control

Tolcapone Naproxen

Sulindac Tolmetin Papaverin

Carboplatin 51

Valproate no tox

Staurosporine 1.71

Mercury 5.05

Phenylbutasone 24

Doxorubicin 0.71

IC50 (mM)

V

iab

le C

ells

Concentration, mM

Antimycin A Control

Neutral lipids Phospholipids

Propranolol B

IC50, mM

Amiodarone 18

Cyclosporin A 1.3

Propranolol 25.7

Chloroquine 6.5

Concentration, mM

Tota

l G

ranula

r Are

a

Granularity Analysis

Rapamycin A

Aflatoxin Control

Aflatoxin B4 1.9

Staurosporine 2.0

Aflatoxin B1 5.4

Idarubicin 7.4

Mitomycin C 19

Retinoic acid 480

IC50 (mM)

Cells/A

ctin

Concentration, mM

Cytoskeleton Integrity

Cytoskeleton integrity was assessed by phalloidin staining. The parameters measured in this assay were count of cells positive for actin staining, total positive cell area, or integrated fluorescent intensity. These measurements can be used for the characterization of concentration dependent hepatotoxicity response. The cytoskeleton integrity assay had an excellent assay window (W>50) and low variance (Z’-value >0.8).

Bottom: Transmitted light image of iCell Hepatocytes after 4 days in culture. Bi-nucleation is indicated by circles and bile canaliculi by arrows.

Figure 4. Top: Images are 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.

Phenotypic characterization of hepatotoxicity by quantitation of the average and total stained cell areas.

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.

Figure 6. Top: Images of iCell Hepatocytes treated with 30 mM of Aflatoxin B1 for 72 hr, then fixed and stained with AlexaFluor-488 -Phalloidin and Hoechst 33258. Bottom: Concentration-dependent responses for several compounds using number of actin positive cells as a read-out.

C

Introduction

Figure 1. Characterization of iCell Hepatocytes.