Name: generation of human neurons and oligodendrocytes from pluripotent stem cells for modeling neuron-oligodendrocyte interactions
Uploaded: 2020-11-09T15:00:00
Description: Watch this Scientific Journal Video about Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions at JoVE.com

这项工作得到了美国国立卫生研究院（R00 AG054616 至 YAH，T32 GM136566 至 K.C.）、斯坦福大学医学院和 Siebel 奖学金（授予 SC）的资助。YAH是布朗转化科学研究所转化神经科学中心的GFL转化教授。

1. 人多能干细胞对人神经元的诱导
<ol><li>慢病毒制备（~5天，详细方案如前所述16）<ol><li>在每个T75烧瓶中接种~100万个HEK293T细胞，使其在进行转染时~40%汇合。用表达四环素诱导型Ngn2和嘌呤霉素抗性基因（PuroR;在相同的TetO启动子对照下）、rtTA和三种辅助质粒pRSV-REV、pMDLg/pRRE和VSV-G（12μg慢病毒载体DNA和6μg辅助质粒DNA）的质粒转染它们。每种慢病毒制剂至少准备三个烧瓶。按照制...

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H., Park, C. H. <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+reprogramming+methods+for+generation+of+induced-oligodendrocyte+precursor+cells.">Comparison of reprogramming methods for generation of induced-oligodendrocyte precursor cells.</a> Biomolecules &amp; Therapeutics (Seoul). 25 (4), 362-366 (2017).</li><li>Ehrlich, 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=Rapid+and+efficient+generation+of+oligodendrocytes+from+human+induced+pluripotent+stem+cells+using+transcription+factors.">Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (11), 2243-2252 (2017).</li><li>Rodrigues, G. M. 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=Defined+and+scalable+differentiation+of+human+oligodendrocyte+precursors+from+pluripotent+stem+cells+in+a+3D+culture+system.">Defined and scalable differentiation of human oligodendrocyte precursors from pluripotent stem cells in a 3D culture system.</a> Stem Cell Reports. 8 (6), 1770-1783 (2017).</li><li>Hu, B. Y., Du, Z. W., Li, X. J., Ayala, M., 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=Human+oligodendrocytes+from+embryonic+stem+cells:+conserved+SHH+signaling+networks+and+divergent+FGF+effects.">Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects.</a> Development. 136 (9), 1443-1452 (2009).</li><li>Izrael, 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=Human+oligodendrocytes+derived+from+embryonic+stem+cells:+Effect+of+noggin+on+phenotypic+differentiation+in+vitro+and+on+myelination+in+vivo.">Human oligodendrocytes derived from embryonic stem cells: Effect of noggin on phenotypic differentiation in vitro and on myelination in vivo.</a> Molecular and Cellular Neuroscience. 34 (3), 310-323 (2007).</li><li>Yamashita, T., 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=Differentiation+of+oligodendrocyte+progenitor+cells+from+dissociated+monolayer+and+feeder-free+cultured+pluripotent+stem+cells.">Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.</a> PLoS One. 12 (2), 0171947 (2017).</li><li>Wang, S., 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+iPSC-derived+oligodendrocyte+progenitor+cells+can+myelinate+and+rescue+a+mouse+model+of+congenital+hypomyelination.">Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination.</a> Cell Stem Cell. 12 (2), 252-264 (2013).</li><li>Chanoumidou, K., Mozafari, S., Baron-Van Evercooren, A., Kuhlmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+derived+oligodendrocytes+to+study+myelin+diseases.">Stem cell derived oligodendrocytes to study myelin diseases.</a> Glia. 68 (4), 705-720 (2020).</li><li>Chetty, S., 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=A+simple+tool+to+improve+pluripotent+stem+cell+differentiation.">A simple tool to improve pluripotent stem cell differentiation.</a> Nature Methods. 10 (6), 553-556 (2013).</li><li>Li, 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=A+transient+DMSO+treatment+increases+the+differentiation+potential+of+human+pluripotent+stem+cells+through+the+Rb+family.">A transient DMSO treatment increases the differentiation potential of human pluripotent stem cells through the Rb family.</a> PLoS One. 13 (12), 0208110 (2018).</li><li>Sambo, D., Li, J., Brickler, T., Chetty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+treatment+of+human+pluripotent+stem+cells+with+DMSO+to+promote+differentiation.">Transient treatment of human pluripotent stem cells with DMSO to promote differentiation.</a> Journal of Visualized Experiments: JoVE. 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除了通过髓鞘形成稳定突触结构和促进盐信号传导的物理和代谢支持外，少突胶质细胞谱系细胞还可以通过与神经元的快速和动态串扰来塑造神经元活动模式5，6，7。虽然在AD病理学中，少突胶质反应最初被认为仅继发于炎症和氧化应激，但现在有有希望的证据表明，髓鞘完整性受损是Aβ聚集和tau过度磷酸化出现<...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61778/generation-human-neurons-oligodendrocytes-from-pluripotent-stem-cells">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions</a>.&#160;The Representative Results section has been updated.
Figure 3 was updated from:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61778/61778fig03.jpg" /> 
