Development and Characterization of Human Ipsc-Derived Neurons for Drug Discovery Applications

Development and Characterization of Human iPSC-derived Neurons for Drug Discovery Applications 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

Target Target Compound Lead Preclinical Clinical Identification Validation Screening Optimization Trials Trials

Abstract Phenotype Characterization Electrophysiology High Content Image-based Assays Cell-based Assays A. B. The human brain represents a complex organ that has A. Evoked Action Potential B. Spontaneous Action Potential A. B. consistently been proven difficult to model in vitro. Current models A. B. 30

20 20 including primary rodent tissue and immortalized cell lines have

10 10

0 0mV served as mainstays in both academic research and the 0

0mV

ubulin t

-10 -

-10 pharmaceutical industry. These models, while providing a means 

-20 III III -20 for numerous landmark discoveries, have suffered from various -30 -30 10mV10mV -40 10mV Figure 10. iCell Neurons display an expected sensitivity to known compounds. iCell Class Class 1sec 5msec 1sec -40 issues including biological relevance, reproducibility and -50 Neurons were cultured for 7-14 days post-thaw on PLO/Laminin pre-coated 96-well plates

-60 scalability. Considerable efforts have been made specifically -50 and exposed to a dilution series of (A) staurosporine and (B) kainic acid. Viability (as ® -60 measured using cellular ATP content) was determined using the CellTiter-Glo within the pharmaceutical industry to reduce late-stage drug Nestin Luminescent Cell Viability Assay (Promega). attrition through the development of more relevant in vitro human Class III -tubulin /Nestin/ Hoechst 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 Figure 5. iCell Neurons exhibit neuron-like action potentials. Action potential tracings ® ® model systems. One area given significant attention has been the Figure 2. A highly pure neuronal population. iCell Neurons represent a highly pure Molecular Devices ImageXpress Micro system and MetaXpress analysis software. BoNT Toxigenicity Testing recorded from a single iCell Neuron using a whole-cell patch clamp methodology. (A) A population as demonstrated by (A) flow cytometry (Day 1 post-thaw) and (B) Magnification = 10x objective. (B) Neurite outgrowth analysis using the Neurite Outgrowth development of platforms than can enable the modeling of human representative evoked action potential from post-thaw day 11 neurons. Evoked action immunocytochemistry (Day 7 post-thaw) for class III -tubulin (positive; neuronal marker) module of MetaXpress Software. potentials from these cells display an average resting membrane potential of -46mV as early A. B. degenerative (e.g. Alzheimer’s and Parkinson’s disease) and and nestin (negative, neural stem/progenitor cell marker). as 9 days post-thaw. (B) Representative spontaneous action potentials from a post-thaw day genetic (i.e. Huntington's disease and muscular dystrophy) Day 5 14 neuron. All action potentials demonstrate an overshoot of the depolarization phase above A. B. Day 10 10 Day 15 0mV and an undershoot of the repolarization phase below baseline before correction to Day 20 diseases as well as neurotoxicity. The recent discovery of induced A. B. Total Outgrowth Adult Human Brain steady-state. pluripotent stem cells (iPSCs) not only overcomes the ethical and 100 1 30000 90 logistical issues associated with human embryonic stem cells, but 80 0.1 70 20000 also provides a flexible platform for generating various 60 antimycin A 0.01 50 + antimycin A staurosporine A. Na Channel Current (I ) – Tetrodotoxin Inhibition 40 differentiated cell types from diseased individuals. Using this Na staurosporine mitomycin C 0.001

