Name: efficient neural differentiation using single-cell culture of human embryonic stem cells
Uploaded: 2020-01-18T13:00:00
Description: Watch this Scientific Journal Video about Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells at JoVE.com

我们感谢卡尔·博尔特纳博士（NIEHS）协助FACS分析。这项研究得到了国家环境卫生科学研究所、国家卫生研究院、Z01-ES-101585至AMJ的校内研究计划的支持。

1. 制备符合hESC资格的地下室膜基质涂层板
<ol><li>在4°C下缓慢解冻hESC认证的基底膜基质（见材料表）溶液至少2~3小时或过夜，以避免形成凝胶。</li>
	<li>要制备基底膜基质涂层板，请将冷 DMEM/F12 中的基质稀释至 2% 的最终浓度。混合好，用1 mL的稀释基质溶液涂覆6孔板。</li>
	<li>在室温 （RT） 下孵育基质涂层板至少 3 小时或 4°C 过夜。 注：在去除基质溶液和使用板之前，具有基底?...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+xeno-free+culture+of+human+embryonic+stem+cells.">Toward xeno-free culture of human embryonic stem cells.</a> The International Journal of Biochemistry &amp; Cell Biology. 38 (7), 1063-1075 (2006).</li><li>Hoffman, L. M., Carpenter, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+culture+of+human+embryonic+stem+cells.">Characterization and culture of human embryonic stem cells.</a> Nature Biotechnology. 23 (6), 699-708 (2005).</li><li>Yan, 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=Efficient+and+rapid+derivation+of+primitive+neural+stem+cells+and+generation+of+brain+subtype+neurons+from+human+pluripotent+stem+cells.">Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells.</a> Stem Cells Translational Medicine. 2 (11), 862-870 (2013).</li><li>Goncalves, J. T., Schafer, S. T., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Neurogenesis+in+the+Hippocampus:+From+Stem+Cells+to+Behavior.">Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior.</a> Cell. 167 (4), 897-914 (2016).</li><li>Watanabe, 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=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nature Biotechnology. 25 (6), 681-686 (2007).</li><li>International Stem Cell Initiative. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+embryonic+stem+cell+lines+by+the+International+Stem+Cell+Initiative.">Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.</a> Nature Biotechnology. 25 (7), 803-816 (2007).</li><li>Hartung, O., Huo, H., Daley, G. Q., Schlaeger, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clump+passaging+and+expansion+of+human+embryonic+and+induced+pluripotent+stem+cells+on+mouse+embryonic+fibroblast+feeder+cells.">Clump passaging and expansion of human embryonic and induced pluripotent stem cells on mouse embryonic fibroblast feeder cells.</a> Current Protocols in Stem Cell Biology. 14 (1), 10 (2010).</li><li>Chen, K. 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=Non-colony+type+monolayer+culture+of+human+embryonic+stem+cells.">Non-colony type monolayer culture of human embryonic stem cells.</a> Stem Cell Research. 9 (3), 237-248 (2012).</li><li>Chen, G., Hou, Z., Gulbranson, D. R., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin-myosin+contractility+is+responsible+for+the+reduced+viability+of+dissociated+human+embryonic+stem+cells.">Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.</a> Cell Stem Cell. 7 (2), 240-248 (2010).</li><li>Li, X., Krawetz, R., Liu, S., Meng, G., Rancourt, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK+inhibitor+improves+survival+of+cryopreserved+serum/feeder-free+single+human+embryonic+stem+cells.">ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells.</a> Human Reproduction. 24 (3), 580-589 (2009).</li><li>Ohgushi, 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=Molecular+pathway+and+cell+state+responsible+for+dissociation-induced+apoptosis+in+human+pluripotent+stem+cells.">Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells.</a> Cell Stem Cell. 7 (2), 225-239 (2010).</li><li>Pakzad, 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=Presence+of+a+ROCK+inhibitor+in+extracellular+matrix+supports+more+undifferentiated+growth+of+feeder-free+human+embryonic+and+induced+pluripotent+stem+cells+upon+passaging.">Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging.</a> Stem Cell Reviews and Reports. 6 (1), 96-107 (2010).</li><li>Chambers, S. 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=Highly+efficient+neural+conversion+of+human+ES+and+iPS+cells+by+dual+inhibition+of+SMAD+signaling.">Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.</a> Nature Biotechnology. 27 (3), 275-280 (2009).</li><li>Jeon, 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=GLIS3+Transcriptionally+Activates+WNT+Genes+to+Promote+Differentiation+of+Human+Embryonic+Stem+Cells+into+Posterior+Neural+Progenitors.">GLIS3 Transcriptionally Activates WNT Genes to Promote Differentiation of Human Embryonic Stem Cells into Posterior Neural Progenitors.</a> Stem Cells. 37 (2), 202-215 (2019).</li><li>Emre, N., 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=The+ROCK+inhibitor+Y-27632+improves+recovery+of+human+embryonic+stem+cells+after+fluorescence-activated+cell+sorting+with+multiple+cell+surface+markers.">The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers.</a> PLoS ONE. 5 (8), e12148 (2010).</li><li>Hanna, 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+cells+with+biological+and+epigenetic+characteristics+similar+to+those+of+mouse+ESCs.">Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (20), 9222-9227 (2010).</li><li>Saha, 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=Surface-engineered+substrates+for+improved+human+pluripotent+stem+cell+culture+under+fully+defined+conditions.">Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (46), 18714-18719 (2011).</li><li>Tsutsui, H., 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=An+optimized+small+molecule+inhibitor+cocktail+supports+long-term+maintenance+of+human+embryonic+stem+cells.">An optimized small molecule inhibitor cocktail supports long-term maintenance of human embryonic stem cells.</a> Nature Communications. 2, 167 (2011).</li><li>Xu, 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=Revealing+a+core+signaling+regulatory+mechanism+for+pluripotent+stem+cell+survival+and+self-renewal+by+small+molecules.">Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (18), 8129-8134 (2010).</li><li>Ungrin, M. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+protocol+that+induces+human+embryonic+stem+cells+to+differentiate+into+neural+cells+in+vitro.">An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro.</a> Cell Biology International. 32 (1), 80-85 (2008).</li><li>Smith, J. R., 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+Activin/Nodal+signaling+promotes+specification+of+human+embryonic+stem+cells+into+neuroectoderm.">Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm.</a> Developmental Biology. 313 (1), 107-117 (2008).</li></ol>