Figure 3: Co-culture of iNs and iOPCs.&#160;(A) Representative bright field image of co-cultured iNs and iOPCs at Day 7, showing a proper density for further maturation. (B) Representative immunofluorescence image of iNs and iOPCs co-cultured for 28 days. Axonal marker neurofilament NF is shown in green and oligodendrocytic marker MBP in red. Right, a segment of iN axon ensheathed by iOL process (MBP+). (C) Synapse formation assayed in 4-week-old co-cultures. Cells were stained for Synapsin 1 (Syn1, green) and MAP2 (red), and synaptic puncta were quantified by confocal analysis of density along the dendritic segments as described17,18. (D) In our co-cultures of iNs and iOPCs (7 days of co-culturing), the expression of astrocyte markers, ALDHL1 and GFAP, is minimal (top), and the expression of microglia markers, TMEM119, TREM2, and CD33, is not detected (N.D.) by qPCR. The contamination from these two glial cell types is thus excluded. <a href="https://www.jove.com/files/ftp_upload/61778/61778fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61778/61778fig3v2.png" /> 
Figure 3: Co-culture of iNs and iOPCs.&#160;(A) Representative bright field image of co-cultured iNs and iOPCs at Day 7, showing a proper density for further maturation. (B) Representative immunofluorescence image of iNs and iOPCs co-cultured for 28 days. Axonal marker neurofilament NF is shown in green and oligodendrocytic marker MBP in red. Right, a segment of iN axon ensheathed by iOL process (MBP+). (C) Synapse formation assayed in 4-week-old co-cultures. Cells were stained for Synapsin 1 (Syn1, green) and MAP2 (red), and synaptic puncta were quantified by confocal analysis of density along the dendritic segments as described17,18. (D) In our co-cultures of iNs and iOPCs (7 days of co-culturing), the expression of astrocyte markers, ALDHL1 and GFAP, is minimal (top), and the expression of microglia markers, TMEM119, TREM2, and CD33, is not detected (N.D.) by qPCR. The contamination from these two glial cell types is thus excluded.&#160;(E) Coculturing iOPC with iN leads to the formation of neuron-OPC synapses. The fluorescence-tagged post-synaptic marker PSD95-mCherry is expressed only in OPCs, and display a diffuse pattern in single cultures (left) but aggregate to form puncta in cocultures (right, indicated by arrows; Tuj1, neuronal marker). (F) The expression of well-characterized oligodendroglial genes that can sense and respond to neuronal activities in the pure cultures of iOPCs at Day 14.&#160;<a href="https://www.jove.com/files/ftp_upload/61778/61778fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The fourth&#160;paragraph was updated from:
Co-culturing of iNs and iOPCs 
This protocol is optimized specifically for co-culturing iNs and iOPCs and allow our real-time monitoring of the inter-cellular communications between these two cell types along the course of neural development. The ideal plating densities for both cell types need to be decided with a series of cell number titration to achieve proper differentiation (Figure 3A). After 4 weeks in co-cultures, the iOPCs are expected to be adequately differentiated into OLs that are positive for specific markers such as MBP and extend processes to ensheath axons (Figure 3B). The co-culture system can robustly boost up the number of synapses, indicating that the iOPCs provide a neuronal support through physical contacts or release of trophic factors (Figure 3C). We can maintain the co-cultures in acceptable health condition for up to 6 weeks and observe that the synapse number and other neuronal attributes plateau around the fifth week. Of note, astrocytes and microglia are not present in our preparations and their absence can be documented by checking the expression of specific markers (Figure 3D).
to:
Co-culturing of iNs and iOPCs 
This protocol is optimized specifically for co-culturing iNs and iOPCs and allow our real-time monitoring of the inter-cellular communications between these two cell types along the course of neural development. The ideal plating densities for both cell types need to be decided with a series of cell number titration to achieve proper differentiation (Figure 3A). After 4 weeks in co-cultures, the iOPCs are expected to be adequately differentiated into OLs that are positive for specific markers such as MBP and extend processes to ensheath axons (Figure 3B). The co-culture system can robustly boost up the number of synapses, indicating that the iOPCs provide a neuronal support through physical contacts or release of trophic factors (Figure 3C). We can maintain the co-cultures in acceptable health condition for up to 6 weeks and observe that the synapse number and other neuronal attributes plateau around the fifth week. Of note, astrocytes and microglia are not present in our preparations and their absence can be documented by checking the expression of specific markers (Figure 3D).&#160;The iOPCs express a good number of well-characterized genes that can potentially respond to and mediate the activity-dependent signals from neighboring neurons, in a paracrine (e.g. neurotrophins and metabolites) and/or a synaptic manner (Figure&#160;3E and 3F).&#160;

