Name: Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons (Video) | JoVE Journal
Uploaded: 2021-08-26T14:30:00.000+00:00
Duration: 11 min 52 s
Description: Learn to establish electrophysiological recordings using human iPSC-derived spinal cord astrocytes and neurons in co-culture. This method enables analysis of neuronal activity influenced by astrocytes for studying ALS and other neurodegenerative diseases.

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5 minutes

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.&#160;We would like to thank Khalil Rust for their assistance with protocol demonstration and filming.

<ol><li>Ferraiuolo, L., Maragakis, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mini-review%3A+Induced+pluripotent+stem+cells+and+the+search+for+new+cell-specific+ALS+therapeutic+targets%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">Mini-review: Induced pluripotent stem cells and the search for new cell-specific ALS therapeutic targets.</a> Neuroscience Letters. 755, 135911(2021).</li>
<li>Taga, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+human-induced+pluripotent+stem+cell-derived+spinal+cord+astrocytes+in+the+functional+maturation+of+motor+neurons+in+a+multi-electrode+array+system%5BTitle%5D)+AND+%22Stem+Cells+Translational+Medicine%22%5BJournal%5D)">Role of human-induced pluripotent stem cell-derived spinal cord astrocytes in the functional maturation of motor neurons in a multi-electrode array system.</a> Stem Cells Translational Medicine. 8 (12), 1272-1285 (2019).</li>
<li>Klapper, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocyte+lineage+cells+are+essential+for+functional+neuronal+differentiation+and+synapse+maturation+in+human+iPSC-derived+neural+networks%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Astrocyte lineage cells are essential for functional neuronal differentiation and synapse maturation in human iPSC-derived neural networks.</a> Glia. 67 (10), 1893-1909 (2019).</li>
<li>Zhao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutant+C9orf72+human+iPSC-derived+astrocytes+cause+non-cell+autonomous+motor+neuron+pathophysiology%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Mutant C9orf72 human iPSC-derived astrocytes cause non-cell autonomous motor neuron pathophysiology.</a> Glia. 68 (5), 1046-1064 (2020).</li>
<li>Almad, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Connexin+43+in+astrocytes+contributes+to+motor+neuron+toxicity+in+amyotrophic+lateral+sclerosis%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Connexin 43 in astrocytes contributes to motor neuron toxicity in amyotrophic lateral sclerosis.</a> Glia. 64 (7), 1154-1169 (2016).</li>
<li>Wainger, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intrinsic+membrane+hyperexcitability+of+amyotrophic+lateral+sclerosis+patient-derived+motor+neurons%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons.</a> Cell Reports. 7 (1), 1-11 (2014).</li>
<li>Tyzack, G., Lakatos, A., Patani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+stem+cell-derived+astrocytes%3A+Specification+and+relevance+for+neurological+disorders%5BTitle%5D)+AND+%22Current+Stem+Cell+Reports%22%5BJournal%5D)">Human stem cell-derived astrocytes: Specification and relevance for neurological disorders.</a> Current Stem Cell Reports. 2, 236-247 (2016).</li>
<li>Imaizumi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controlling+the+regional+identity+of+hPSC-derived+neurons+to+uncover+neuronal+subtype+specificity+of+neurological+disease+phenotypes%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">Controlling the regional identity of hPSC-derived neurons to uncover neuronal subtype specificity of neurological disease phenotypes.</a> Stem Cell Reports. 5 (6), 1010-1022 (2015).</li>
<li>Odawara, A., Matsuda, N., Ishibashi, Y., Yokoi, R., Suzuki, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toxicological+evaluation+of+convulsant+and+anticonvulsant+drugs+in+human+induced+pluripotent+stem+cell-derived+cortical+neuronal+networks+using+an+MEA+system%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Toxicological evaluation of convulsant and anticonvulsant drugs in human induced pluripotent stem cell-derived cortical neuronal networks using an MEA system.</a> Scientific Reports. 8 (1), 10416(2018).</li>
<li>Haidet-Phillips, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+profiling+of+human+induced+pluripotent+stem+cell-derived+astrocyte+progenitors+following+spinal+cord+engraftment%5BTitle%5D)+AND+%22Stem+Cells+Translational+Medicine%22%5BJournal%5D)">Gene profiling of human induced pluripotent stem cell-derived astrocyte progenitors following spinal cord engraftment.</a> Stem Cells Translational Medicine. 3 (5), 575-585 (2014).</li>
<li>Roybon, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+stem+cell-derived+spinal+cord+astrocytes+with+defined+mature+or+reactive+phenotypes%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Human stem cell-derived spinal cord astrocytes with defined mature or reactive phenotypes.</a> Cell Reports. 4 (5), 1035-1048 (2013).</li>
<li>Shimojo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((simple+motor+neuron+differentiation+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Molecular+Brain%22%5BJournal%5D)">simple motor neuron differentiation from human pluripotent stem cells.</a> Molecular Brain. 8 (1), 79(2015).</li>
<li>Chandrasekaran, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+2D+and+3D+neural+induction+methods+for+the+generation+of+neural+progenitor+cells+from+human+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells.</a> Stem Cell Reports. 25, 139-151 (2017).</li>
<li>Krencik, R., Weick, J. P., Liu, Y., Zhang, Z. J., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specification+of+transplantable+astroglial+subtypes+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Specification of transplantable astroglial subtypes from human pluripotent stem cells.</a> Nature Biotechnology. 29 (6), 528-534 (2011).</li>
<li>Li, X. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coordination+of+sonic+hedgehog+and+Wnt+signaling+determines+ventral+and+dorsal+telencephalic+neuron+types+from+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells.</a> Development. 136 (23), 4055-4063 (2009).</li>
<li>Liu, H., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specification+of+neuronal+and+glial+subtypes+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cellular+and+Molecular+Life+Sciences%22%5BJournal%5D)">Specification of neuronal and glial subtypes from human pluripotent stem cells.</a> Cellular and Molecular Life Sciences. 68 (24), 3995-4008 (2011).</li>
<li>Borghese, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+notch+signaling+in+human+embryonic+stem+cell-derived+neural+stem+cells+delays+G1%2FS+phase+transition+and+accelerates+neuronal+differentiation+in+vitro+and+in+vivo%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Inhibition of notch signaling in human embryonic stem cell-derived neural stem cells delays G1/S phase transition and accelerates neuronal differentiation in vitro and in vivo.</a> Stem Cells. 28 (5), 955-964 (2010).</li>
<li>Shi, Y., Kirwan, P., Smith, J., Robinson, H. P., Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+cerebral+cortex+development+from+pluripotent+stem+cells+to+functional+excitatory+synapses%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses.</a> Nature Neuroscience. 15 (3), 477-486 (2012).</li>
</ol>

