Development and Characterization of Human iPSC-derived Neurons for Drug Discovery Applications
www.cellulardynamics.com Madison, WI USA +1 (608) 310-5100
Target
Identification
Target
Validation
Compound
Screening
Lead
Optimization
Preclinical
Trials
Clinical
Trials
Post-Thaw Morphology
The human brain represents a complex organ that has
consistently been proven difficult to model in vitro. Current models
including primary rodent tissue and immortalized cell lines have
served as mainstays in both academic research and the
pharmaceutical industry. These models, while providing a means
for numerous landmark discoveries, have suffered from various
issues including biological relevance, reproducibility and
scalability. Considerable efforts have been made specifically
within the pharmaceutical industry to reduce late-stage drug
attrition through the development of more relevant in vitro human
model systems. One area given significant attention has been the
development of platforms than can enable the modeling of human
degenerative (e.g. Alzheimer’s and Parkinson’s disease) and
genetic (i.e. Huntington's disease and muscular dystrophy)
diseases as well as neurotoxicity. The recent discovery of induced
pluripotent stem cells (iPSCs) not only overcomes the ethical and
logistical issues associated with human embryonic stem cells, but
also provides a flexible platform for generating various
differentiated cell types from diseased individuals. Using this
platform, we have developed a highly consistent and scalable
protocol to differentiate and cryopreserve purified human iPSC-
derived neurons, called iCell® Neurons. Phenotypically, these
cells are >90% pure as measured using flow cytometry for
presence of the neuronal marker class III beta tubulin (TuJ1) and
absence of the progenitor marker nestin. Within 24hrs of thawing,
these neurons display a typical neuronal morphology including a
dense network of neurites. Detailed phenotypic analyses reveal
that these neurons are comprised of a mix of predominantly
GABAergic and Glutamatergic subtypes as measured at both the
gene expression and protein levels and form characteristic
synaptic connections. Functionally, these cells reveal typical
electrophysiological characteristics as measured using single-cell
patch clamp to detect both spontaneous and evoked action
potentials as well as functional ion channels. Finally, when applied
to high throughput applications, including cytotoxicity assays, iCell
Neurons reveal characteristic pharmacological responses to
known toxic compounds. The results demonstrate not only a
novel cell model for use in various academic and pharmaceutical
applications, but they also support the use of the iPSC technology
as a platform capable of generating neurons against diverse
genetic backgrounds.
Abstract Cell-based Assays Electrophysiology
Summary
A. Evoked Action Potential B. Spontaneous Action Potential
Figure 3. iCell Neurons display characteristic subtype protein expression. Post-thaw
iCell Neurons were plated in iCell Neurons Maintenance Medium with iCell Neurons
Supplement on poly-L-ornithine/laminin-coated tissue culture plates and immunostained on
day 14 for (A) the synaptic markers vGAT (vesicular GABA transporter) and vGLUT2
(vesicular glutamate transporter 2), markers of GABAergic and Glutamatergic neurons,
respectively; and (B) MAP2 (microtubule-associated protein 2) and GABA (gamma-
aminobutyric acid). Magnification = 20x objective.
Figure 10. iCell Neurons display an expected sensitivity to known compounds. iCell
Neurons were cultured for 7-14 days post-thaw on PLO/Laminin pre-coated 96-well plates
and exposed to a dilution series of (A) staurosporine and (B) kainic acid. Viability (as
measured using cellular ATP content) was determined using the CellTiter-Glo®
Luminescent Cell Viability Assay (Promega).
Figure 4. Gene expression analysis of iCell Neurons. Day 15 post-thaw iCell Neurons
were analyzed against a focused panel of TaqMan Gene Expression Assays. The data
demonstrate that iCell Neurons represent a population with largely a forebrain identify (top),
are predominantly glutamatergic and GABAergic neuronal subtypes (middle), and express a
number of characteristic receptors (bottom).