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61778/generation-human-neurons-oligodendrocytes-from-pluripotent-stem-cells">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions</a>.&#160;The Representative Results section has been updated.

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

少突胶质细胞谱系细胞（包括少突胶质细胞前体细胞 （OPC）、髓鞘少突胶质细胞和介于两者之间的过渡型细胞）构成了人类脑细胞的主要群体1，它们积极参与许多关键功能，在整个神经发育和衰老过程中正常运作和维护我们的中枢神经系统2，3，4.虽然少突胶质细胞以产生髓磷脂以促进神经元活动传递并支持白质中的轴突健康而闻名，但OPC在髓鞘形成稀缺的灰质中含量丰富（~5%），并执行活动依赖性信号传导功能以控制学习行为和记忆形成5，6，7，8.少突胶质细胞在阿尔茨海默病（AD）和其他年龄相关神经退行性疾病的发病机制中的功能和功能障碍的研究不足9。适当的模型系统的不足和指导实验路径前进的一般知识的不足是造成这一差距的主要原因。
鉴于从多能干细胞（包括胚胎干（ES）和诱导多能干细胞（iPS）细胞）中提取人脑细胞的最新突破，这种细胞模型与现代基因编辑工具相结合已成为处理大脑中细胞相互作用的复杂联系的强大工具，并且能够证明人类特有的疾病表现10，11.考虑到单个脑细胞类型在面对相同的AD促进条件时可以表现出不同甚至相互矛盾的效果12，13，这种干细胞方法独特地提供了以前使用已建立的体内或体外模型错过的细胞类型特异性信息，这些模型仅提供来自脑细胞类型集合的汇总读数。在过去的十年中，已经开发了大量可靠的协议，以通过ES / iPS细胞的转分化或从其他终末分化细胞类型（例如成纤维细胞）的直接转化中产生人类神经元14，15。特别是，将关键的神经源性转录因子（例如，神经原蛋白2，Ngn2）16应用于人多能干细胞，可以为纯培养物产生表征良好的神经元细胞类型的均质群体，而无需与神经胶质细胞共培养12，17，18。对于诱导的人少突胶质细胞，有一些已发布的方案可以产生与其主要对应物高度相似的功能细胞，具有广泛的效率和时间和资源需求19，20，21，22，23，24，25，26，27，28.迄今为止，这些方案均未应用于研究少突胶质细胞如何响应和影响AD发病机制。
在这里，我们描述了我们改进的人类诱导神经元（iNs）和OPC /少突胶质细胞（iOPC / iOLs）的单一和混合培养方案。这里描述的iN协议基于广泛使用的Ngn2方法16，并且具有无胶质细胞的附加特征。所得iN是同质的，与皮质层2/3兴奋性神经元高度相似，具有特征性的锥体形态、基因表达模式和电生理特征17，18（图1）。为了克服多能干细胞定向分化的一些基本障碍，我们开发了一种简单有效的低剂量二甲基亚砜（DMSO）预处理方法29，30，并报告了人ES / iPS细胞转分化为iOPC和iOLs31的增强倾向，基于Douvaras和Fossati32广泛适应的方案.我们进一步简化了方案，并掺入了一种强大的分化促进化合物克立姆定7，33，34，以加速少突胶质细胞成熟过程。结果（图2），iOPCs可以在2周内产生（标记物O4的~95%阳性），iOLs可以在四周内产生（表达成熟的标志物MBP和PLP1）。有趣的是，我们发现单独iOPCs分泌大量淀粉样蛋白-β（Aβ），与独立的转录组学数据一致，数据显示淀粉样蛋白前体蛋白（APP）和加工蛋白酶β分泌酶（BACE1）在少突胶质细胞谱系细胞中的丰富表达35，36。此外，我们的iN-iOPC共培养系统通过MBP阳性iOL过程促进轴突的鞘化，并为突触形成提供重要支持（图3）。因此，我们在下面详细介绍的方案与先前编目的神经元-少突胶质共培养方法相比具有技术和生物学优势，并有望更好地模拟AD中的神经变性。