The authors have nothing to disclose.

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...

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 can recapitulate hallmarks of ALS pathobiology such as astrocyte-mediated neurotoxicity4,5 and neuronal hyper-excitability6. Additionally, with advancements in differentiation protocols, human induced pluripotent stem cells (hiPSC) can be differentiated into regionally-specific neural subtypes, including cortical and spinal cord hiPSC-A and hiPSC-N7,8. These strategies provide the potential for modeling cortical and spinal motor neuron pathology in ALS as well as the astrocytic influence on both. However, this requires that there is a reproducible functional assay to determine these effects.
Recently it was shown that multi-electrode array (MEA) recording is particularly suitable for the electrophysiological characterization of neuron-astrocyte co-cultures2. As opposed to single-cell electrophysiological analyses, these high-density electrode arrays passively record extracellular field potentials from large populations of neurons without disrupting culture conditions and preserving cell membranes' integrity. These platforms are particularly useful for recording the cellular and network activity of cultures over time and in response to pharmacologic manipulation. Finally, when the presence of astrocytes is a culture variable, MEA recordings can provide functional insights into astrocyte-neuron bidirectional interactions2,9.
Presented here is an optimized protocol for the differentiation of hiPSC into spinal cord hiPSC-A and hiPSC-motor neurons (MN) that has been previously validated2. The spinal cord hiPSC-A differentiation protocol consistently results in astrocyte cultures, which are positive for S100 calcium-binding protein B (S100&#946;), glial fibrillary acidic protein (GFAP), and Homeobox B4 (HOXB4) in up to 80%, 50%, and 90% of cells, respectively, indicating a maturing glial and spinal cord specification2,10. The hiPSC-MN differentiation protocol generates neurons that are &gt;90% positive for choline acetyltransferase (ChAT), suggestive of a mature alpha-motor neuron identity2. Additionally, the protocol describes techniques for the generation of hiPSC-A/MN co-cultures previously demonstrated to result in neurons with enhanced morphological complexity by Scholl analysis and immunofluorescent microscopy when compared to neuronal cultures without astrocytes or with rodent astrocytes2. While these descriptions are specific to spinal cord hiPSC-A and hiPSC-N, a unique advantage is that the initial independent culture of astrocytes and neurons followed by steps to co-culture at later time points can be translated to study the effects of neuron-astrocyte interactions from other specific regions as well as disease-specific cells7,8. Finally, the protocol describes how to grow these cultures on MEA plates so that functional activity as a factor of co-culture composition can be studied over time with the ability to manipulate cellular composition as well as culture conditions.
The goal of these protocols is to provide a functional assay to investigate astrocyte-neuron interactions, examine disease-specific changes, and test drugs with therapeutic potential in the field of ALS. Video instructions are provided for the most challenging steps of this protocol.