将hESCs分化成各种谱系和产生足够数量的分化细胞的可扩展和有效方法是药物筛选和干细胞治疗的重要标准。已经发表了各种单细胞传递方法，其中细胞在ROCK抑制剂或其他小分子的存在下培养，以提高存活率，但这些培养方法的最终产物是菌群型hESCs17，18，19，20，21。单细?...

人类胚胎干细胞（hESCs）有可能分化成三种主要生殖层，然后分化成各种多能的祖细胞系。这些血统随后引起人体的所有细胞类型。体外hESC培养系统已经改变了研究人类胚胎发育的能力，并已成为获得关于如何在生物和分子水平上对这些过程进行调控的新见解的宝贵工具。同样，从人类患者中分离出来的诱导多能干细胞（iPSCs）的研究提供了对各种疾病的新见解。此外，从hESCs衍生的祖细胞和分化细胞可用于干细胞治疗和药物筛选1、2、3、4的研究。
hESCs 可以诱导分化成神经祖细胞 （NPC），这是具有广泛自我更新能力的多电位细胞。随后，这些细胞可以分化成神经元、星形细胞和寡核苷酸细胞5，6。NPC还为神经发育生物学和各种神经系统疾病的体外研究提供了一个细胞系统。然而，目前涉及hESC及其分化为NPC的菌群培养方法效率不高，往往涉及共栽以及胚胎体（EB）和玫瑰花形成5、7、8、9。这些协议表现出较低的存活率和自发分化，并且更耗时。
这里介绍的是一个改进和强大的培养系统，易于扩展，并使用高密度单细胞类型培养hESCs10。在hESC10、11、12、13、14的单细胞类型培养中，加入Roh-激酶（ROCK）抑制剂有助于显著提高生存效率。在此文化系统中，hESC 可以轻松维护和扩展。此外，该议定书还提出了一种从hESCs的单细胞类型培养物生成NPC的有效方法，它允许产生高度纯的NPC。 用ALK抑制剂抑制BMP/TGF+/activin信号通路，有效地诱导单细胞型hESC分化为NPC15、16，然后可以诱导分化成功能神经谱系，如多巴胺能神经元和星形。
总之，使用hESCs的单细胞类型培养协议为研究这些细胞分化为包括NPC在内的各种谱系提供了一个有吸引力的模型。该协议易于扩展，因此适合生成细胞，用于涉及再生治疗和药物筛选的研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm m-dishes</td>
			<td>ibidi</td>
			<td>81156</td>
			<td>Cell culture dish</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104-500</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&#38;D system</td>
			<td>338-AC-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement (-Vit A)</td>
			<td>Thermo Fisher</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Applied Biological Materials</td>
			<td>Z100065</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Peprotech</td>
			<td>100-18C</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>DAMON/ICE</td>
			<td>428-6759</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher</td>
			<td>4110</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning hESC-qulified Matrix (Magrigel)</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Basement membrane matrix (used for most of the protocol here)</td>
		</tr>
		<tr>
			<td>Cryostor CS 10</td>
			<td>Stemcell Technologies</td>
			<td>7930</td>
			<td>Cell freezing solution</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Stemcell Technologies</td>
			<td>7923</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Tocris</td>
			<td>3093</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>AF-100-16A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>SH3007003HI</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF8</td>
			<td>Applied Biological Materials</td>
			<td>Z101705</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Applied Biological Materials</td>
			<td>Z101057</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex matrix</td>
			<td>Thermo Fisher</td>
			<td>A1569601</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Thermo Fisher</td>
			<td>35050061</td>
			<td>Glutamine supplement, 100X</td>
		</tr>
		<tr>
			<td>H9 (WA09) human embryonic stem cell line</td>
			<td>WiCell</td>
			<td>WA09</td>
			<td></td>
		</tr>
		<tr>
			<td>Heregulin b-1</td>
			<td>Peprotech</td>
			<td>100-3</td>
			<td></td>
		</tr>
		<tr>
			<td>IGF</td>
			<td>Peprotech</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Thermo Fisher</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Thermo Fisher</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>Stemcell Technologies</td>
			<td>85850</td>
			<td>hESC culture medium</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermo Fisher</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Thermo Fisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Thermo Fisher</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>Sigma Aldrich</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr>
			<td>SHH</td>
			<td>Applied Biological Materials</td>
			<td>Z200617</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemdiff Neural Progenitor medium</td>
			<td>Stemcell Technologies</td>
			<td>5833</td>
			<td>NPC expansion medium</td>
		</tr>
	</tbody>
</table>

