Name: establishment of an electrophysiological platform for modeling als with regionally-specific human pluripotent stem cell-derived astrocytes and neurons
Uploaded: 2021-08-26T14:30:00
Description: Watch this Scientific Journal Video about Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons at JoVE

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.

1. Cell culture media preparation
<ol>
	<li>Prepare the individual cell culture media using the compositions mentioned in Table 1.</li>
	<li>Mix and sterile filter the media in 500 mL filtered bottles, and store protected from light at 4 &#176;C for up to 2 weeks.</li>
</ol>
2. Maintaining and passaging non-confluent human induced Pluripotent Stem Cells (hiPSC)
<ol>
	<li>Thaw the basement membrane matrix (stored in aliquots at -80 &#176;C) in a 2-8 &#176;C refrigerator overnig...

<ol>
	<li>Ferraiuolo, L., Maragakis, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-review:+Induced+pluripotent+stem+cells+and+the+search+for+new+cell-specific+ALS+therapeutic+targets.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+lineage+cells+are+essential+for+functional+neuronal+differentiation+and+synapse+maturation+in+human+iPSC-derived+neural+networks.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+C9orf72+human+iPSC-derived+astrocytes+cause+non-cell+autonomous+motor+neuron+pathophysiology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+43+in+astrocytes+contributes+to+motor+neuron+toxicity+in+amyotrophic+lateral+sclerosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+membrane+hyperexcitability+of+amyotrophic+lateral+sclerosis+patient-derived+motor+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+stem+cell-derived+astrocytes:+Specification+and+relevance+for+neurological+disorders.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+regional+identity+of+hPSC-derived+neurons+to+uncover+neuronal+subtype+specificity+of+neurological+disease+phenotypes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicological+evaluation+of+convulsant+and+anticonvulsant+drugs+in+human+induced+pluripotent+stem+cell-derived+cortical+neuronal+networks+using+an+MEA+system.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+profiling+of+human+induced+pluripotent+stem+cell-derived+astrocyte+progenitors+following+spinal+cord+engraftment.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+stem+cell-derived+spinal+cord+astrocytes+with+defined+mature+or+reactive+phenotypes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=simple+motor+neuron+differentiation+from+human+pluripotent+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+2D+and+3D+neural+induction+methods+for+the+generation+of+neural+progenitor+cells+from+human+induced+pluripotent+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+transplantable+astroglial+subtypes+from+human+pluripotent+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+sonic+hedgehog+and+Wnt+signaling+determines+ventral+and+dorsal+telencephalic+neuron+types+from+human+embryonic+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+neuronal+and+glial+subtypes+from+human+pluripotent+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cerebral+cortex+development+from+pluripotent+stem+cells+to+functional+excitatory+synapses.">Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses.</a> Nature Neuroscience. 15 (3), 477-486 (2012).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm sterile culture plates</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>25 cm2 sterile culture flasks</td>
			<td>Falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (&#946;-ME)</td>
			<td>Thermofisher</td>
			<td>21985023</td>
			<td>Working concentration 110 &#181;M</td>
		</tr>
		<tr>
			<td>500 mL 0.2 &#181;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 &#956;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 dH2O to get 0.4mg/ml stock. Sterile filter through a 0.22 &#181;m filter, aliquot and freeze at -80 &#186;C. Dilute at 1:1000 for use. (working concentration 0.4 &#181;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 &#956;M</td>
		</tr>
		<tr>
			<td>Ciliary neurotrophic factor (CNTF)</td>
			<td>Peprotech</td>
			<td>450-13</td>
			<td>Dissolve 100 &#181;g in 1mL of sterile PBS, and then add 9 mL of sterile 0.1% BSA-PBS to 10 &#181;g/mL. Aliquot and freeze at -80 &#186;C. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).</td>
		</tr>
		<tr>
			<td>CO2 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 &#181;g in 2.0387 mL of DMSO to get a 250 &#181;M stock. Aliquot and freeze at -80 &#186;C. Dilute 1:2000 for use. (working concentration 125 nM).</td>
		</tr>
		<tr>
			<td>Cyanquixaline&#160; (CNQX)</td>
			<td>Sigma Aldrich</td>
			<td>C239</td>
			<td>Working concentration 50 &#956;M</td>
		</tr>
		<tr>
			<td>Dihydrokainic acid (DHK)</td>
			<td>Tocris</td>
			<td>111</td>
			<td>Working concentration 50 &#956;M and 300 &#956;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)&#160; 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 &#181;g in 1mL of PBS to 100 &#181;g/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 &#181;g/mL. Aliquot and freeze at -80 &#186;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 &#181;L tubes. Dilute 1:1000 for use. (working concentration 2 &#181;g/mL).</td>
		</tr>
		<tr>
			<td>Humidity controlled Cell culture incubator</td>
			<td>ThermoFisher</td>
			<td>370</td>
			<td>set to 37 &#186;C, 5 % CO2</td>
		</tr>
		<tr>
			<td>Insulin-like growth factor 1 (IGF-1)</td>
			<td>R&#38;D systems</td>
			<td>291-G1-200</td>
			<td>Dissolve 200 &#181;g in 2 mL of PBS to 100 &#181;g/mL, then add 18 mL of 0.1%BSA-PBS to make 10 &#181;g/mL stock and store at -80 &#186;C. Aliquot and freeze at -80 &#186;C. Dilute 10 &#181;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 &#956;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 &#181;g/mL (coating), 1 &#181;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 &#181;L of Chloroform to get 10 mM stock. Aliquot this and freeze at -80 &#186;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 &#181;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 &#181;L tubes. (working concentration 100 &#181;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 &#186;C. Dilute 1:1000 for use. (working concentration 1 &#181;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 &#181;g in 1 mL of PBS to 100 &#181;g/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 &#181;g/mL. Aliquot and freeze at -80 &#186;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 &#181;L/tube and freeze at -80 &#186;C. Take 200 &#181;L of 100 mM stock and dilute 10x (add 1.8 mL of DMSO) to make 10 mM stock. Aliquot 50 &#181;L/tube&#160;and store at -80 &#186;C. Dilute at 1:10000 for use. (working concentration 2 &#181;M).</td>
		</tr>
		<tr>
			<td>ROCK-I nhibitor</td>
			<td>Peprotech</td>
			<td>1293823</td>
			<td>Dissolve 5 mg in 1480 &#181;L of dH2O to get 10 mM stock, aliquot and freeze at -80 &#186;C. Dilute at 1: 500 for use. (working concentration 20 &#181;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 &#186;C. Dilute at 1:1000 for use. (working con 10 &#181;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&#160;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 ddH2O to get 10 mg/mL stock. Aliquot and store at 4 &#186;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