<table><tbody><tr><td>10 cm sterile culture plates</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; sterile culture flasks</td><td>Falcon</td><td>353136</td><td></td></tr><tr><td>2-Mercaptoethanol (&amp;beta;-ME)</td><td>Thermofisher</td><td>21985023</td><td>Working concentration 110 &amp;micro;M</td></tr><tr><td>500 mL 0.2 &amp;micro;m CA Filter System</td><td>Corning</td><td>430769</td><td></td></tr><tr><td>5 mL pipette</td><td>Falcon</td><td>357543</td><td></td></tr><tr><td>6 well sterile culture plates</td><td>Falcon</td><td>3046</td><td></td></tr><tr><td>Amphotericine B</td><td>Gibco</td><td>15290018</td><td>Working concentration 2.0 &amp;mu;g/mL</td></tr><tr><td>Anionic detergent with protease enzyme - Terg-A-Zyme</td><td>Sigma-Aldrich</td><td>Z273287</td><td>Working concentration 1% m/v</td></tr><tr><td>Ascorbic acid (ASAC)</td><td>Sigma</td><td>A4403</td><td>Dissolve 100 mg into 250 mL of dH&lt;sub&gt;2&lt;/sub&gt;O to get 0.4mg/ml stock. Sterile filter through a 0.22 &amp;micro;m filter, aliquot and freeze at -80 &amp;ordm;C. Dilute at 1:1000 for use. (working concentration 0.4 &amp;micro;g/mL).</td></tr><tr><td>Axion CytoView MEA 24 plates (M384-tMEA-24W)</td><td>Axion</td><td>OPT-24</td><td></td></tr><tr><td>Axion Edge MEA platform</td><td>Axion</td><td>Maestro Edge</td><td></td></tr><tr><td>Basement Membrane matrix - Matrigel</td><td>Corning</td><td>354277</td><td>Details in the protocol</td></tr><tr><td>Benchtop microscope (sterile under cell culture hood)</td><td>Zeiss</td><td>415510-1100-000</td><td>Primo Vert</td></tr><tr><td>Bicuculline</td><td>Sigma Aldrich</td><td>14340</td><td>Working concentration10 &amp;mu;M</td></tr><tr><td>Ciliary neurotrophic factor (CNTF)</td><td>Peprotech</td><td>450-13</td><td>Dissolve 100 &amp;micro;g in 1mL of sterile PBS, and then add 9 mL of sterile 0.1% BSA-PBS to 10 &amp;micro;g/mL. Aliquot and freeze at -80 &amp;ordm;C. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; tanks and regulator for Axion Edge</td><td>AirGas/Harris</td><td>9296NC</td><td></td></tr><tr><td>Compound E</td><td>Abcam</td><td>ab142164</td><td>Dissolve 250 &amp;micro;g in 2.0387 mL of DMSO to get a 250 &amp;micro;M stock. Aliquot and freeze at -80 &amp;ordm;C. Dilute 1:2000 for use. (working concentration 125 nM).</td></tr><tr><td>Cyanquixaline&amp;nbsp; (CNQX)</td><td>Sigma Aldrich</td><td>C239</td><td>Working concentration 50 &amp;mu;M</td></tr><tr><td>Dihydrokainic acid (DHK)</td><td>Tocris</td><td>111</td><td>Working concentration 50 &amp;mu;M and 300 &amp;mu;M</td></tr><tr><td>DMEM/F12</td><td>Thermofisher</td><td>113300</td><td>Working concentration 1x</td></tr><tr><td>Essential 8 Medium + Essential 8 Supplement</td><td>Thermofisher</td><td>A1517001</td><td>Combine 10 mL of Essential 8 (10x)&amp;nbsp; supplement with 500 mL of Essential 8 Growth Medium (1x)</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermofisher</td><td>16140071</td><td>Working concentration 1x</td></tr><tr><td>Glial cell line-derived neurotrophic (GDNF)</td><td>Peprotech</td><td>450-10</td><td>Dissolve 100 &amp;micro;g in 1mL of PBS to 100 &amp;micro;g/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 &amp;micro;g/mL. Aliquot and freeze at -80 &amp;ordm;C. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).</td></tr><tr><td>Hemocytometer</td><td>Election Microscopy Sciences</td><td>63510-20</td><td></td></tr><tr><td>Heparin</td><td>Millipore-sigma</td><td>H3149-100KU</td><td>Dissolve 200 mg in 100 mL of PBS to get 2 mg/mL stock solution. Aliquot the stock in 15 mL and 500 &amp;micro;L tubes. Dilute 1:1000 for use. (working concentration 2 &amp;micro;g/mL).</td></tr><tr><td>Humidity controlled Cell culture incubator</td><td>ThermoFisher</td><td>370</td><td>set to 37 &amp;ordm;C, 5 % CO2</td></tr><tr><td>Insulin-like growth factor 1 (IGF-1)</td><td>R&amp;amp;D systems</td><td>291-G1-200</td><td>Dissolve 200 &amp;micro;g in 2 mL of PBS to 100 &amp;micro;g/mL, then add 18 mL of 0.1%BSA-PBS to make 10 &amp;micro;g/mL stock and store at -80 &amp;ordm;C. Aliquot and freeze at -80 &amp;ordm;C. Dilute 10 &amp;micro;g/mL stock at 1: 1000 for use. (working concentration 10 ng/mL).