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。按照制造商的说明使用PEI进行转染。16小时后更换培养基并丢弃。</li>
		<li>通过每天收集培养基来收获释放的病毒颗粒，并用新鲜培养基替换3天。汇集收集的含有病毒颗粒的培养基进行纯化。通过0.22μm过滤器过滤病毒，并以49，000× g 离心90分钟。将沉淀重悬于适当体积的PBS-葡萄糖（~150μL）中。</li>
	</ol></li>
	<li>神经元诱导（~5天） 注意：该诱导方案（图1A;流程图）对于经过验证的多能性的iPS和ES细胞都非常有效（可以通过对表征良好的多能性标志物进行免疫组织化学染色来测定; 图 1B）。<ol><li>在52代时使用市售的H1人ES细胞（参见材料表）。使用ES细胞维持培养基（参见材料表）在包被的6孔板（每6孔板~0.5mg基质溶液;参见材料表）的细胞外基质溶液上培养细胞，并将板在37°C与5%CO2孵育。</li>
		<li>在第-2天，用1mL细胞分离溶液（参见 材料表）分离ES细胞（80%汇合），并在室温下孵育10分钟。将细胞转移到试管中;用 2 mL 培养基清洗孔并在同一管中混合。以300× g 离心5分钟，将沉淀重悬于培养基中，并以每孔1 x 105 个细胞的接种密度将细胞接种到基质包被的6孔板上。</li>
		<li>在第-1天，将表达Ngn2的慢病毒加上PuroR和rtTA与聚溴乙烯（8μg/ ml）一起添加到新鲜ES细胞维持培养基中的ES细胞中（参见 材料表）。病毒的确切数量应由实际滴度或滴定确定。我们通常在 6 孔板中每孔添加 5 μL 每种病毒。</li>
		<li>在第 0 天，在 DMEM-F12 培养基中加入多西环素（2 μg/mL，以激活 Ngn2 表达），加入不含形态原的 N2 补充剂。</li>
		<li>在第 1 天，在 DMEM-F12 加 N2 和多西环素的新鲜培养基中加入嘌呤霉素，至终浓度为 1 μg/mL 培养基。选择嘌呤霉素中的转导细胞至少24小时，如果病毒滴度低，可能需要更高的嘌呤霉素浓度（高达5μg/ mL）和更长的选择期（长达48小时）以充分去除转导不足的细胞。</li>
		<li>在第2天，用细胞分离溶液分离分化神经元（见 材料表），并将它们重新铺在涂有基质溶液的24孔板（80，000-200，000个细胞/孔之间）上（见 材料表），并将它们保存在不含强力霉素的NBA / B27培养基中。播种密度至关重要。</li>
		<li>在这个阶段，分离的神经元可以在专门的商业冷冻培养基中冷冻（见 材料表）并在液氮中储存长达3个月。纯神经元可以铺板，占解冻后典型的~15%-20%细胞死亡，单独培养或与其他脑细胞类型共培养（参见步骤3.2.3.与OPC共培养）。</li>
		<li>按照制造商的说明，在涂有细胞外基质溶液的平板上培养纯iN（参见 材料表）。特征性锥体形态应在第 4 天（和第 6 天; 图 1C）。突触形成最早可在第 14 至 16 天检测到，并在第 24 天通过用标准突触前和突触后标志物进行免疫组织化学染色而突出。（图1D;用突触前标记物突触1和树突标记物Map2标记）。</li>
	</ol></li>
</ol>2. 多能干细胞诱导人少突胶质细胞前体细胞（OPCs）和少突胶质细胞成熟
<ol><li>神经祖细胞（NPC）生成：单层方案（~7天）。流程图见 图2A 。<ol><li>如前所述培养H1人ES细胞（参见步骤1.2.1.），并通过称为双SMADi的既定方法将其转分化为神经祖细胞（NPC），并具有用于多种信号通路的小分子抑制剂。在这里，我们使用广泛接受的商业试剂盒，并遵循制造商提供的单层协议（见 材料表）。</li>
		<li>在第-1天，在涂有生长因子还原基质溶液（参见材料表;每6孔板~0.5mg基质溶液）的6孔板中板0.5–1 x 106个细胞，并用ES细胞维持培养基（参见材料表）。该生长因子还原基质溶液用于涂覆将在以下步骤中使用的所有板。</li>
		<li>在第0天，用补充2%DMSO的ES细胞维持培养基（参见 材料表）处理细胞24小时。</li>
		<li>在第1-6天，用含有商业试剂盒中SMAD抑制剂的温热（37°C）神经诱导培养基更换全培养基（见 材料表）。如果细胞在第7天之前分裂并达到汇合，则传代至0.5-1 x 106的接种密度，如前面的步骤2.1.2中所述。</li>
		<li>在第7天，使用细胞分离溶液（参见 材料表）传代NPC，并以1-2×105个细胞 /孔的接种密度在24孔板中接种。</li>
		<li>通过免疫组织化学 （IHC） 染色测定缺乏多能性标志物（例如 OCT4）和 NPC 标志物（例如 PAX6、Nestin 和 Sox1）的分化效率。</li>