efficient neural differentiation using single-cell culture of human embryonic stem cells

人类胚胎干细胞（hESCs）的体外分化改变了研究人体在生物学和分子水平上发育的能力，并为再生应用提供了用于再生的细胞。hESC 培养的标准方法使用菌落类型培养来保持未分化的 hESC 和胚胎体 （EB） 和玫瑰形形成，以分化成不同的胚层，效率低且耗时。此处介绍的是使用 hESC 而不是殖民地类型的区域性的单细胞培养方法。此方法允许维护未分化 hESC 的特征，包括与菌群类型 hESC 相媲美的 hESC 标记的表达。此外，该协议还提出了一种从单细胞型hESC生成神经祖细胞（NPC）的有效方法，在1周内产生NPC。这些细胞高度表达几个NPC标记基因，并可以分化成各种神经细胞类型，包括多巴胺能神经元和星形细胞。这种用于hESC的单细胞培养系统将有助于研究这些过程的分子机制、某些疾病的研究以及药物发现屏幕。

这里介绍的是一个改进的协议，用于维护和扩展hESC的单细胞类型培养及其有效分化为神经祖细胞，后者随后分化成各种下游神经谱系，包括多巴胺能神经元和星形细胞。
代表性相位对比图像显示细胞形态在适应单细胞类型培养过程中不同步骤的细胞形态（图1A）。通过单细胞培养条件，发现适应的hESC能够维持在高密度，然后在达到汇合时?...

Watch this Scientific Journal Video about Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells at JoVE.com

