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

该手稿得到以下支持：2019 MSCRFF 5119 （AT）。K08NS102526 NIH/NINDS （CWH），2020 年多丽丝杜克慈善基金会临床科学家职业发展奖 （CWH）。1R01NS117604-01NIH/NINDS （NJM）， DOD ALSRP W81XWH2010161 （NJM）， 2019 MSCRFD 5122 （NJM）.我们感谢Raha Dastgheyb博士和Norman Haughey博士提供MEA平台和数据分析软件，我们用它来验证所描述的电生理平台。我们要感谢Khalil Rust在协议演示和拍摄方面的协助。

1. 细胞培养基制备
<ol><li>使用 表1中提到的组合物制备单个细胞培养基。</li>
	<li>在500mL过滤瓶中混合并无菌过滤培养基，并在4°C避光下储存长达2周。</li>
</ol>2. 维持和传代非融合人诱导多能干细胞 （hiPSC）
<ol><li>将基底膜基质（在-80°C下等分储存）在2-8°C冰箱中解冻过夜。</li>
	<li>在冷磷酸盐缓冲盐水（PBS）或Dulbecco的改良鹰培养基（DMEM）中以1：100（v / v）稀释?...

<ol>
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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. 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迄今为止，基于hiPSC和MEA的星形胶质细胞-神经元共培养电生理记录方法在ALS6领域的应用有限，并且仍然没有在完全人类平台上应用，相比之下，它们更广泛地用于癫痫9的体外建模。然而，该平台有可能解决ALS研究中的病理生理学相关问题，例如神经元过度兴奋的机制，星形胶质细胞对神经毒性的贡献或网络活动在疾病进展中的作用。此外，该平台允许?...

人类多能干细胞衍生的星形胶质细胞（hiPSC-A）和神经元（hiPSC-N）是体 外 模拟肌萎缩侧索硬化症（ALS）病理生理学的强大工具，并为药物发现策略提供了转化范式1。研究人员已经证明，hiPSC-A 与 hiPSC-N 的共培养增强了两种细胞类型的形态、分子、电生理和药理成熟，产生复杂的神经元网络和星形胶质细胞-神经元相互作用，类似于 体内 对应物2，3。类似的共培养实验可以概括ALS病理生物学的特征，例如星形胶质细胞介导的神经毒性4，5 和神经元过度兴奋性6。此外，随着分化方案的进步，人诱导多能干细胞（hiPSC）可以分化为区域特异性神经亚型，包括皮质和脊髓hiPSC-A和hiPSC-N7，8。这些策略为模拟ALS中的皮质和脊髓运动神经元病理学以及星形胶质细胞对两者的影响提供了潜力。然而，这需要有可重现的功能测定来确定这些影响。
最近研究表明，多电极阵列（MEA）记录特别适用于神经元-星形胶质细胞共培养物的电生理表征2。与单细胞电生理分析相反，这些高密度电极阵列被动记录来自大量神经元的细胞外场电位，而不会破坏培养条件并保持细胞膜的完整性。这些平台对于记录培养物随时间推移的细胞和网络活性以及对药物操作的响应特别有用。最后，当星形胶质细胞的存在是一个培养变量时，MEA记录可以提供星形胶质细胞-神经元双向相互作用的功能见解2，9。
这里介绍的是将hiPSC分化为脊髓hiPSC-A和hiPSC运动神经元（MN）的优化方案，该方案先前已经过验证2。脊髓 hiPSC-A 分化方案一致导致星形胶质细胞培养，分别在多达 80%、50% 和 90% 的细胞中对 S100 钙结合蛋白 B （S100β）、神经胶质纤维酸性蛋白 （GFAP） 和同源盒 B4 （HOXB4） 呈阳性，表明神经胶质细胞和脊髓规格成熟2，10.hiPSC-MN 分化方案产生的神经元胆碱乙酰转移酶 （ChAT） 呈 >90% 阳性，提示成熟的 α 运动神经元身份2。此外，该协议描述了用于生成hiPSC-A / MN共培养的技术，与没有星形胶质细胞或啮齿动物星形胶质细胞的神经元培养物相比，先前通过Scholl分析和免疫荧光显微镜证明导致神经元具有增强的形态复杂性2。虽然这些描述特定于脊髓hiPSC-A和hiPSC-N，但一个独特的优势是，星形胶质细胞和神经元的初始独立培养，然后在以后的时间点进行共培养的步骤可以转化为研究来自其他特定区域以及疾病特异性细胞的神经元 - 星形胶质细胞相互作用的影响7，8.最后，该协议描述了如何在MEA平板上培养这些培养物，以便随着时间的推移，可以通过操纵细胞组成和培养条件的能力来研究作为共培养组成因子的功能活性。
这些协议的目标是提供一种功能测定，以研究星形胶质细胞 - 神经元相互作用，检查疾病特异性变化，并测试在ALS领域具有治疗潜力的药物。为该协议最具挑战性的步骤提供了视频说明。

