作者想感谢Y.笹井，慷慨提供FOXG1抗体。这项工作是由康涅狄格州干细胞研究资助（08-SCB-UCHC- 022和11-SCB24）和痉挛性截瘫基金会。

1。代的人类多能干细胞聚集体（D1-D4） <ol><li>人类多能干细胞对小鼠胚胎成纤维细胞（MEF）的送料器，在胚胎干细胞的存在下与碱性成纤维细胞生长因子（bFGF，4纳克/毫升）的培养基中培养。经过5-7天的培养时，菌落大，但仍然未分化的，他们是为下一步做好准备。 </li><li>应先来制备的酶溶液。在一个50毫升的试管中，溶解在1毫克/毫升的浓度成DMEM/F12培养基分散酶（或胶原酶）。由于这些解决方案，混合最好的，受热后，将管中介质和酶，37℃水浴10-15分钟，然后它使用Steriflip过滤器进行消毒。 </li><li>吸介质的细胞，将其冲洗DMEM/F12，并吸取这一关。 3-5分钟为胚胎干细胞（iPS细胞）最多10分钟，加入1毫升分离酶在孵化器中的各孔和地点。的细胞在显微镜下看;边缘微微蜷缩/看起来有点暗，细胞，为下一步做好准备。最好是，检查细胞后3分钟，如有必要，将它们放回。当首次使用此协议，它是最好的开始与在同一时间只有一个板的细胞，因为它重要的是尽可能快地从分离酶以除去细胞。 </li><li>吸液从细胞中分离酶。轻轻地（在这个阶段的细胞是非常脆弱的，并会在板相对容易）加入1.5毫升的DMEM/F12培养基，每孔以冲洗掉分离酶。吸液DMEM/F12和1.5毫升的人类胚胎干细胞培养基添加到各孔中。 </li><li>要分离，打破了细胞，将10毫升的玻璃吸管的尖端对右下角的良好（触摸细胞）和向上移液管和上下（垂直） - 向上的行程应该是在接近该法律程序的向下行程。一旦达到的井，另一侧，重复在水平可怕ction。 </li><li>一旦所有的细胞是在悬浮液中，将它们放置在15ml试管中，并在200×g离心2分钟离心。应该有一个细胞在试管底部沉淀。吸液介质从上面的粒料。 </li><li>应当被用来保持中的聚集体的细胞培养烧瓶中的大小取决于原始细胞的数量。 6口井（板）的胚胎干细胞，组织培养处理的T75烧瓶，用50毫升的人类胚胎干细胞培养基中应使用。从步骤1.6将细胞转移到烧瓶中并孵育在37℃下</li><li>为了摆脱的细胞碎片，细胞培养基和烧瓶中约12小时后，应更换。要做到这一点，将所有的介质/细胞到一个50毫升的试管中，向下旋转的细胞（200×g离心2分钟），并吸移的旧培养基。接下来，加50毫升的新鲜胚胎干细胞培养基中的细胞，并放置到一个新的T75烧瓶。跟随此相同的方法（直到第5天），然后，这些细胞可以每天供给。 </li></ol><ol><li>细胞/培养基转移到一个50毫升的试管中，离心细胞，以200×g的2分钟。接着，抽吸除去上清液，将细胞转移到一个新的（非组织培养处理）的T75烧瓶中的神经诱导培养基（NIM）用50ml。 </li><li>一旦细胞在NIM，有一半的介质应更换每一天。应该变得足够大，以在那里他们将下沉到自己的50毫升锥形管的底部，几分钟后（没有离心）的聚集体。经过2-3天NIM（D7-D8总），细胞板的准备。层粘连蛋白或牛胎儿血清（FBS），可以被用来帮助细胞附着到板。 </li><li>组织培养处理的数量取决于所需要的6孔板中的细胞密度为每个聚合应该是能够生长在平板上的多个天，而不会接触任何其他。平均来说，从一个细胞T75烧瓶，将产生约4-6 6孔板价值的细胞。 </li><li><ol class="ualpha"><li>如果使用的层粘连蛋白，它在NIM稀释至10-20μg/ ml到涂覆的6孔板中（〜0.5毫升/孔）。涂布后的2-3小时，板到6孔板中（〜20-30的总量每孔）的细胞聚集。后聚集附加，添加1.5毫升的NIM。 </li><li>如果使用FBS，制备90％的NIM和10％FBS（1.5毫升/孔）的混合物。的细胞，每孔加入1.5毫升中，轻轻摇动板来回传播的聚集，使细胞附着在孵化器的O / N。第二天早上一定要改变介质（2毫升/ NIM）删除FBS。 </li></ol></li><li>经过一两天的电镀，每个聚集会蔓延到形成单分子膜。中心在一两天内，这些细胞形态出现类似的柱状细胞（拉长）。在此期间，改变NIM每一天。有些PSC线（尤其是LY IPSC），可能会形成柱状细胞的效率较低。如果发生这种情况，除了在第2阶段的BMP和TGF的抑制剂（Dorsomorphin 29，2μMSB431542 19），的能有所帮助。 </li><li>大约14-17天，柱状细胞会出现更加紧凑。有时，这些细胞形成，可见肠腔内的隆起或环。除了 ​​柱状形态，在此阶段的细胞表达神经上皮的标记物，如PAX6和SOX1 12,26。 </li><li>的隔离就可以开始之前，必须先检查，以确定是否有任何非神经上皮细胞的细胞。可以利用的客观指标，以指示非神经上皮菌落。然后可以消除刮了一套吸头。 </li></ol> 3。在前脑祖细胞的生成（D17-D24） <ol><li>菌落的中心包含神经上皮细胞，和应分离的集落的其余部分的。这是可以做到使用微管和过滤，无菌1毫升移液枪枪头。当少量的压力被施加到的菌落的中央部，它通常很容易地升空。要小心不要让外部分的殖民地脱离。如果中心固执，不会脱落在他们自己的，他们可以被删除，他们在无菌罩在显微镜下用针切除。转移到一个15毫升的试管的菌落的中心部，和自旋在200×g离心2分钟。 </li><li>介质应该吸气的细胞。两个板价值的细胞可以被转移到一个非组织培养处理的T25烧瓶中含有10毫升的NIM与B27的添加（不包括维生素A，1:50）。使用5-10毫升培养基，每一个板的细胞。 </li><li>当细胞被查看过翌日，他们应该有一个球体状外观。这些细胞转移到一个新的烧瓶中（在存在的NIM和B27，1:50）是必要的，因为它往往是周边小区忙里偷闲，附着在烧瓶的底部。 </li><li>然后悬浮培养的神经球一个星期用NIM补充B27（1:50）。为了促进细胞的耐力和增殖，环AMP（cAMP，1μM）以及胰岛素生长因子（IGF-1，10毫微克/毫升），可以添加到培养基中悬浮培养过程中。此时，神经球包含在前脑祖细胞（FOXG1 +）和准备终末分化的盖玻片的培养皿上。 </li></ol> 4。进一步分化为大脑（D25 +） <ol><li><ol class="ualpha"><li>准备的poly-ornithine/laminin涂布盖玻片（24孔板），电镀细胞。盖玻片首先清除，然后，涂覆有伎俩-鸟氨酸（0.1毫克/毫升，37℃下，O / N，并储存在冷冻机）。 聚鸟氨酸涂层： <ol><li>消毒盖玻片：在将盖玻片烧杯中含有硝酸，并轻轻摇动的通风柜中一小时。下运行至少15分钟，去离子水，倒出来的硝酸，用去离子水冲洗几次，并留下盖玻片。