		<li>在此阶段，分离的NPC可以在专门的商业NPC冷冻介质中冷冻（见 材料表）并在液氮中储存长达3个月。在一次冻融后，NPC仍然保留多能性，以产生具有可靠协议的神经元，星形胶质细胞和OPC。</li>
	</ol></li>
	<li>少突胶质细胞前体细胞（OPC）生成（~7天）。流程图请参见 图 2A 。<ol><li>在第7天，使用细胞分离溶液（参见 材料表）传代NPC，并以每孔1-2×10 5个细胞的 接种密度将它们接种在24孔板中，在温暖（37°C）的神经诱导培养基中加上来自商业试剂盒的SMAD抑制剂（参见 材料表）。</li>
		<li>在第8天，在OPC分化培养基中制备1%DMSO的溶液，并将铺板的NPC处理24小时。OPC 分化培养基由：DMEM/F12 培养基、1% N2 补充剂、1% B27 添加剂、20 ng/mL 的 bFGF、1 μM 的 SAG、10 ng/mL 的 PDGF-AA 组成（参见 材料表）。</li>
		<li>在第9天，用不含DMSO的新鲜OPC分化培养基替换培养基。每隔一天喂一次细胞，直到第15天。如果细胞在第15天之前达到汇合，则按照步骤2.2.1中所述，将它们传代至每孔1-2×105 个细胞的接种密度。</li>
		<li>在第 14 天，将 OPC 以 1-2 x 105 个细胞 /孔的密度在 OPC 分化培养基中接种 24 孔板。</li>
		<li>在此阶段（第 15 天），通过 IHC 染色或 qPCR 测试细胞是否存在 OPC 特异性标志物（例如，O4、Olig1/2、CSPG4/Ng2、NKX2.2、PDGFRa; 图2B）以及缺乏鼻咽癌标志物（Pax6或Nestin; 图 2D）。我们通常在第15天检测到超过95%的细胞中的O4免疫反应性。与阿尔茨海默病特别相关的是， APP （淀粉样蛋白前体蛋白）、 BACE1 （加工蛋白酶β-秒化酶1）和肽淀粉样蛋白-β（Aβ）的表达在OPCs中含量丰富（图2F）。</li>
	</ol></li>
	<li>少突胶质细胞 （OL） 成熟（~7-20 天）<ol><li>在第15天，用OL成熟培养基替换培养基：神经基础-A培养基，2%B27补充剂，1μM cAMP，200ng / mL T3三碘甲状腺原氨酸和1μM的氯马斯汀（参见 材料表）。如有必要，每隔一天或每天更换培养基。</li>
		<li>当细胞达到90%汇合时，以1：3的比例分裂至最多2代或直到细胞分裂显着减慢。如果OPCs分裂太快并在不到3天的时间内达到汇合，则以2-5μM的浓度添加Ara-C（参见 材料表）1-3天。活性增殖表明成熟效率降低。</li>
		<li>通过评估OL标志物的表达来检查少突胶质细胞成熟的效率，例如CLDN11，PLP1，通过qPCR，IHC染色或免疫印迹的MBP。高度复杂结构的特征形态（图2C）和OL标志物的表达（图2E）应在第28天之前轻松检测到。</li>
	</ol></li>
</ol>3. 人诱导神经元 （iN） 和少突胶质细胞前体细胞 （iOPC） 的共培养
<ol><li>iOPC电镀（~3天）<ol><li>在第14天以1 x 105个细胞 的密度在OPC分化培养基中的24孔板（如上文步骤2.2.4所述）中接种iOPC。</li>
	</ol></li>
	<li>iN-iOPC共培养建立<ol><li>在第15天，在嘌呤霉素选择后第2天的步骤中分离诱导的人神经元（如步骤1.2.6中所述）与细胞分离溶液（参见 材料表）。</li>
		<li>将神经元添加到培养的OPC上，以每孔2 x 105 个细胞的接种密度在具有生长OPC的24孔板中接种（从步骤3.1.1开始）。使用含有神经基础-A 培养基、2% B27 补充剂和 100 ng/mL T3 三碘甲状腺原氨酸的共培养基。第二天更换培养基，之后每隔一天更换一次。如果OPCs增殖太快并在不到3天的时间内达到汇合，则以2-5μM的浓度添加Ara-C。7天后神经元共培养生长的iN和iOPC的代表性图像如图 3A所示。</li>
		<li>使用上述步骤1.2.7中制备的冷冻神经元与OPC共培养。以每孔 3 x 105 个细胞的更高密度冻融神经元。</li>
		<li>在共培养的第14-16天之后，可以通过突触前和突触后标记物的IHC染色来观察iN中的突触形成，到第21天，突触点应该丰富（图3C），并且可以可靠地记录神经元活动。</li>
		<li>从第 21 天开始，测试细胞的 OL 特异性标志物（例如，MBP 和 PLP1）。到第28天，我们通常观察到iOL过程对iN轴突的鞘现象，通过IHC染色标记特定标志物（图3B;iN轴突的神经丝NF和iOPC过程的MBP）。</li>
	</ol></li>
</ol>