<table>
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>ThermoFisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>ThermoFisher</td>
			<td>PHG 0266</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>MilliporeSigma</td>
			<td>A9501</td>
			<td></td>
		</tr>
		<tr>
			<td>Clemastine</td>
			<td>MilliporeSigma</td>
			<td>SML0445</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>STEMCELL Technologies</td>
			<td>36254</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>ThermoFisher</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>MilliporeSigma</td>
			<td>D3072</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ScienCell</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>H1 human ES cells</td>
			<td>WiCell</td>
			<td>WA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR plus</td>
			<td>STEMCELL Technologies</td>
			<td>5825</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>ThermoFisher</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal A medium</td>
			<td>ThermoFisher</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>ThermoFisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-AA</td>
			<td>R&#38;D Systems</td>
			<td>221-AA-010</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>VWR</td>
			<td>71002-812</td>
			<td></td>
		</tr>
		<tr>
			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>MilliporeSigma</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-REV</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>ThermoFisher</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Tocris</td>
			<td>4366</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Neural Progenitor Freezing Media</td>
			<td>STEMCELL Technologies</td>
			<td>5838</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff SMADi Neural Induction Kit</td>
			<td>STEMCELL Technologies</td>
			<td>8581</td>
			<td></td>
		</tr>
		<tr>
			<td>T3 triiodothyronine</td>
			<td>MilliporeSigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Tempo-iOlogo: Human iPSC-derived OPCs</td>
			<td>Tempo BioScience</td>
			<td>SKU102</td>
			<td></td>
		</tr>
		<tr>
			<td>TetO-Ng2-Puro</td>
			<td>Addgene</td>
			<td>52047</td>
			<td></td>
		</tr>
		<tr>
			<td>VSV-G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of human neurons and oligodendrocytes from pluripotent stem cells for modeling neuron-oligodendrocyte interactions

