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©2013 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-Throughput Assays for Hepatotoxicity and Cardiac
Physiology Using iPSC-Derived Cell ModelsOksana Sirenko1, Jayne Hesley1, Carole Crittenden1, Vanessa Ott2, Blake Anson2, Ivan Rusyn3, and Evan F. Cromwell1
1Molecular Devices, LLC, 1311 Orleans Drive, Sunnyvale, CA 94089, 2Cellular Dynamics International, 525 Science Drive, Madison WI 537113 University of North Carolina, Chapel Hill, NC 27599
Introduction
Materials & Methods
Summary• We developed and validated assays for measuring impact of pharmacological
compounds on beating rate of stem cell derived cardiomyocytes with FLIPR Tetra
System or SpectraMax i3 and multi-parametric analysis of beating profiles. The
estimated predictive value of the assay was greater than 80%, while the multi-
parametric analysis has demonstrated prediction long QT syndrome and other
types of cardiotoxicity.
• 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.
• We demonstrate utility of these in vitro assay models for toxicity screening and
understanding potential hepatotoxic effects early in the drug development process.
Hepatotoxicity Library ScreeningA 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. 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. The assay was used to asses toxicity of
compounds in the library (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
Toxicity Cell CountOther
Parameters
Sensitivity 40% 60%
Specificity 97% 91%
Predictive value 69% 75%
• 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.
•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 (
11)
Kno
wn
toxi
ns (
13)
Ant
i-His
tam
ines
(7)
0102030405060708090
100
Ass
ay s
ensi
tivity
(%
)
We tested the Screen-Well™ Cardiotoxicity Library (ENZO Life Sciences): 107 compounds
representing different types of cardiotoxic compounds, as well as 24 non-toxic substances. We
measured the concentration-response effects of these compounds after 30 min or 24 hrs of
incubation; 4-6 concentrations were tested. Overall, 69 compounds affected beating frequency, as
well as other parameters, greater than 3 SDs from DMSO controls in at least one concentration.
Only one of the negative control compounds caused significant deviations of beating patterns at the
highest concentration. Among positive compounds were cardiac glycosides, anti-arrythmic
compounds, α- and β-adrenoreceptor blockers, Ca2+ and K+ channel blockers, dopamine receptor
antagonists, tricyclic anti-depressants, and anti-cancer drugs. The estimated predictive value of the
assay was greater than 80%, while the multi-parametric analysis has demonstrated great value in
prediction long QT syndrome and other types of arrhythmic and non-arrhythmic cardiotoxicity.
Left: Results from screening a library of cardiotoxic
(top) and “safe” (bottom) compounds. Color code
shows deviations from DMSO controls. Clustering
order set using “peak width” at 30 min.
Right: Principal component analysis using chemical
descriptors (top), or responses in the
cardiomyocyte beating assay at 30 min and 24 hrs.
Calcein AM, MitoTracker Orange, Hoechst
3µM 10µMcontrol
Cardiotoxicity Library Screening
1 2 3 4
Control Epinephrine Cisapride
1
23
4
Inte
nsity
Time
Beats per Minute
BPM + Peak Width
High Content Toxicity
Sensitivity 66% 86% 58%Specificity 100% 93% 92%Predictivity 82% 89% 75%
Cardiomyocyte Beating Assay using Intracellular Ca2+ Flux
iPSC derived iCell® Cardiomyocytes
•Cells were received frozen from Cellular Dynamics International (CDI). Cells were thawed
and plated according to CDI protocol at 20K/96well plate or 4 K/384 well on gelatin coated
plates and incubated for typically 7 days. The presence of strong synchronous contractions of
cells in the wells under the light microscope was confirmed prior to running experiments.
FLIPR® Tetra System Ca2+ Flux Assay
•The assays employ FLIPR® Calcium 5 dye (Molecular Devices), a calcium sensitive dye, to
monitor changes in Ca2+ fluxes synchronous with cell beating. Reagents were added to the
plates and incubated 30 min at 37oC in 5% CO2.
•Experimental plates were loaded and a pre-drug read was acquired. Acquisition was set up
for FLIPR Calcium 5 dye (Ex 485nm, Em 530nm). Data acquired at ~8 fps
•FLIPR Tetra System reads were acquired during compound addition and at prescribed times
after addition (read time ~ 2 min).
•Data was analyzed using FLIPR Screenworks® Peak Pro™ software to automatically detect
and characterize individual contraction events.
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 appropriate
compounds for 72 hr.
Cardiotoxicity Using SpectraMax® i3 System with MiniMax Option
Isoproterenol10 µµµµM
Control Haloperidol 10 µµµµM
Doxorubicin 30 µµµµM
Cell viability determination via imaging with live-cell stains can provide complementary information to that
obtained by the cardiac beating assay. We have developed capabilities on the new SpectraMax i3 multi-
mode plate reader to do fast kinetic fluorescence measurements of contracting cardiomyocytes. Beat rate
and peak parameters are automatically calculated by the Peak Pro algorithms contained in the SoftMax Pro
plate reader control software. In addition the SpectraMax MiniMax imaging cytometer option allows images
to be acquired of the same wells to provide phenotypic information as well as concentration response of cell
viability.
Development of highly predictive in vitro assays is extremely important for improving the
drug development process and reducing drug attrition due to toxicity. iPSC-derived cell
models are rapidly being adopted by the pharmaceutical industry for pre-clinical toxicity
studies. iPSC-derived cells provide relevant human biology and are increasingly being
shown to accurately predict drug-induced toxicity. Human iPSC-derived hepatocytes and
cardiomyocytes show great promise with respect to primary tissue-like phenotype,
unlimited availability, and a potential to establish cells from different individuals. To enable
the full potential of iPSC-derived cell models, it is necessary to develop highly predictive in
vitro assays that can be performed in a high throughput manner.
We have developed several high content imaging (HCI) assays to assess drug-induced
hepatotoxicity and cardiotoxicity. First, we used HCI and iPSC-derived hepatocytes to assess
general and mechanism-specific liver toxicity. We have also developed a physiological assay
for measuring the impact of pharmacological compounds on the beating rate of human
iPSC-derived cardiomyocytes with fast kinetic fluorescence imaging systems.
Cardiomyocyte contraction rate and pattern was characterized via changes in intracellular
Ca2+ measured using calcium sensitive dyes. These assays have been adapted for 384 well
plates and automated protocols were developed to use for small library screens.
Top: Time lapse series of images of contracting cardiomyocytes loaded with a Ca2+ sensitive dye.
Bottom: Integrated intensity versus time showing beating behavior for three different conditions.
Relative timing of images with respect to a contraction event is indicated numerically.
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.
Images and beat traces of iCell Cardiomyocytes
treated as indicated for 30 min. Data acquired
on SpectraMax i3 system with MiniMax option.
Compound Profiling
JC -10Granules Calcein AM NuclearPositive
cell countJC -10
Ln (
fold
ch
ange
vs
DM
SO
)
-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
Tot
al
Tot
al A
rea
Ave
Are
a
Inte
nsity
Ave
Are
aA
ve I
nten
sity
Cal
cein
AM
Mito
Tra
cker
Tot
al c
ells
No/
cell
Tot
al
Assay Sensitivity by Compound Class
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