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-&#946;) pathways, respectively. A monolayer-ba...

Watch this Scientific Journal Video about Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons at JoVE

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

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) ...

The technique we present today allows for the analysis of spinal cord neuron activity as a result of intrinsic neuronal factor and the influence of surrounding astrocytes. This technique reliably generates both human neurons and human astrocytes that are spinal cord specific and uses a functional output measure that can be scaled for reproducibility. The technique we present allows to investigate spinal cord neurons specific diseases such as amyotrophic lateral sclerosis, using neurons from patients with either sporadic or genetic disease.To begin with, examine the human iPSC plates for sufficiently dense colonies such that the center of the colonies displays scattered white areas. Then to start packaging the cells, draw grid lines on the bottom outer surface of the plates with a marker to facilitate accurate coverage of the entire plate. Use a phase contrast microscope of 10x magnification in a culturehood to detach differentiated colonies by manually scraping with a 200 microliter pipette tip.Next, rinse the plates twice with PBS. And then add five milliliters of tissue dissociation protease pre-warmed at 37 degrees Celsius. Incubate the cells at 37 degrees Celsius for four to seven minutes until the edges of the colonies start to round up, prior to lifting colonies from the plate.Carefully aspirate the dissociation protease and gently rinse the plate twice with PBS without dislodging the colonies. Add five milliliters of pre-warmed human iPSC medium to the culture plate. Then scratch the entire plate with a tip of a five milliliter pipette using a back and forth motion from top to bottom and lift the colonies while breaking them up into smaller cell aggregates.For differentiation of human iPSC into neural progenitor cells. Once human iPSC forms a monolayer and becomes 90%confluent, start differentiation as described in the manuscript. On the 12th day, after trypsin treatment, if the cells are not in suspension, detached cells mechanically by ejecting neural induction medium from a 1000 microliter pipette tip onto the cells with a circular motion to cover the whole surface.If the neural progenitor cell cultures were frozen, thaw them. After medium exchange and PBS rinsing as described in the manuscript, change the medium with a motor neuron differentiation medium containing 0.02 micromolar cytosine arabinoside then incubate the cells for 48 hours at 37 degrees Celsius and 5%carbon dioxide when glial committed progenitors emerge as single proliferating flat cells under post mitotic motor neuron progenitors, aggregated in cell clusters. Later, performed medium exchange as indicated in the manuscript.On the day of plating or before, start coating the 24 well MEA plates by first diluting PLO in water or PBS. Then add 15 to 20 microliters of PLO to each well forming a droplet on the center of the well, covering the electrode area ensuring not to damage electrodes with the pipette tip. Add water to the compartments surrounding wells, ensuring sufficient humidity.And incubate PLO at 37 degrees Celsius for one to two hours. Then using a plastic micro pipette tip, aspirate as much PLO as possible without touching the electrodes. Next, wash the well with 250 microliters of water three times.Using a pipette tip, remove as much water as possible and let the surface dry with the lid removed. Next, add 15 to 20 microliters of diluted laminin to cover each electrode array. After adding water to the humidity compartments and replacing the lid, incubate at 37 degrees Celsius for a minimum of two hours up to overnight.On the day of plating, after rinsing the motor neurons and astrocyte cultures once with PBS, add 0.05%trypsin and incubate at 37 degrees Celsius for about five minutes to lift cells. Collect cells into a 15 milliliter conical tube containing trypsin inhibitor. And wash plates with medium or base to ensure that all the cells are collected.Pellet down the cells by centrification. And