放置在95％乙醇和盖玻片要么振摇30分钟（立即如果使用），或存储在乙醇中，在RT。 </li><li>在无菌罩中，倒入含有灭菌盖玻片的6孔​​板中的盖的50ml管中的内容。用灭菌镊子，拿起一个盖玻片的时间，并设置在一个单一的24孔板直立。如此重复，各孔有盖玻片。 </li><li>他们完全干燥后，请点选板，直到的盖玻片已经落空，每孔。下一步加入100μl聚鸟氨酸（0.1毫克/毫升的无菌蒸馏水2 O），每个盖玻片和孵化板O / N，37°C。 </li><li>第二天，取出板从培养箱中，并让他们冷却至室温。吸液聚-鸟氨酸每个盖玻片（从边缘吨他盖玻片）确保在这个过程中不要触摸或划伤盖玻片。允许盖玻片洗涤前干燥约30分钟。 </li><li>加入1毫升无菌蒸馏水2 O，每孔，让我们坐了10分钟，从每口井的水吸出。重复清洗步骤两次。后板完全干燥，（休假覆盖在引擎盖）将盖子上，盖上铝箔，并储存在-20°C </li></ol></li><li><ol><li>与层粘连蛋白涂覆盖玻片，神经分化培养基和层粘连蛋白（终浓度为20微克/毫升）的混合物中加入50μl的每个聚-鸟氨酸处理盖玻片小心只有触摸盖玻片本身（的液体和不漏关闭）。在37°C孵育1-2小时，然后准备附件的细胞。 </li></ol></li></ol></li><li>收集和神经球在200 XG离心2分钟在15毫升的试管。吸液上清液。 </li> &lt;李&gt; <ol class="ualpha"><li>冲洗DMEM-F12细胞的细胞再次离心。如果神经球太大正确附加，它们可以是在此步骤中与Accutase分离。 </li><li>如果使用Accutase：洗涤后的细胞用DMEM-F12中，神经球解离的通过添加Accutase（〜2毫升）和3分钟，在37℃下孵育细胞的小簇。接下来，添加相同量的胰蛋白酶抑制剂（〜2毫升）和离心处理，以使细胞在200×g离心3分钟。然后，将细胞重新悬浮在NDM破碎使用P200的吸移管。小群，然后镀上预包被的盖玻片。 </li></ol></li><li>吸干关闭大多数的培养基中，并将其放置到6厘米的陪替氏培养皿上的细胞在介质上，以便选择它们变得更容易几滴的。新增约4-5神经球到每个预包被的盖玻片。有没有必要第一吸入关闭的层粘连蛋白。 </li><li>让我们的神经球贴壁至少2-4小时。一旦他们有了一个ttached，更介质（0.5毫升/盖玻片）应予以补充。它应该包括有B27（1:50）的神经分化培养基中，cAMP（1μM），和神经营养因子（BDNF，GDNF，和胰岛素样生长因子，所有在10纳克/毫升）。此时介质的一半可以隔日更换，这些细胞可以维持数月。 </li></ol>

<ol>
	<li>Takahashi, K., Tanabe, 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Thomson, J. A., Itskovitz-Eldor, 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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Yu, J., Vodyanik, M. 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=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Jessell, 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=Neuronal+specification+in+the+spinal+cord:+inductive+signals+and+transcriptional+codes.">Neuronal specification in the spinal cord: inductive signals and transcriptional codes.</a> Nat. Rev. Genet. 1, 20-29 (1038).</li><li>Wilson, S. I., Edlund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+induction:+toward+a+unifying+mechanism.">Neural induction: toward a unifying mechanism.</a> Nat. Neurosci. 4, 1161-1168 (2001).</li><li>Reubinoff, B. E., Itsykson, P., 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=Neural+progenitors+from+human+embryonic+stem+cells.">Neural progenitors from human embryonic stem cells.</a> Nat. Biotechnol. 19, 1134-1140 (2001).</li><li>Zhang, S. C., Wernig, M., Duncan, I. D., Brustle, O., 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=In+vitro+differentiation+of+transplantable+neural+precursors+from+human+embryonic+stem+cells.">In vitro differentiation of transplantable neural precursors from human embryonic stem cells.</a> Nat. Biotechnol. 19, 1129-1133 (2001).</li><li>Hu, B. Y., Weick, J. P., 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=Neural+differentiation+of+human+induced+pluripotent+stem+cells+follows+developmental+principles+but+with+variable+potency.">Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 4335-4340 (2010).