A. B.
iCell® Neurons represent a robust, consistent and commercially
available population of human neurons for basic biological and drug
discovery applications. This highly pure population of cells (Figure 2)
displays a robust and stable neuronal morphology (Figure 1) and is
comprised of largely Glutamatergic and GABAergic neuronal
subtypes (Figures 3 and 4). iCell Neurons display evoked and
spontaneous neuron-like action potentials (Figure 5) and possess
functional sodium, potassium and calcium channels (Figure 6). In
addition, these cells are amenable to various assay systems including
high content image-based assays (Figures 7-9), standard cell-based
assays (Figure 10) and toxigenicity testing (Figure 11). The results
demonstrate a convenient, novel human cell model for neuroscience
research which supports the use of iPSC technology as a platform
capable of generating neurons from disease relevant genetic
backgrounds.
Na+ Channel Current (INa) – Tetrodotoxin Inhibition
K+ Channel Current (IK) – Tetraethylammonium Inhibition
Figure 6. iCell Neurons respond to ion channel blockers. Addition of classical neuron ion
channel antagonists tetrodotoxin (TTX,100nM), tetraethylammonium (TEA, 30mM) and
nifedipine (10µM) blocks inward sodium outward potassium, and inward calcium currents,
respectively, as measured using a single-cell patch clamp. (A) Sodium channel antagonist
TTX blocks the inward current of post-thaw day 13 neuron from a holding potential of -70mV.
(B) Potassium channel antagonist TEA blocks outward current of post-thaw day 12 neuron
from a holding potential of -80mV. (C) Calcium channel antagonist nifedipine partially blocks
inward calcium current of post-thaw day 19 neuron from a holding potential of -90mV.
Calcium current was isolated by addition of TEA (50mM) and TTX (300nM) to block
potassium and sodium currents, respectively.
Day 1 Day 8 Day 14 Day 28
Cla
ss I
II
-tu
bu
lin
Nestin
Class III -tubulin /Nestin/ Hoechst
A. B.
Figure 2. A highly pure neuronal population. iCell Neurons represent a highly pure
population as demonstrated by (A) flow cytometry (Day 1 post-thaw) and (B)
immunocytochemistry (Day 7 post-thaw) for class III -tubulin (positive; neuronal marker)
and nestin (negative, neural stem/progenitor cell marker).
Figure 1. Post-thaw iCell Neurons exhibit a typical neuronal morphology. Cryopreserved iCell
Neurons were thawed and plated in iCell Neurons Maintenance Medium with iCell Neurons
Supplement on poly-L-ornithine/laminin-coated tissue culture plates. Reanimated neurons develop
branched networks within 24 hours and remain viable and adherent for an extended period in
culture (14 days).
vGAT / vGLUT2 MAP2 / GABA / Hoechst
Phenotype Characterization
Lucas Chase1, Monica Strathman1, Jeff Grinager1, David Majewski1, Regina Whitemarsh2, Sabine Pellett2, Oksana Sirenko3, Jayne Hesley3, Penny Tavormina3,
Casey Stankewicz1, Matt George1, Ning Liu1, Nathan Meyer1, Matthew Riley1, Xuezhu Feng1, Eric Johnson2, Wen Bo Wang1 and Brad Swanson1 1Cellular Dynamics International, Inc., Madison, WI 53711; 2Department of Bacteriology, University of Wisconsin, Madison, WI 53706; 3Molecular Devices, Sunnyvale, CA 94089
0.000001