<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

人多能干细胞衍生的星形胶质细胞（hiPSC-A）和神经元（hiPSC-N）为 体外肌萎缩侧索硬化症（ALS）病理生理学建模提供了强大的工具。多电极阵列（MEA）记录是一种记录大量神经元的电场电位并分析网络活动随时间变化的方法。先前已经证明，与没有hiPSC-A或在存在啮齿动物星形胶质细胞的情况下培养的hiPSC-A相比，使用促进脊髓星形胶质细胞表型的技术分化的hiPSC-A的存在改善了区域特异性脊髓hiPSC-运动神经元（MN）的成熟和电生理活性。这里描述的是一种将脊髓hiPSC-A与hiPSC-MN共培养并使用MEA记录记录电生理活性的方法。虽然这里描述的分化方案特定于星形胶质细胞和脊髓区域特异性的神经元，但共培养平台可应用于星形胶质细胞和神经元，这些分化技术特定于其他命运，包括皮质hiPSC-A和hiPSC-N。这些协议旨在提供电生理测定，以告知神经胶质神经元相互作用，并为测试ALS中具有治疗潜力的药物提供平台。

用于生成hiPSC-MN和脊髓hiPSC-A的脊髓模式方案如图 1所示。在该协议中，hiPSCs作为非融合集落维持和传代（图2A）。通过添加LDN193189和SB431542的双重SMAD抑制启动神经发生（神经诱导），分别灭活骨形态发生蛋白（BMP）和转化生长因子-β（TGF-β）途径。此步骤使用基于单层的方法，其中将hiPSC接种到贴壁基质（即基底膜基质）上，以产生神经上皮样2D培养物?...