10000 30 Expression Relative GAPDH) (vs. mitomycin C MK571 Total outgrowth Total 20 platform, we have developed a highly consistent and scalable Voltage (mV) MK571 significantgrowth cells 0.0001 I 10 STX1A STX1B VAMP1 VAMP2 VAMP3 SV2A SV2B SV2C SNAP25 SYT1 SYT2 Na -50 -30 -10 10 30 50 70 % 0 0 protocol to differentiate and cryopreserve purified human iPSC- 200 0.001 0.01 0.1 1 10 100 1000 0.001 0.01 0.1 1 10 100 1000 100 ® Concentration, uM Concentration, uM Control Concentration (mM) Concentration (mM) derived neurons, called iCell Neurons. Phenotypically, these 0 4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2 Total neurite % cells with4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2 BoNT/A BoNT/B -100 10nM TTX Antimycin A (Antimycin A: Concentration vs Mean... 2.48e+04 4.63 13.3 2.25e+03 0.936 outgrowth Antimycinoutgrowth A (Antimycin A: Concentration vs Mean... 90.9 28.6 14.5 16.4 0.987 C. -200 Staurosporin (Staurosporin: Concentration vs Mea... 2.6e+04 2.26 0.943 1.63e+03 0.983 Staurosporin (Staurosporin: Concentration vs Mea... 85.3 1.47 1.77 12.3 0.978 cells are >90% pure as measured using flow cytometry for vGAT / vGLUT2 MAP2 / GABA / Hoechst Compound IC50 (µM) MK IC50(MK: Concentration (µM) vs MeanValue) 90 29.3 3.94 12 0.982 30nM TTX MK (MK: Concentration vs MeanValue) 2.67e+04 2.27 3.95 -532 0.988 -300 antimycin A 13 Mitomycin14 C (Mitomycin A: Concentration vs Mean... 87.9 27.4 0.667 16.1 0.996 Mitomycin C (Mitomycin A: Concentration vs Mean... 2.67e+04 3.53 0.732 1.06e+03 0.992 ______-400 100nm TTX ______staurosporine 0.9 1.7 presence of the neuronal marker class III beta tubulin (TuJ1) and (pA) Current Weighting: Fixed Weighting: Fixed Figure 3. iCell Neurons display characteristic subtype protein expression. Post-thaw -500 300nM TTX mitomycin C 0.7 0.7 absence of the progenitor marker nestin. Within 24hrs of thawing, 150pA -600 C. MK571 4.1 3.9 iCell Neurons were plated in iCell Neurons Maintenance Medium with iCell Neurons 1ms -700 these neurons display a typical neuronal morphology including a Supplement on poly-L-ornithine/laminin-coated tissue culture plates and immunostained on -800 0.01mM 1mM 10mM day 14 for (A) the synaptic markers vGAT (vesicular GABA transporter) and vGLUT2 dense network of neurites. Detailed phenotypic analyses reveal (vesicular glutamate transporter 2), markers of GABAergic and Glutamatergic neurons, respectively; and (B) MAP2 (microtubule-associated protein 2) and GABA (gamma- that these neurons are comprised of a mix of predominantly + aminobutyric acid). Magnification = 20x objective. B. K Channel Current (IK) – Tetraethylammonium Inhibition GABAergic and Glutamatergic subtypes as measured at both the BoNT/C BoNT/E gene expression and protein levels and form characteristic IK 800 synaptic connections. Functionally, these cells reveal typical 700 Regional Gene Expression 600 Control 10.000000 electrophysiological characteristics as measured using single-cell 500 0.1mM TEA 400 1.000000 0.3mM TEA patch clamp to detect both spontaneous and evoked action 300 0.100000 1mM TEA Current (pA) Current 200 Figure 8. High content image-based assay for neurite outgrowth. (A and B) iCell (vs. GAPDH) (vs. 0.010000 potentials as well as functional ion channels. Finally, when applied 100 3mM TEA Neurons treated with increasing concentrations of antimycin A, staurosporine, mitomycin 0.001000 0 C and MK571 were stained for nuclei (DAPI) and class III -tubulin and analyzed using the to high throughput applications, including cytotoxicity assays, iCell -50 -40 -30 -20 -10 0 10 20 30 40 0.000100 Voltage (mV) Molecular Devices ImageXpress Micro system and MetaXpress software. The resultant Figure 11. BoNT target characterization and toxigenicity testing. (A) TaqMan gene

Neurons reveal characteristic pharmacological responses to Expression Relative 0.000010 dose response curves to cytotoxic compounds for 2 parameters: (A) total outgrowth and expression assays were used to detect Botulinum neurotoxin (BoNT) receptor and target 0.000001 (B) % cells with significant growth, were generated. (C) Images of cells treated with known toxic compounds. The results demonstrate not only a LHX2 DACH1 FOXG1 OTX2 GBX2 EN1 PAX2 OLIG2 HOXB4 protein expression in iCell Neurons cultured for 5-20 days post-thaw. Adult human brain was novel cell model for use in various academic and pharmaceutical increasing concentrations of mitomycin C and stained with class III -tubulin. Magnification used as a positive control. (B) Protein expression of BoNT receptors and target proteins was C. Ca+ Channel Current (I ) – Nifedipine Inhibition = 20x objective. analyzed via Western blot for iCell Neurons cultured for 4-21 days post-thaw. Rat spinal cord Subtype Gene Expression Ca applications, but they also support the use of the iPSC technology B. Control cells (RSC) were used as a positive control. (C) iCell Neurons (4 or 7 days post-thaw) and rat as a platform capable of generating neurons against diverse 10.000000 I A. spinal cord cells were exposed to serial dilutions of BoNT/A, /B, /C, or /E for 48 hrs. Cell 1.000000 Ca Voltage (mV) lysates were analyzed for respective SNARE target protein cleavage by Western blot. Data genetic backgrounds. 0.100000 -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 from three Western blots were quantified by densitometry and dose-response curves were