</td></tr><tr><td>Kainc acid</td><td>Abcam</td><td>ab144490</td><td>Working concentration 5 &amp;mu;M</td></tr><tr><td>Knockout Serum Replacement (KSR)</td><td>Thermofisher</td><td>10828</td><td>Working concentration 1x</td></tr><tr><td>Laminin</td><td>Thermofisher</td><td>23017-015</td><td>Stock solution 1 mg/mL, working concentration 10 &amp;micro;g/mL (coating), 1 &amp;micro;g/mL (cell media)</td></tr><tr><td>LDN193189</td><td>Stemgent</td><td>04-0074</td><td>Dissolve 2 mg of LDN into 500 &amp;micro;L of Chloroform to get 10 mM stock. Aliquot this and freeze at -80 &amp;ordm;C. 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 &amp;micro;M working solution</td></tr><tr><td>L-Glutamine</td><td>Thermofisher</td><td>25030</td><td>Working concentration 100x</td></tr><tr><td>MEA glass plates</td><td>MultiChannel Systems</td><td>60MEA200/30iR-Ti-gr</td><td></td></tr><tr><td>Multichannel Pipet P200</td><td>Gilson</td><td>PJ22224</td><td></td></tr><tr><td>Neurobasal</td><td>Thermofisher</td><td>21103049</td><td>Working concentration 1x</td></tr><tr><td>Non-Essential Amino Acids (NEAA)</td><td>Thermofisher</td><td>11140050</td><td>Working concentration 100x</td></tr><tr><td>Pencillin/Streptomycin</td><td>Thermofisher</td><td>15140122</td><td>Working concentration 100x</td></tr><tr><td>Polyornithine (PLO)</td><td>Sigma-Aldrich</td><td>P3655</td><td>Dissolve 100 mg in 1 mL of ddW to get 100 mg/mL stock solution. Aliquot the stock in 100 &amp;micro;L tubes. (working concentration 100 &amp;micro;g/mL)</td></tr><tr><td>Potassium chloride (KCl)</td><td>NA</td><td>NA</td><td>Working concentration100 mM</td></tr><tr><td>Purmorphamine (PMN)</td><td>Millipore-Sigma</td><td>540223</td><td>Dissolve 5 mg in 9.6 mL of DMSO to get 1 mM solution. Aliquot and freeze at -80 &amp;ordm;C. Dilute 1:1000 for use. (working concentration 1 &amp;micro;M).</td></tr><tr><td>Recombinant human-brain-derived neurotrophic factor (BDNF)</td><td>Peprotech</td><td>450-02</td><td>Centrifuge briefly before reconstitution. Dissolve 100 &amp;micro;g in 1 mL of PBS to 100 &amp;micro;g/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 &amp;micro;g/mL. Aliquot and freeze at -80 &amp;ordm;C. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).</td></tr><tr><td>Retinoic acid (RA)</td><td>Sigma</td><td>R2625</td><td>Dissolve 100 mg into 3.3 mL of DMSO to get 100 mM stock solution. Aliquot the stock 100 &amp;micro;L/tube and freeze at -80 &amp;ordm;C. Take 200 &amp;micro;L of 100 mM stock and dilute 10x (add 1.8 mL of DMSO) to make 10 mM stock. Aliquot 50 &amp;micro;L/tube&amp;nbsp;and store at -80 &amp;ordm;C. Dilute at 1:10000 for use. (working concentration 2 &amp;micro;M).</td></tr><tr><td>ROCK-I nhibitor</td><td>Peprotech</td><td>1293823</td><td>Dissolve 5 mg in 1480 &amp;micro;L of dH&lt;sub&gt;2&lt;/sub&gt;O to get 10 mM stock, aliquot and freeze at -80 &amp;ordm;C. Dilute at 1: 500 for use. (working concentration 20 &amp;micro;M).</td></tr><tr><td>SB431542</td><td>Sigma</td><td>S4317</td><td>Dissolve 5mg into 1.3 mL of DMSO to get 10 mM stock solution. Aliquot and freeze at -80 &amp;ordm;C. Dilute at 1:1000 for use. (working con 10 &amp;micro;M)</td></tr><tr><td>Sterile cell culture hoods</td><td>Baker Company</td><td>SG-600</td><td></td></tr><tr><td>Supplement B - B27 Supplement</td><td>Thermofisher</td><td>21985023</td><td>Working concentration 50x</td></tr><tr><td>Supplement N - N2 Supplement</td><td>Thermofisher</td><td>17502048</td><td>Working concentration 100x</td></tr><tr><td>Table top cell culture centrifuge</td><td>ThermoFisher</td><td>75004261</td><td>Sorvall Legend X1R</td></tr><tr><td>Thermoplastic&amp;nbsp;film - Parafilm</td><td>PARAFILM</td><td>P7793</td><td></td></tr><tr><td>Tissue dissociation protease - Dispase</td><td>StemCell Technologies</td><td>7923</td><td>Working concentration 1x</td></tr><tr><td>Trypsin Inhibitor</td><td>Sigma</td><td>T6522-1G</td><td>Dissolve 1g in 100mL ddH&lt;sub&gt;2&lt;/sub&gt;O to get 10 mg/mL stock. Aliquot and store at 4 &amp;ordm;C. Dilute 1:10 of trypsin volume for use. (working concentration 1 mg/mL).</td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>Thermofisher</td><td>2530054</td><td>Working concentration 1x</td></tr><tr><td>Waterbath</td><td>ThermoFisher</td><td>2332</td><td>Isotemp</td></tr></tbody></table>