</li><li>Singh Roy, N., Nakano, 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=Enhancer-specified+GFP-based+FACS+purification+of+human+spinal+motor+neurons+from+embryonic+stem+cells.">Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells.</a> Exp. Neurol. 196, 224-234 (2005).</li><li>Lee, H., Shamy, G. 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=Directed+differentiation+and+transplantation+of+human+embryonic+stem+cell-derived+motoneurons.">Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons.</a> Stem Cells. 25, 1931-1939 (2007).</li><li>Boulting, G. L., Kiskinis, E., 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+functionally+characterized+test+set+of+human+induced+pluripotent+stem+cells.">A functionally characterized test set of human induced pluripotent stem cells.</a> Nat. Biotechnol. 29, 279-286 (2011).</li><li>Li, X. J., Du, Z. W., 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=Specification+of+motoneurons+from+human+embryonic+stem+cells.">Specification of motoneurons from human embryonic stem cells.</a> Nat. Biotechnol. 23, 215-221 (2005).</li><li>Perrier, A. L., Tabar, V., 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=Derivation+of+midbrain+dopamine+neurons+from+human+embryonic+stem+cells.">Derivation of midbrain dopamine neurons from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 12543-12548 (2004).</li><li>Roy, N. S., Cleren, 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=Functional+engraftment+of+human+ES+cell-derived+dopaminergic+neurons+enriched+by+coculture+with+telomerase-immortalized+midbrain+astrocytes.">Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes.</a> Nat Med. 12, 1259-1268 (2006).</li><li>Yan, Y., Yang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+dopaminergic+neuronal+subtypes+from+human+embryonic+stem+cells.">Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells.</a> Stem Cells. 23, 781-790 (2005).</li><li>Meyer, J. S., Shearer, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+early+retinal+development+with+human+embryonic+and+induced+pluripotent+stem+cells.">Modeling early retinal development with human embryonic and induced pluripotent stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 16698-16703 (2009).</li><li>Osakada, F., Ikeda, 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=Toward+the+generation+of+rod+and+cone+photoreceptors+from+mouse,+monkey+and+human+embryonic+stem+cells.">Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.</a> Nat Biotechnol. 26, 215-224 (2008).</li><li>Carpenter, M. K., Inokuma, M. 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=Enrichment+of+neurons+and+neural+precursors+from+human+embryonic+stem+cells.">Enrichment of neurons and neural precursors from human embryonic stem cells.</a> Exp. Neurol. 172, 383-397 (2001).</li><li>Chambers, S. M., Fasano, C. 