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

我们描述了一种区分脊髓人诱导的多能源星形胶质细胞和神经元及其共培养以进行电生理记录的方法。

建立区域特异性人多能干细胞衍生星形胶质细胞和神经元模拟ALS的电生理平台

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

我们今天介绍的技术允许分析由于内在神经元因素和周围星形胶质细胞的影响而导致的脊髓神经元活动。该技术可靠地产生脊髓特异性的人类神经元和人类星形胶质细胞，并使用可以缩放以实现可重复性的功能输出测量。我们提出的技术允许使用散发性或遗传性疾病患者的神经元来研究脊髓神经元的特定疾病，例如肌萎缩侧索硬化症。首先，检查人iPSC平板中是否有足够密集的菌落，使得菌落的中心显示分散的白色区域。然后开始包装细胞，用记号笔在板的底部外表面上绘制网格线，以便准确覆盖整个板。在培养罩中使用放大倍数为 10 倍的相衬显微镜，通过用 200 微升移液器吸头手动刮擦来分离分化的菌落。接下来，用PBS冲洗板两次。然后加入5毫升组织解离蛋白酶在37摄氏度下预热。将细胞在 37 摄氏度下孵育四到七分钟，直到菌落的边缘开始变圆，然后再从平板上提起菌落。小心吸出解离蛋白酶，并用PBS轻轻冲洗板两次，而不会移位菌落。向培养板中加入5毫升预热的人iPSC培养基。然后用五毫升移液管的尖端从上到下来回划伤整个板，并提起菌落，同时将它们分解成更小的细胞聚集体。用于将人iPSC分化为神经祖细胞。一旦人类iPSC形成单层并达到90%汇合，就可以按照手稿中的描述开始分化。在第12天，胰蛋白酶处理后，如果细胞未悬浮，则通过以圆周运动将神经诱导培养基从1000微升移液器尖端喷射到细胞上以覆盖整个表面来机械分离细胞。如果神经祖细胞培养物被冷冻，请解冻它们。如手稿中所述进行培养基交换和PBS冲洗后，用含有0.02微摩尔胞嘧啶阿拉伯糖苷的运动神经元分化培养基更换培养基，然后将细胞在37摄氏度和5%二氧化碳下孵育48小时，当神经胶质承诺祖细胞在有丝分裂后运动神经元祖细胞下出现为单个增殖扁平细胞时， 聚合在细胞簇中。之后，按照手稿中的说明进行了介质交换。在电镀当天或之前，首先将PLO稀释在水或PBS中，开始涂覆24孔MEA板。然后向每个孔中加入15至20微升PLO，在孔的中心形成液滴，覆盖电极区域，确保不会损坏移液器尖端的电极。向井周围的隔间加水，确保足够的湿度。并将PLO在37摄氏度下孵育一到两个小时。然后使用塑料微量移液器吸头，在不接触电极的情况下尽可能多地吸出PLO。接下来，用250微升水清洗井三次。使用移液器吸头，尽可能多地去除水，并在取下盖子的情况下让表面干燥。接下来，加入 15 至 20 微升稀释的层粘连蛋白以覆盖每个电极阵列。向湿度室加水并盖上盖子后，在 37 摄氏度下孵育至少两个小时直至过夜。在铺板当天，用PBS冲洗运动神经元和星形胶质细胞培养物一次后，加入0.05%胰蛋白酶并在37摄氏度下孵育约5分钟以提升细胞。将细胞收集到含有胰蛋白酶抑制剂的15毫升锥形管中。并用培养基或碱清洗板，以确保收集所有细胞。通过离心沉淀细胞。然后使用1000微升移液管，用含有20微摩尔ROCK抑制剂的共培养基重悬运动神经元和星形胶质细胞，以产生1毫升单细胞悬液。使用血细胞计数器，计数运动神经元和星形胶质细胞。在计数时，保持细胞悬浮液并将它们放置在四摄氏度的聚苯乙烯泡沫塑料架中。以300倍G离心一毫升两种细胞悬浮液五分钟。计算所需的体积并重悬颗粒。以相等的比例合并所需体积的神经元和星形胶质细胞悬浮液，并通过移液两次轻轻混合以彻底混合。然后从板的每个孔中取出层粘连蛋白，并将10微升最终组合的细胞悬液转移到每个孔中，形成覆盖电极阵列的小液滴。将平板与未受干扰的细胞液滴在 37 摄氏度下孵育 20 至 30 分钟，以在平板上形成初始附着。然后将250微升含有ROCK抑制剂的温热共培养基移液到每个孔的壁上，并在每个孔的对壁上移取相同体积的样品，并将板在37摄氏度下孵育24至36小时。第二天，检查板是否有碎片或死细胞。如果需要，用含有ROCK抑制剂的新鲜共培养基交换培养基。否则，从共培养第二天开始，使用不含 ROCK 抑制剂的共培养基每周进行两次半培养基置换。要进行记录，请在共培养第一天后尽快开始，将温度设置为 37 摄氏度，将二氧化碳设置为 5%，然后将板转移到记录机中并在记录前平衡至少五分钟。每隔一天或每周记录一次基线活动，超过1至15分钟，具体取决于实验设计。为了研究靶向神经元或星形胶质细胞的化合物的瞬时电生理效应，请取下板盖，但保持机器盖关闭并记录基线活动至少一分钟。手动打开机器盖而不停止电生理记录，并与适当的药物载体交换25微升培养基。然后手动盖上盖子，并在需要时记录额外的分钟数。同样，测试感兴趣的药物。在该协议中，hiPSCs作为非融合集落得以维持和传代。神经发生是通过灭活BMP和TGF-β途径通过双重SMAD抑制启动的。由此产生的神经上皮干细胞分裂并产生多层上皮。早期暴露于神经元生长因子和胶质生成睫状神经营养因子产生了混合的祖细胞群。神经元从这个混合群体中自发出现。胶质细胞开关通过激活JAK-STAT途径诱导星形胶质细胞分化。经Notch抑制剂处理和分化的运动神经元细胞的脊髓特性由高胆碱乙酰转移酶表达水平支持。在体外培养后第90天，人iPSC星形胶质细胞显示出成熟的表型，如S100 β和GFAP表达所示，而其脊髓区域身份之前由鹰的表达支持。共培养的细胞自发地与星形胶质细胞重新排列，在底部形成喂养层，在顶部创建连接网络的神经元。单独培养时神经元聚集在大细胞簇中，而人iPSC运动神经元与人iPSC星形胶质细胞共培养导致均匀分布的单层。人iPSC星形胶质细胞增强了人iPSC运动神经元的电生理成熟，如共培养物中更高程度的尖峰和爆发活性所示。根据我们的经验，干细胞培养和早期鼻咽癌分化是最困难的，对技术错误最敏感。在此阶段，不得以一致和准确的方式执行这些技术，并且几乎没有故障排除的余地。这里描述的共培养技术也可用于其他细胞类型特定的模型。

Research

JoVE Journal

Neuroscience

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

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

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

科研

神经科学

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

从人类多能干细胞的规范在前脑谷氨酸能神经元

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

从人多能干细胞定向多巴胺能神经元分化

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

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

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

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

96井格式人诱导多潜能干细胞源神经元高速拉伸损伤的方法

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

预组装塑料微流体芯片中人类干细胞衍生神经元的分段化

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

分化后再蛋白的人类多能干细胞衍生神经元，用于高含量的内尿成生和突合性成熟筛查

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

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

利用 PiggyBac 载体将人诱导的多能干细胞 (Ipsc) 转化为功能性脊柱和颅内运动神经元

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

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

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

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

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