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

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Control

10nM TTX

30nM TTX

100nm TTX

300nM TTX

Curr

ent (

pA

)

Voltage (mV)

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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

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50

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70

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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

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0

50

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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.