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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Abstract

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

Introduction

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) are powerful tools for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro and provide a translational paradigm for drug discovery strategies1. Researchers have demonstrated that the co-culture of hiPSC-A with hiPSC-N enhances the morphological, molecular, electrophysiological, and pharmacological maturation of both cell types, generating complex neuronal networks and astrocyte-neuron interactions that resemble their in vivo counterparts2,3. Similar co-culture experiments c....

Protocol

1. Cell culture media preparation

  1. Prepare the individual cell culture media using the compositions mentioned in Table 1.
  2. Mix and sterile filter the media in 500 mL filtered bottles, and store protected from light at 4 °C for up to 2 weeks.

2. Maintaining and passaging non-confluent human induced Pluripotent Stem Cells (hiPSC)

  1. Thaw the basement membrane matrix (stored in aliquots at -80 °C) in a 2-8 °C refrigerator overnig.......

Representative Results

The spinal cord patterning protocol for the generation of hiPSC-MN and spinal cord hiPSC-A is outlined in Figure 1. In this protocol, hiPSCs are maintained and passaged as non-confluent colonies (Figure 2A). Neurogenesis is initiated (neural induction) through dual SMAD inhibition by the addition of LDN193189 and SB431542, inactivating the bone morphogenetic protein (BMP) and the transforming growth factor-beta (TGF-β) pathways, respectively. A monolayer-ba.......

Discussion

To date, hiPSC- and MEA-based methods for electrophysiological recordings of astrocyte-neuron co-cultures have found limited application in the field of ALS6 and still not in fully human platforms, in contrast to their more widespread use for in vitro modeling of epilepsy9. This platform, however, has the potential to address pathophysiologically relevant questions in ALS research, such as the mechanisms of neuronal hyperexcitability, astrocyte contribution to neur.......

Acknowledgements

This manuscript was supported by the following: 2019 MSCRFF 5119 (AT). K08NS102526 NIH/NINDS (CWH), 2020 Doris Duke Charitable Foundation Clinical Scientist Career Development Award (CWH). 1R01NS117604-01NIH/NINDS (NJM), DOD ALSRP W81XWH2010161 (NJM), 2019 MSCRFD 5122 (NJM). We thank Dr. Raha Dastgheyb and Dr. Norman Haughey for providing the MEA platform and data analysis software we have utilized to validate the described electrophysiological platform. We would like to thank Khalil Rust for their assistance with protocol demonstration and filming.

....