0.000010
0.000100
0.001000
0.010000
0.100000
1.000000
10.000000
GR
IA1
GR
IA2
GR
IK1
GR
IK3
GR
IN1
GR
IN2
B
GR
M1
GR
M5
GR
M2
GR
M3
GR
M4
CH
RM
1
CH
RM
4
GA
BR
A1
GA
BR
B1
GA
BB
R1
GA
BR
R1
DR
D1
DR
D2
AMPA Kainate NMDA mGluRs mAChRs GABARs DopRs
Re
lati
ve E
xpre
ssio
n (
vs. G
AP
DH
)
Receptor Gene Expression
High Content Image-based Assays
-60
-50
-40
-30
-20
-10
0
10
20
30
10mV
5msec
0mV
10mV
1sec
-60
-50
-40
-30
-20
-10
0
10
20
0mV
10mV
1sec
-800
-700
-600
-500
-400
-300
-200
-100
0
100
200
-50 -30 -10 10 30 50 70
Control
10nM TTX
30nM TTX
100nm TTX
300nM TTX
Curr
ent (
pA
)
Voltage (mV)
0
100
200
300
400
500
600
700
800
-50 -40 -30 -20 -10 0 10 20 30 40
Control
0.1mM TEA
0.3mM TEA
1mM TEA
3mM TEA
Curr
ent (
pA
)
Voltage (mV)
INa
IK
150pA
1ms
Figure 5. iCell Neurons exhibit neuron-like action potentials. Action potential tracings
recorded from a single iCell Neuron using a whole-cell patch clamp methodology. (A) A
representative evoked action potential from post-thaw day 11 neurons. Evoked action
potentials from these cells display an average resting membrane potential of -46mV as early
as 9 days post-thaw. (B) Representative spontaneous action potentials from a post-thaw day
14 neuron. All action potentials demonstrate an overshoot of the depolarization phase above
0mV and an undershoot of the repolarization phase below baseline before correction to
steady-state.
Figure 7. High content image analysis. (A) An example overlay image of post-thaw
iCell Neurons stained with class III -tubulin (green) and DAPI stain (blue) using the
Molecular Devices ImageXpress® Micro system and MetaXpress® analysis software.
Magnification = 10x objective. (B) Neurite outgrowth analysis using the Neurite Outgrowth
module of MetaXpress Software.
A. B.
A.
0.01mM 1mM 10mM
% c
ell
s s
ign
ific
an
t g
row
th
Concentration (mM) Concentration, uM
0.001 0.01 0.1 1 10 100 1000
0
10000
20000
30000
Total Outgrowth
4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2
Antimycin A (Antimycin A: Concentration vs Mean... 2.48e+04 4.63 13.3 2.25e+03 0.936
Staurosporin (Staurosporin: Concentration vs Mea... 2.6e+04 2.26 0.943 1.63e+03 0.983
MK (MK: Concentration vs MeanValue) 2.67e+04 2.27 3.95 -532 0.988
Mitomycin C (Mitomycin A: Concentration vs Mean... 2.67e+04 3.53 0.732 1.06e+03 0.992__________
Weighting: Fixed
Concentration, uM
0.001 0.01 0.1 1 10 100 10000
10
20
30
40
50
60
70
80
90
100
4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2
Antimycin A (Antimycin A: Concentration vs Mean... 90.9 28.6 14.5 16.4 0.987
Staurosporin (Staurosporin: Concentration vs Mea... 85.3 1.47 1.77 12.3 0.978
MK (MK: Concentration vs MeanValue) 90 29.3 3.94 12 0.982
Mitomycin C (Mitomycin A: Concentration vs Mean... 87.9 27.4 0.667 16.1 0.996__________
Weighting: Fixed
To
tal o
utg
row
th
Concentration (mM)
antimycin A
staurosporine
MK571
mitomycin C
B.
Figure 9. High content image-based assay for mitochondrial integrity. (A) iCell
Neurons treated with increasing concentrations of antimycin A and valinomycin and were
stained for class III -tubulin and JC-10. Images were analyzed using the Molecular
Devices ImageXpress Micro system and MetaXpress software for JC-10 aggregates.
