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

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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 -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 ExpressionRelative GAPDH) (vs. mitomycin C MK571 Total outgrowth Total 20 platform, we have developed a highly consistent and scalable Voltage (mV) MK571 significant cells growth 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 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 novel cell model for use in various academic and pharmaceutical + 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
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  • Calcium Control of Neurotransmitter Release

    Calcium Control of Neurotransmitter Release

    Downloaded from http://cshperspectives.cshlp.org/ on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Calcium Control of Neurotransmitter Release Thomas C. Su¨dhof Department of Molecular and Cellular Physiology, and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305 Correspondence: [email protected] Upon entering a presynaptic terminal, an action potential opens Ca2þ channels, and transiently increases the local Ca2þ concentration at the presynaptic active zone. Ca2þ then triggers neurotransmitter release within a few hundred microseconds by activating synaptotagmins Ca2þ. Synaptotagmins bind Ca2þ via two C2-domains, and transduce the Ca2þ signal into a nanomechanical activation of the membrane fusion machinery; this acti- vation is mediated by the Ca2þ-dependent interaction of the synaptotagmin C2-domains with phospholipids and SNARE proteins. In triggering exocytosis, synaptotagmins do not act alone, but require an obligatory cofactor called complexin, a small protein that binds to SNARE complexes and simultaneously activates and clamps the SNARE complexes, thereby positioning the SNARE complexes for subsequent synaptotagmin action. The con- served function of synaptotagmins and complexins operates generally in most, if not all, Ca2þ-regulated forms of exocytosis throughout the body in addition to synaptic vesicle exo- cytosis, including in the degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release. ynaptic transmission is initiated when an in the brainstem (Fig. 1B; reviewed in Mein- Saction potential invades a nerve terminal, renken et al. 2003). Overall, these high-resolution opening Ca2þ channels, which gate a highly electrophysiological studies on neurotrans- localized, transient increase in intracellular mitter release revealed that a presynaptic action Ca2þ at the active zone (Fig.