在阿尔茨海默病（AD）和其他神经退行性疾病中，少突胶质细胞衰竭是一种常见的早期病理特征，但它如何促进疾病的发展和进展，特别是在大脑的灰质中，仍然在很大程度上是未知的。少突胶质细胞谱系细胞的功能障碍以髓鞘形成缺陷和少突胶质细胞前体细胞（OPCs）自我更新受损为特征。这两种缺陷至少部分是由神经元和少突胶质细胞之间沿着病理学积累的相互作用的破坏引起的。OPC在中枢神经系统发育过程中产生髓鞘少突胶质细胞。在成熟的大脑皮层中，OPC是主要的增殖细胞（占总脑细胞的~5%），并以神经活动依赖性的方式控制新的髓磷脂形成。由于缺乏适当的工具，这种神经元到少突胶质细胞的通讯研究明显不足，特别是在AD等神经退行性疾病的背景下。近年来，我们的小组和其他人在改进目前可用的方案方面取得了重大进展，以从人类多能干细胞中单独生成功能性神经元和少突胶质细胞。在这篇手稿中，我们描述了我们的优化程序，包括建立一个共培养系统来模拟神经元 - 少突胶质细胞连接。我们的说明性结果表明，OPCs/少突胶质细胞对脑淀粉样变性和突触完整性做出了意想不到的贡献，并强调了该方法在AD研究中的实用性。这种还原论方法是从大脑内部固有的复杂性中剖析特定异细胞相互作用的有力工具。我们在这里描述的方案有望促进未来对神经变性发病机制中少突胶质细胞缺陷的研究。

从人多能干细胞直接产生人诱导神经元 非常重要的是，起始人多能干细胞表现出高度的多能性，以成功生成iN或iOPC / iOLs。因此，在开始本手稿中描述的任一诱导方案之前，应针对特定标记物（例如Oct4和SOX2）对细胞进行染色（图1A）。按照Zhang等人先前发表的方案，使用人H1细胞获得诱导的兴奋性前脑神经元，并进行一些修改（图1C）<sup ...

Watch this Scientific Journal Video about Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions at JoVE.com

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

由于工具和方法不足，神经退行性变中的神经元-神经胶质相互作用尚未得到很好的理解。在这里，我们描述了从人类多能干细胞中获得诱导神经元，少突胶质细胞前体细胞和少突胶质细胞的优化方案，并提供这些方法在理解阿尔茨海默病细胞类型特异性贡献方面的价值示例。