Resultant data was used to generate dose-response curves. (B) Images of control and
30-minute antimycin A-treated iCell Neurons, causing a block of oxidative respiration.
antimycin A valinomycin
1e-5 1e-4 0.001 0.01 0.1 1 10
0
10
20
30
Gra
nu
lari
ty A
rea
/Ce
ll
Concentration (mM)
Figure 8. High content image-based assay for neurite outgrowth. (A and B) iCell
Neurons treated with increasing concentrations of antimycin A, staurosporine, mitomycin
C and MK571 were stained for nuclei (DAPI) and class III -tubulin and analyzed using the
Molecular Devices ImageXpress Micro system and MetaXpress software. The resultant
dose response curves to cytotoxic compounds for 2 parameters: (A) total outgrowth and
(B) % cells with significant growth, were generated. (C) Images of cells treated with
increasing concentrations of mitomycin C and stained with class III -tubulin. Magnification
= 20x objective.
Control
antimycin A (3µM)
Ca+ Channel Current (ICa) – Nifedipine Inhibition
Figure 11. BoNT target characterization and toxigenicity testing. (A) TaqMan gene
expression assays were used to detect Botulinum neurotoxin (BoNT) receptor and target
protein expression in iCell Neurons cultured for 5-20 days post-thaw. Adult human brain was
used as a positive control. (B) Protein expression of BoNT receptors and target proteins was
analyzed via Western blot for iCell Neurons cultured for 4-21 days post-thaw. Rat spinal cord
cells (RSC) were used as a positive control. (C) iCell Neurons (4 or 7 days post-thaw) and rat
spinal cord cells were exposed to serial dilutions of BoNT/A, /B, /C, or /E for 48 hrs. Cell
lysates were analyzed for respective SNARE target protein cleavage by Western blot. Data
from three Western blots were quantified by densitometry and dose-response curves were
plotted using GraphPad Prism 5. Protein expression of BoNT receptors and target proteins
and BoNT toxigenicity assay data was generated by Regina Whitemarsh and Dr. Sabine
Pellett, University of Wisconsin-Madison (Dr. Eric Johnson Lab).
BoNT Toxigenicity Testing
BoNT/A BoNT/B
BoNT/C BoNT/E
-250
-200
-150
-100
-50
0
50
-60 -50 -40 -30 -20 -10 0 10 20 30 40 50
Cu
rren
t (p
A)
Voltage (mV)
Control
0.0001
0.001
0.01
0.1
1
10
STX1A STX1B VAMP1 VAMP2 VAMP3 SV2A SV2B SV2C SNAP25 SYT1 SYT2
Re
lati
ve E
xpre
ssio
n (
vs. G
AP
DH
)
Day 5 Day 10 Day 15 Day 20 Adult Human Brain
B. A.
C.
A. B.
ICa
C.
antimycin A
staurosporine
MK571
mitomycin C
A. B.
Total neurite
outgrowth
% cells with
outgrowth
Compound IC50 (µM) IC50 (µM)
antimycin A 13 14
staurosporine 0.9 1.7
mitomycin C 0.7 0.7
MK571 4.1 3.9
Compound IC50 (nM)
antimycin A 46
valinomycin 0.15
0.000001
0.000010
0.000100
0.001000
0.010000
0.100000
1.000000
10.000000
TH DAT VMAT2 VGLUT1 VGLUT2 VGAT GAD67 GAD65 SERT PET1 TPH2 ADRA2B DBH CHAT VACHT
Dopaminergic Glutamatergic GABAergic Serotinergic Adrenergic Cholinergic
Re
lati
ve E
xpre
ssio
n (
vs. G
AP
DH
)
Subtype Gene Expression
0.000001
0.000010
0.000100
0.001000
0.010000
0.100000
1.000000
10.000000
LHX2 DACH1 FOXG1 OTX2 GBX2 EN1 PAX2 OLIG2 HOXB4
Re
lati
ve E
xpre
ssio
n (v
s. G
AP
DH
)
Regional Gene Expression
A.
B.
C.