establishment of an electrophysiological platform for modeling als with regionally-specific human pluripotent stem cell-derived astrocytes and neurons

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

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.

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-based m...

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

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

establishment-an-electrophysiological-platform-for-modeling-als-with

Research

JoVE Journal

Neuroscience

The video demonstrates a method to generate astrospheres from progenitors from human pluripotent stem cells. It involves the growth and proliferation of stem cells with nutrient media containing growth factors and kinase inhibitors. These cells then aggregate and differentiate into astrocytes, forming astrospheres.

Generating and Maintaining Astrospheres Using Human Pluripotent Stem Cells

This video showcases a method for differentiating spinal cord neural progenitor cells (NPCs) into motor neurons. It details the culturing process of the NPCs on a coated culture plate using specialized media enriched with specific inhibitors aimed at facilitating NPC differentiation into mature motor neurons.

Differentiation of Spinal Cord Neural Progenitor Cells into Motor Neurons

This video demonstrates establishing a neuron-astrocyte co-culture for recording neuronal network activity. Upon establishing the co-culture of motor neurons and astrocytes on a multi-electrode array (MEA) plate, the synchronous rhythmic electrical activity of the neuronal network was recorded extracellularly using the MEA.

Recording Electrophysiological Activity of the Neuronal Network in a Neuron-Astrocyte Co-culture

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human induced Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered "living scaffolds", exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer’s disease.

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Learn to establish electrophysiological recordings using human iPSC-derived spinal cord astrocytes and neurons in co-culture. This method enables analysis of neuronal activity influenced by astrocytes for studying ALS and other neurodegenerative diseases.

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons (Video) | JoVE Journal

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

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