Materials

NameCompanyCatalog NumberComments
10 cm sterile culture platesFalcon353003
25 cm2 sterile culture flasksFalcon353136
2-Mercaptoethanol (β-ME)Thermofisher21985023Working concentration 110 µM
500 mL 0.2 µm CA Filter SystemCorning430769
5 mL pipetteFalcon357543
6 well sterile culture platesFalcon3046
Amphotericine BGibco15290018Working concentration 2.0 μg/mL
Anionic detergent with protease enzyme - Terg-A-ZymeSigma-AldrichZ273287Working concentration 1% m/v
Ascorbic acid (ASAC)SigmaA4403Dissolve 100 mg into 250 mL of dH2O to get 0.4mg/ml stock. Sterile filter through a 0.22 µm filter, aliquot and freeze at -80 ºC. Dilute at 1:1000 for use. (working concentration 0.4 µg/mL).
Axion CytoView MEA 24 plates (M384-tMEA-24W)AxionOPT-24
Axion Edge MEA platformAxionMaestro Edge
Basement Membrane matrix - MatrigelCorning354277Details in the protocol
Benchtop microscope (sterile under cell culture hood)Zeiss415510-1100-000Primo Vert
BicucullineSigma Aldrich14340Working concentration10 μM
Ciliary neurotrophic factor (CNTF)Peprotech450-13Dissolve 100 µg in 1mL of sterile PBS, and then add 9 mL of sterile 0.1% BSA-PBS to 10 µg/mL. Aliquot and freeze at -80 ºC. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).
CO2 tanks and regulator for Axion EdgeAirGas/Harris9296NC
Compound EAbcamab142164Dissolve 250 µg in 2.0387 mL of DMSO to get a 250 µM stock. Aliquot and freeze at -80 ºC. Dilute 1:2000 for use. (working concentration 125 nM).
Cyanquixaline  (CNQX)Sigma AldrichC239Working concentration 50 μM
Dihydrokainic acid (DHK)Tocris111Working concentration 50 μM and 300 μM
DMEM/F12Thermofisher113300Working concentration 1x
Essential 8 Medium + Essential 8 SupplementThermofisherA1517001Combine 10 mL of Essential 8 (10x)  supplement with 500 mL of Essential 8 Growth Medium (1x)
Fetal Bovine Serum (FBS)Thermofisher16140071Working concentration 1x
Glial cell line-derived neurotrophic (GDNF)Peprotech450-10Dissolve 100 µg in 1mL of PBS to 100 µg/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 µg/mL. Aliquot and freeze at -80 ºC. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).
HemocytometerElection Microscopy Sciences63510-20
HeparinMillipore-sigmaH3149-100KUDissolve 200 mg in 100 mL of PBS to get 2 mg/mL stock solution. Aliquot the stock in 15 mL and 500 µL tubes. Dilute 1:1000 for use. (working concentration 2 µg/mL).
Humidity controlled Cell culture incubatorThermoFisher370set to 37 ºC, 5 % CO2
Insulin-like growth factor 1 (IGF-1)R&D systems291-G1-200Dissolve 200 µg in 2 mL of PBS to 100 µg/mL, then add 18 mL of 0.1%BSA-PBS to make 10 µg/mL stock and store at -80 ºC. Aliquot and freeze at -80 ºC. Dilute 10 µg/mL stock at 1: 1000 for use. (working concentration 10 ng/mL).
Kainc acidAbcamab144490Working concentration 5 μM
Knockout Serum Replacement (KSR)Thermofisher10828Working concentration 1x
LamininThermofisher23017-015Stock solution 1 mg/mL, working concentration 10 µg/mL (coating), 1 µg/mL (cell media)
LDN193189Stemgent04-0074Dissolve 2 mg of LDN into 500 µL of Chloroform to get 10 mM stock. Aliquot this and freeze at -80 ºC. For using, dilute the stock 1 to 10 into DMSO [to 1 mM] first, then dilute 1:5000 of 1 mM into the desired media to get 0.2 µM working solution
L-GlutamineThermofisher25030Working concentration 100x
MEA glass platesMultiChannel Systems60MEA200/30iR-Ti-gr
Multichannel Pipet P200GilsonPJ22224
NeurobasalThermofisher21103049Working concentration 1x
Non-Essential Amino Acids (NEAA)Thermofisher11140050Working concentration 100x
Pencillin/StreptomycinThermofisher15140122Working concentration 100x
Polyornithine (PLO)Sigma-AldrichP3655Dissolve 100 mg in 1 mL of ddW to get 100 mg/mL stock solution. Aliquot the stock in 100 µL tubes. (working concentration 100 µg/mL)
Potassium chloride (KCl)NANAWorking concentration100 mM
Purmorphamine (PMN)Millipore-Sigma540223Dissolve 5 mg in 9.6 mL of DMSO to get 1 mM solution. Aliquot and freeze at -80 ºC. Dilute 1:1000 for use. (working concentration 1 µM).
Recombinant human-brain-derived neurotrophic factor (BDNF)Peprotech450-02Centrifuge briefly before reconstitution. Dissolve 100 µg in 1 mL of PBS to 100 µg/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 µg/mL. Aliquot and freeze at -80 ºC. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).
Retinoic acid (RA)SigmaR2625Dissolve 100 mg into 3.3 mL of DMSO to get 100 mM stock solution. Aliquot the stock 100 µL/tube and freeze at -80 ºC. Take 200 µL of 100 mM stock and dilute 10x (add 1.8 mL of DMSO) to make 10 mM stock. Aliquot 50 µL/tube and store at -80 ºC. Dilute at 1:10000 for use. (working concentration 2 µM).
ROCK-I nhibitorPeprotech1293823Dissolve 5 mg in 1480 µL of dH2O to get 10 mM stock, aliquot and freeze at -80 ºC. Dilute at 1: 500 for use. (working concentration 20 µM).
SB431542SigmaS4317Dissolve 5mg into 1.3 mL of DMSO to get 10 mM stock solution. Aliquot and freeze at -80 ºC. Dilute at 1:1000 for use. (working con 10 µM)
Sterile cell culture hoodsBaker CompanySG-600
Supplement B - B27 SupplementThermofisher21985023Working concentration 50x
Supplement N - N2 SupplementThermofisher17502048Working concentration 100x
Table top cell culture centrifugeThermoFisher75004261Sorvall Legend X1R
Thermoplastic film - ParafilmPARAFILMP7793
Tissue dissociation protease - DispaseStemCell Technologies7923Working concentration 1x
Trypsin InhibitorSigmaT6522-1GDissolve 1g in 100mL ddH2O to get 10 mg/mL stock. Aliquot and store at 4 ºC. Dilute 1:10 of trypsin volume for use. (working concentration 1 mg/mL).
Trypsin-EDTA (0.05%)Thermofisher2530054Working concentration 1x
WaterbathThermoFisher2332Isotemp

References

  1. Ferraiuolo, L., Maragakis, N. J. Mini-review: Induced pluripotent stem cells and the search for new cell-specific ALS therapeutic targets. Neuroscience Letters. 755, 135911 (2021).
  2. Taga, A., et al.

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Human IPSCsAstrocytesNeuronsSpinal CordAmyotrophic Lateral Sclerosis ALSNeural Progenitor CellsDifferentiationElectrophysiologyCell CultureDissociationMedium ExchangeTrypsinPBSPipetteCell AggregatesCell Monolayer37 C Incubation

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