(vs. GAPDH) (vs. 50 0.010000 plotted using GraphPad Prism 5. Protein expression of BoNT receptors and target proteins 0.001000 0 and BoNT toxigenicity assay data was generated by Regina Whitemarsh and Dr. Sabine

30 0.000100 -50 Pellett, University of Wisconsin-Madison (Dr. Eric Johnson Lab). 0.000010 -100 Control Compound IC50 (nM)

0.000001 antimycin A 46

Relative Expression Relative

Current (pA) Current -150 TH DAT VMAT2 VGLUT1 VGLUT2 VGAT GAD67 GAD65 SERT PET1 TPH2 ADRA2B DBH CHAT VACHT 20 valinomycin 0.15 Dopaminergic Glutamatergic GABAergic Serotinergic Adrenergic Cholinergic -200 -250 10 antimycin A (3µM) Receptor Gene Expression Summary Post-Thaw Morphology ® 10.000000 iCell Neurons represent a robust, consistent and commercially 1.000000

Granularity Area/Cell Granularity 0 0.100000 available population of human neurons for basic biological and drug Day 1 Day 8 Day 14 Day 28 0.010000 Figure 6. iCell Neurons respond to ion channel blockers. Addition of classical neuron ion 1e-5 1e-4 0.001 0.01 0.1 1 10 (vs. GAPDH) (vs. 0.001000 discovery applications. This highly pure population of cells (Figure 2) 0.000100 channel antagonists tetrodotoxin (TTX,100nM), tetraethylammonium (TEA, 30mM) and Concentration (mM) 0.000010 nifedipine (10µM) blocks inward sodium outward potassium, and inward calcium currents, displays a robust and stable neuronal morphology (Figure 1) and is

0.000001

respectively, as measured using a single-cell patch clamp. (A) Sodium channel antagonist antimycin A valinomycin comprised of largely Glutamatergic and GABAergic neuronal

DRD1 DRD2

GRIK1 GRIK3

GRIA1 GRIA2

GRM1 GRM5 GRM2 GRM3 GRM4

GRIN1

CHRM1 CHRM4

GRIN2B TTX blocks the inward current of post-thaw day 13 neuron from a holding potential of -70mV.

GABRB1 GABBR1 GABRR1

GABRA1 subtypes (Figures 3 and 4). iCell Neurons display evoked and Relative Expression Relative AMPA Kainate NMDA mGluRs mAChRs GABARs DopRs (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 spontaneous neuron-like action potentials (Figure 5) and possess inward calcium current of post-thaw day 19 neuron from a holding potential of -90mV. Figure 9. High content image-based assay for mitochondrial integrity. (A) iCell functional sodium, potassium and calcium channels (Figure 6). In Calcium current was isolated by addition of TEA (50mM) and TTX (300nM) to block Neurons treated with increasing concentrations of antimycin A and valinomycin and were addition, these cells are amenable to various assay systems including potassium and sodium currents, respectively. Figure 4. Gene expression analysis of iCell Neurons. Day 15 post-thaw iCell Neurons stained for class III -tubulin and JC-10. Images were analyzed using the Molecular high content image-based assays (Figures 7-9), standard cell-based were analyzed against a focused panel of TaqMan Gene Expression Assays. The data Devices ImageXpress Micro system and MetaXpress software for JC-10 aggregates. Figure 1. Post-thaw iCell Neurons exhibit a typical neuronal morphology. Cryopreserved iCell assays (Figure 10) and toxigenicity testing (Figure 11). The results demonstrate that iCell Neurons represent a population with largely a forebrain identify (top), Resultant data was used to generate dose-response curves. (B) Images of control and Neurons were thawed and plated in iCell Neurons Maintenance Medium with iCell Neurons are predominantly glutamatergic and GABAergic neuronal subtypes (middle), and express a 30-minute antimycin A-treated iCell Neurons, causing a block of oxidative respiration. demonstrate a convenient, novel human cell model for neuroscience Supplement on poly-L-ornithine/laminin-coated tissue culture plates. Reanimated neurons develop number of characteristic receptors (bottom). branched networks within 24 hours and remain viable and adherent for an extended period in research which supports the use of iPSC technology as a platform culture (14 days). capable of generating neurons from disease relevant genetic backgrounds.