从多能干细胞生成人类神经元和少突胶质细胞，用于模拟神经元-少突胶质细胞相互作用

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

The protocols described here, are expected to facilitate future studies on oligodendroglial defects, in the pathogenesis of neuro degeneration. This is an improved method that can be used to generate functional human oligodendrocyte lineage cells in only three to four weeks, and establish a co-culture system to model the neuron oligodendrocyte connections. The protocol can be used to study not only Alzheimer's disease, but any other disease associated aspect of neuron oligodendrocyte interactions.To begin, plate approximately one million HEK293T cells in each T75 flask to achieve 40%confluency, then transfect them with plasmids expressing tetracycline inducible NGN2 and puromycin resistant tetracycline transactivator gene, along with the three helper plasmids. Use PEI for transfection. Change the media after 16 hours.Harvest released viral particles by collecting culture media every day and replacing it with fresh media for three days. Pool the collected media for purification, then filter the virus through a 0.22 micrometer filter and centrifuge at 49, 000 times G for 90 minutes. After centrifugation, re-suspend the pellet in the appropriate volume of PBS glucose.Use commercially available H1 human ES cells to perform neuron induction. Culture the cells on extracellular matrix solution coated 6-well plates with ESL maintenance medium and incubate the plates at 37 degrees Celsius with 5%carbon dioxide. Two days before induction of differentiation, detach ES cells by adding one milliliter of cell detachment solution and incubating at room temperature for 10 minutes.Transfer the cells to a tube, then wash the well with two milliliters of media and add it to the same tube. Centrifuge the cells at 300 times G for five minutes. Then re-suspend the pellet in media and plate the cells onto matrix coated 6-well plates at the seating density of one times 10 to the fifth cells per well.On the next day, add lentiviruses, expressing NGN2 plus puro. R, and tetracycline transactivator together with polybrene in fresh ES cell maintenance medium. On day zero, add two micrograms per milliliter doxycycline in DMEM/F-12 medium supplemented with N2 and without morphogens.On day one, add puromycin in fresh DMEM/F-12, plus N2 and doxycycline to the final concentration of one microgram per milliliter. Select the transduced cells in puromycin for at least 24 hours. On day two, detach differentiating neurons with cell detachment solution and re-plate them on 24-well plates coated with matrix solution.Maintain them in MBA B27 medium without doxycycline. Culture pure induced neurons on the plates, coated with extracellular matrix-based solutions. To perform neural progenitor cell generation, culture H1 human ES cells and transdifferentiate them into neural progenitor cells, as described in the text manuscript.On day negative one, seed 0.5 to one million cells in ES cell maintenance medium into each well of a 6-well plate coated with growth factor reduced matrix solution. On day zero, treat cells for 24 hours with ES cell maintenance medium supplemented with 2%DMSO. On days one through six, change the full media with warm neural induction medium containing some add inhibitors from a commercial kit.On day seven, passage NPCs using cell detachment solution and plate them at a seating density of one to two times 10 to the fifth cells per well of a 24-well plate. Assay the differentiation efficiency by immunohistochemical staining for absence of pluripotentcy marker and presence of NPC markers. To generate the oligodendrocyte precursor cells, plate the NPCs in warm neural induction medium with some add inhibitors from a commercial kit.On day eight, prepare a solution of 1%DMSO in the OPC differentiation medium, and treat the plated NPCs for 24 hours. On day nine, replace the media with fresh OPC differentiation medium without DMSO, then feed the cells every other day until day 15. If the the cells reach confluence before day 15, passage them to the seating density of one to two times 10 to the fifth cells per well.On day 14, plate OPCs in OPC differentiation medium at a density of one to two times 10 to the fifth cells per well, in a 24-well plate. Test cells for the presence of OPC specific markers by IHC staining or qPCR, and for the absence of NPC markers. On day 15, replace media with OL maturation medium.Change the medium every other day or every day. When cells reach 90%confluence, split them at a one to three ratio for up to two passages or until cell division slows down substantially. If OPCs divide too fast and reach confluencey in less than three days, add Ara-C at a concentration of two to five micromolar for one to three days.On day 14, plate the IOPCs at a density of one times 10 to the fifth cells per well in a 24-well plate in OPC differentiation medium. On the next day, detach the induced human neurons with cell detachment solution. Add neurons onto the cultured OPCs, plating at the seating density of two times 10 to the fifth cells per well.Use co-culture medium containing neurobasal A medium, 2%B27 supplement, and 100 nanograms per milliliter T3 triiodothyronine. Change the medium on the next day and then every other day. If OPCs proliferate too fast and reach confluency in less than three days, add two to five micromolar Ara-C.It is important that the starting human pluripotent stem cells exhibit a high degree of pluripotentcy for the successful generation of induced neurons, or OPCs and oligodendrocytes. Therefore, cells should be stained for specific markers, such as OCT-4 and SOX2, before starting either of the induction protocols. Human H1 cells were used to obtain induced excitatory forebrain neurons.Pure neurons in culture started expressing synapse in one at day 14 to 16. IOPCs were generated in two weeks and mature oligodendrocytes in four to five weeks. DMSO treatment enriched the number of cells in the early G1 phase for better signaling Integration.Specific OPC markers were detected as early as two weeks after plating of ES H1 cells. The IOPC population at two weeks was fairly homogenous, with over 95%of cells positive for 04 staining and a high level of immunoreactivity for other markers. After initiation of oligodendrocyte maturation, specific oligodendrocyte markers were detected starting at day 28.The ideal plating densities for induced neurons, and IOPCs, need to be determined with a series of cell number titrations to achieve proper differentiation. After four weeks, in co-cultures, the IOPCs should be adequately differentiated into oligodendrocytes that are positive for specific markers such as MBP. This co-culture system can robustly boost the number of synapses, indicating that the IOPCs provide a neuronal support through physical contact or release of trophic factors.This described procedure can be further sophisticated by adding different brain cell types. For example, stem cells derived human astrocytes to model novel aspects of neuron glial interactions in health and disease.

Research

JoVE Journal

Neuroscience

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

科研

神经科学

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 ...

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 ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

人类神经的聚集体cGMP的适用展开法干细胞和祖细胞来源从多能干细胞或胎儿脑组织

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

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 ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

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 ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

人类诱导多能干细胞脑微血管内皮细胞的衍生、扩张、冷冻保存和特征化

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

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

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) ...