then using a 1000 microliter pipette, resuspend motor neurons and astrocytes with a co-culture medium containing 20 micromolar of ROCK inhibitor to generate one milliliter of a single cell suspension. Using a hemocytometer, count the motor neurons and astrocytes.And while counting, kept the cell suspensions and place them in a styrofoam rack at four degrees Celsius. Centrifuge one milliliter of both cell suspensions at 300 times G for five minutes. Calculate the desired volume and resuspend the pellets.Combine the required volume of neuron and astrocyte suspensions in equal ratios, and mix gently by pipetting twice to combine thoroughly. Then remove laminin from each well of the plate and transfer 10 microliters of the final combined cell suspension to each well, forming a small droplet covering the electrode array. Incubate the plates with an undisturbed cell droplet at 37 degrees Celsius for 20 to 30 minutes to form initial attachments on the plates.Then pipette 250 microliters of warm co-culture media containing ROCK inhibitor down on the wall of each well and pipette the same volume on the opposite wall of each well and incubate the plate at 37 degrees Celsius for 24 to 36 hours. The next day, examine the plates for debris or dead cells. And if required, exchange the medium with a fresh co-culture medium containing ROCK inhibitor.Otherwise, perform half medium exchanges twice a week using a co-culture medium without a ROCK inhibitor starting on co-culture day two. To perform recording, start as soon as possible after co-culture day one by setting the temperature to 37 degrees Celsius and carbon dioxide at 5%Then transfer plates to the recording machine and equilibrate for at least five minutes before recording. Record baseline activity every other day or weekly over one to 15 minutes depending on the experimental design.To investigate the transient electrophysiological effects of compounds targeting either neurons or astrocytes, remove the plate lid but keep the machine lid closed and record baseline activity for a minimum of one minute. Manually open the machine lid without stopping the electrophysiological recording and exchange 25 microliters of medium with the appropriate drug vehicle. Then close the lid manually and record for additional minutes if required.Similarly, test the drug of interest. In this protocol, hiPSCs were maintained and passaged as non-confluent colonies. Neurogenesis was initiated through dual SMAD inhibition by inactivating BMP and TGF-beta pathways.The resulting neuro epithelial stem cells divided and generated a multi-layered epithelium. The early exposure to neuronal growth factors and a gliogenic ciliary neurotrophic factor generated a mixed population of progenitor cells. Neurons emerged spontaneously from this mixed population.The gliogenic switch induced astrocyte differentiation through activation of the JAK-STAT pathway. The spinal cord identity of notch inhibitor treated and differentiated motor neuron cells was supported by high choline acetyltransferase expression levels. On the 90th day post in vitro culture, human iPSC astrocytes displayed maturing phenotype as indicated by S100 beta and GFAP expression while their spinal cord regional identity was supported by expression of hawks before.Co-cultured cells rearranged spontaneously with astrocytes creating a feeding layer at the bottom and neurons connecting networks at the top. Neurons aggregated in large cell clusters when cultured alone, while the co-culture of human iPSC motor neurons with human iPSC astrocytes resulted in evenly distributed monolayers. Human iPSC astrocytes enhanced the electrophysiological maturation of human iPSC motor neurons as shown by higher degrees of spiking and bursting activity in the co-cultures.In our experience, the stem cell culture and early NPC differentiation are the most difficult and sensitive to technical error. At this stage, the techniques must not be performed with consistency and accuracy with little margin for troubleshooting. The co-culture techniques described here can be used in other cell type specific models as well.

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Research

JoVE Journal

Neuroscience

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...