Multi-Parameter Cytotoxicity Assay Specific Toxicity Assays Compound Library Screening ·...
1
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners. 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, 1 Cellular Dynamics International, 525 Science Drive, Madison WI 53711, 2 Univeristy 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 IC 50 values given in μM. 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 _ Aflatoxin 1 10.7 Idarubicin 8.9 Retinoic acid 351 Staurosporine 3.1 Mitomycin C 19.8 Aflatoxin 4 3.4 0.1 1 10 100 1000 38 48 58 68 78 88 98 Concentration, μM Nuclear Area IC 50 (μM) 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 1e-7 1e-6 1e-5 1e-4 0.001 0.01 0.1 1 10 100 1000 0 10 20 30 Granules/cell Concentration, μM IC50, μM 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 IC 50 values for phospholipidosis. Specific Toxicity Assays 3μM 10μM 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 75% 69% Predictive value 91% 97% Specificity 60% 40% Sensitivity Other Parameters Cell Count Toxicity 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 ±3σ from DMSO controls. •Number of compounds in group shown in parentheses ACE inhibitors (7) Nucleoside analog RTIs (10) Non-hepatotoxic (32) Other classes (30) Oral hypoglycemics (4) Anti-Inflammatory (23) Antibiotics (32) Statins (cholesterol) (11) Neuroleptics (27) Hormonal (7) Anti-Cancer (14) Vascular (7) Anti-fungal (8) Cardiac drugs (11) Known toxins (13) Anti-Histamines (7) 0 10 20 30 40 50 60 70 80 90 100 Assay sensitivity (%) JC -10 Granules Calcein AM Nuclear Positive cell count JC -10 Ln (fold change vs DMSO) -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/cell Total Total Area Ave Area Intensity Ave Area Ave Intensity CalceinAM MitoTracker Total cells No/cell Total 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 IC 50 (μM) Viable Cells Concentration, μM Antimycin A Control Neutral lipids Phospholipids Propranolol B IC 50 , μM Amiodarone 18 Cyclosporin A 1.3 Propranolol 25.7 Chloroquine 6.5 Concentration, μM Total Granular Area 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 IC 50 (μM) Cells/Actin Concentration, μM 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 μM 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 μM 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 μM 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.
Transcript of Multi-Parameter Cytotoxicity Assay Specific Toxicity Assays Compound Library Screening ·...
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.
Predictive High-Content/High-Throughput Assays for Hepatotoxicity Using
Induced Pluripotent Stem Cell (iPSC)-Derived HepatocytesOksana 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.
References1. 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 cytotoxicitymeasured 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 hepatocytefunctions.
• 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 AssayMulti-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 µM.
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 ScreeningScreen-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
__________
Aflatoxin 1 10.7Idarubicin 8.9Retinoic acid 351Staurosporine 3.1Mitomycin C 19.8Aflatoxin 4 3.4
0.1 1 10 100 100038
48
58
68
78
88
98
Concentration, µM
Nuc
lear
Are
a
IC50 (µM)
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 47Valinomycin 1
Tolcapone 25.7Nefazodone 33.6Leflunomide 34.3Ketoconazole 71.4Isoniazid -Fusariotoxin 132Flutamide 22.2
1e-7 1e-6 1e-5 1e-4 0.001 0.01 0.1 1 10 100 1000
0
10
20
30
Gra
nule
s/c
ell
Concentration, µM
IC50, µMIC50, 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)
MethodsCell 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 & PhospholipidosisSelective 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 LipidTOXreagent 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
3µM 10µMcontrol
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
75%69%Predictive value
91%97%Specificity
60%40%Sensitivity
Other ParametersCell CountToxicity
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 ±3σ from DMSO controls.
•Number of compounds in group shown in parentheses
AC
E in
hibi
tors
(7)
Nuc
leos
ide
anal
og R
TIs
(10)
Non
-hep
atot
oxic
(32
)
Oth
er c
lass
es (
30)
Ora
l hyp
ogly
cem
ics
(4)
Ant
i-Inf
lam
mat
ory
(23)
Ant
ibio
tics
(32)
Sta
tins
(cho
lest
erol
) (1
1)
Neu
role
ptic
s(2
7)
Hor
mon
al (
7)
Ant
i-Can
cer
(14)
Vas
cula
r (7
)
Ant
i-fun
gal (
8)
Car
diac
dru
gs (1
1)
Kno
wn
toxi
ns (
13)
Ant
i-His
tam
ines
(7)
010
203040
506070
8090
100
Ass
ay s
ensi
tivity
(%
)
JC -10Granules Calcein AM NuclearPositive
cell countJC -10
Ln(f
old
chan
ge
vsD
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/
cell
Tota
l
Tota
l Are
a
Ave
Are
a
Inte
nsity
Ave
Are
aA
ve In
tens
ity
Cal
cein
AM
Mito
Tra
cker
Tota
l cel
ls
No/
cell
Tota
l
ColchicineAtorvastatin
Imipramine
Control
TolcaponeNaproxen
SulindacTolmetin Papaverin
Carboplatin 51Valproate no toxStaurosporine 1.71Mercury 5.05Phenylbutasone 24Doxorubicin 0.71
IC50 (µM)
Via
ble
Cel
ls
Concentration, µM
Antimycin AControl
Neutral lipidsPhospholipids
PropranololB
IC50, µMAmiodarone 18Cyclosporin A 1.3Propranolol 25.7Chloroquine 6.5
Concentration, µM
Tota
l G
ranula
r Are
a
Granularity Analysis
RapamycinA
AflatoxinControl
Aflatoxin B4 1.9Staurosporine 2.0Aflatoxin B1 5.4Idarubicin 7.4Mitomycin C 19Retinoic acid 480
IC50 (µM)
Cells/A
cti
n
Concentration, µM
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 µM 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 µM 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 µM 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.