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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. (149), (2019).</li><li>Douvaras, P., Fossati, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+isolation+of+oligodendrocyte+progenitor+cells+from+human+pluripotent+stem+cells.">Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells.</a> Nature Protocols. 10 (8), 1143-1154 (2015).</li><li>Mei, F., 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=Micropillar+arrays+as+a+high-throughput+screening+platform+for+therapeutics+in+multiple+sclerosis.">Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis.</a> Nature Medicine. 20 (8), 954-960 (2014).</li><li>Madhavan, 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=Induction+of+myelinating+oligodendrocytes+in+human+cortical+spheroids.">Induction of myelinating oligodendrocytes in human cortical spheroids.</a> Nature Methods. 15 (9), 700-706 (2018).</li><li>Zhang, Y., 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=Purification+and+characterization+of+progenitor+and+mature+human+astrocytes+reveals+transcriptional+and+functional+differences+with+mouse.">Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse.</a> Neuron. 89 (1), 37-53 (2016).</li><li>Grubman, 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=A+single-cell+atlas+of+entorhinal+cortex+from+individuals+with+Alzheimer's+disease+reveals+cell-type-specific+gene+expression+regulation.">A single-cell atlas of entorhinal cortex from individuals with Alzheimer's disease reveals cell-type-specific gene expression regulation.</a> Nature Neuroscience. 22 (12), 2087-2097 (2019).</li><li>Goldman, S. A., Kuypers, 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=How+to+make+an+oligodendrocyte.">How to make an oligodendrocyte.</a> Development. 142 (23), 3983-3995 (2015).</li><li>Behrendt, G., 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=Dynamic+changes+in+myelin+aberrations+and+oligodendrocyte+generation+in+chronic+amyloidosis+in+mice+and+men.">Dynamic changes in myelin aberrations and oligodendrocyte generation in chronic amyloidosis in mice and men.</a> Glia. 61 (2), 273-286 (2013).</li><li>Patzke, 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=Neuromodulator+signaling+bidirectionally+controls+vesicle+numbers+in+human+synapses.">Neuromodulator signaling bidirectionally controls vesicle numbers in human synapses.</a> Cell. 179 (2), 498-513 (2019).</li><li>Piao, 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=Human+embryonic+stem+cell-derived+oligodendrocyte+progenitors+remyelinate+the+brain+and+rescue+behavioral+deficits+following+radiation.">Human embryonic stem cell-derived oligodendrocyte progenitors remyelinate the brain and rescue behavioral deficits following radiation.</a> Cell Stem Cell. 16 (2), 198-210 (2015).</li><li>Keirstead, H. 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+embryonic+stem+cell-derived+oligodendrocyte+progenitor+cell+transplants+remyelinate+and+restore+locomotion+after+spinal+cord+injury.">Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury.</a> Journal of Neuroscience. 25 (19), 4694-4705 (2005).</li><li>Kim, D. 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=Rapid+generation+of+OPC-like+cells+from+human+pluripotent+stem+cells+for+treating+spinal+cord+injury.">Rapid generation of OPC-like cells from human pluripotent stem cells for treating spinal cord injury.</a> Experimental &amp; Molecular Medicine. 49 (7), 361 (2017).</li></ol>

作者没有什么可透露的。

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 ...

generation-human-neurons-oligodendrocytes-from-pluripotent-stem-cells

Research

JoVE Journal

Neuroscience

4.5K 次观看.  Brown University. 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。

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

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

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

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.

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

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.

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 ...

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

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

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

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.

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

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 accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

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

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.

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

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

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

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.

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

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) ...

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

用于线粒体疾病建模的人脑类器官的生成

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...