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

这里介绍的是人类胚胎干细胞单细胞培养及其随后分化为神经祖细胞的一个方案。该协议简单、可靠、可扩展，适用于药物筛选和再生医学应用。

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

人类胚胎干细胞可以诱导分化成神经祖细胞，然后分化成神经元星细胞和寡头细胞。然而，人类胚胎干细胞的类型培养方法及其分化成神经祖细胞是非常低效和开放的体内共培养系统在这里，我们提出了一个改进和强大的培养系统，很容易扩展使用高密度单细胞类型培养与人类胚胎干细胞。人类胚胎干细胞的单细胞类型培养提供了一个快速高效的系统，用于研究沿各种和不同分化的血统（包括神经祖细胞及其随后分化为其他神经系）调节多步分化的分子机制。首先准备人类胚胎干细胞合格的地下室膜基质涂层板。在四摄氏度下慢慢解冻基底膜基质溶液至少两到三个小时或过夜。解冻后，将冷DMEM/F-12中的基质稀释至2%混合，用一毫升稀释溶液覆盖六井板的每个井。在室温下孵育涂层板至少三小时或在四摄氏度下过夜。通过生长在地下室膜基质上的H9型人类胚胎干细胞的分离培养物，从孔中吸出培养基，用一毫升DMBS洗涤一次。在每个井中加入一毫升 Dispase 溶液，并在 37 摄氏度下孵育板 20 分钟。取出 Dispase，用两毫升 DMEM/F-12 轻轻清洗细胞一次。洗涤后，取出介质，向每井添加两毫升 DMEM/F-12。通过上下移液轻轻分离菌落，将其转移到15毫升的管子中。将管子以370倍g离心两分钟。吸气介质。然后加入两毫升细胞分离溶液，在37摄氏度下孵育10分钟，将细胞颗粒分离成单细胞。重复离心并拆下分离溶液。通过上下轻轻移液，在mTeSR-1人类ESC介质中重新发送细胞。为了将群体型人类胚胎干细胞适应单个细胞型培养，将大约150万至200万个细胞板入地下室膜基质涂层板的每个孔中，放入含有10微摩尔 ROCK抑制剂的两毫升mTeSR-1中。24 小时后，用新鲜的 mTeSR-1 更换介质，不带 ROCK 抑制剂。允许细胞作为单个细胞类型生长三天，每天改变介质。在第四天，分离分离溶液中的细胞，并重新镀上它们，如前所述。根据手稿指示重新沉淀和孵育细胞后，将小胚胎体转移到15毫升管中，让它们沉淀到底部，然后用移液器轻轻取出介质。将它们转移到EB介质中，并允许它们在低附着菜中扩展七天。为了诱导神经祖细胞或NPC分化，用一毫升分离溶液分离单细胞型人类胚胎干细胞，并在37摄氏度下孵育10分钟。以370倍g将细胞离心两分钟。拆下分离溶液上一样。然后在DMEM/F-12中重新暂停细胞。在两毫升mTeSR-1和10微摩尔 ROCK抑制剂的两毫升中，将基底膜基质上的细胞以每孔2倍10至第五孔的密度涂覆六孔板。24小时后，用神经诱导介质代替培养基，辅以1微摩尔多索莫非辛和5微摩尔SB431542。在神经感应的头四天中，每隔一天更换一次介质，然后每天更换一次，直到第七天达到汇合。经过七天的神经感应后，通过加入一毫升分离溶液，在37摄氏度下孵育10分钟，使细胞分离。将细胞以 370 倍 g 离心，并取出分离溶液上一液。加入一毫升的NPC膨胀介质，轻轻移液细胞上下重新暂停。然后板1倍10至第五细胞每孔在两毫升的NPC膨胀介质在地下室膜基质上涂上六孔板。当培养达到90%的汇合时，必要时通过细胞，在前三到四个通道中加入10微摩尔 ROCK抑制剂。在扩展期间每隔一天更换一次介质。要准备 PLO 层压涂层板，将 PLO 库存溶液稀释到 PBS 或水中，最终浓度为每毫升 10 微克，混合好，然后用溶液的六孔板在每孔中涂上一毫升溶液。在37摄氏度下孵育板至少两小时，或在4摄氏度下过夜。接下来，用 PBS 清洗每一个井，并在涂覆每个井后立即按照手稿说明准备一毫升层压溶液。将盘子保持在四摄氏度，长达一周。为了将NPC分化成多巴胺神经元，在24小时后以大约50%的密度将PLLA层涂层的菜中的细胞板入NPC膨胀介质中，将介质更改为DA1，并在此后每隔一天更换一次。要通过培养物，当它们达到汇合时，用一毫升分离溶液分离细胞，并在37摄氏度下孵育10分钟。然后以 370 倍 g 离心，取出分离溶液上一样。将细胞在一毫升的DA1介质中，将10倍的板在PLP层压涂层的六孔板上的两毫升介质中，将第五细胞重新暂停。为了将NPC区分为星细胞，在24小时后将介质以50%的密度将细胞板板到PLC层压板和NPC膨胀介质，然后通过前面描述的细胞。当群体型人类胚胎干细胞适应单细胞型培养时，发现细胞能够保持高密度，然后在对汇接时容易有效地亚培养。这些细胞保留了人类胚胎干细胞群的典型细胞周期特征，如短G1阶段和高比例细胞在S阶段。定量PCR分析还证实，它们表示的ESC标记水平与群体型细胞相当。此外，研究表明，单细胞人类胚胎干细胞能够形成胚胎体，包含来自所有三个胚胎层的细胞：内皮、中皮和外皮。结果表明，单细胞型人类胚胎干细胞有效分化成NPC，这表现为典型的人类胚胎干细胞形态的丧失和NPC形态的出现。差异得到签名NPC标记的增强表达的支持，并得到免疫污染和FACS分析的证实。同样的分析还显示，超过90%的细胞对SOX1、PAX6和NCAM蛋白的染色呈阳性。此外，衍生的NPC能够分化成多巴胺神经元和星形细胞，如特征形态的出现和血统特异性标记的表达所示。在尝试这种程序时，必须高密度地对人类胚胎干细胞进行板状，以保持健康、无差别的人类胚胎干细胞培养。该协议为简单、可靠和可扩展的祖细胞和分化细胞的生产提供了一个平台，这些细胞将可扩展用于基础研究、药物筛选和神经退行性疾病的应用。

Research

JoVE Journal

Developmental Biology

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

科研

发育生物学

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...