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=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> Nat. Biotechnol. 27, 275-280 (2009).</li><li>Chambers, S. M., Qi, 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=Combined+small-molecule+inhibition+accelerates+developmental+timing+and+converts+human+pluripotent+stem+cells+into+nociceptors.">Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors.</a> Nat. Biotechnol. 30, 715-720 (2012).</li><li>Kawasaki, H., Mizuseki, 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=Induction+of+midbrain+dopaminergic+neurons+from+ES+cells+by+stromal+cell-derived+inducing+activity.">Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.</a> Neuron. 28, 31-40 (2000).</li><li>Rubenstein, J. L., Beachy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+of+the+embryonic+forebrain.">Patterning of the embryonic forebrain.</a> Curr. Opin. Neurobiol. 8, 18-26 (1998).</li><li>Hevner, R. F., Hodge, R. D., Daza, R. A., Englund, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+in+glutamatergic+neurogenesis:+conserved+programs+in+neocortex,+cerebellum,+and+adult+hippocampus.">Transcription factors in glutamatergic neurogenesis: conserved programs in neocortex, cerebellum, and adult hippocampus.</a> Neurosci. Res. 55, 223-233 (2006).</li><li>Watanabe, K., Kamiya, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+telencephalic+precursors+from+embryonic+stem+cells.">Directed differentiation of telencephalic precursors from embryonic stem cells.</a> Nat. Neurosci. 8, 288-296 (2005).</li><li>Watanabe, K., Ueno, 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=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Pankratz, M. T., Li, X. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neural+differentiation+of+human+embryonic+stem+cells+via+an+obligated+primitive+anterior+stage.">Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage.</a> Stem Cells. 25, 1511-1520 (2007).</li><li>Li, X. J., Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+sonic+hedgehog+and+Wnt+signaling+determines+ventral+and+dorsal+telencephalic+neuron+types+from+human+embryonic+stem+cells.">Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells.</a> Development. 136, 4055-4063 (2009).</li><li>Zeng, H., Guo, 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=Specification+of+region-specific+neurons+including+forebrain+glutamatergic+neurons+from+human+induced+pluripotent+stem+cells.">Specification of region-specific neurons including forebrain glutamatergic neurons from human induced pluripotent stem cells.</a> PLoS One. 5, e11853 (2010).</li><li>Kim, D. S., Lee, J. 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=Robust+enhancement+of+neural+differentiation+from+human+ES+and+iPS+cells+regardless+of+their+innate+difference+in+differentiation+propensity.">Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity.</a> Stem Cell Rev. 6, 270-281 (2010).</li><li>Zhang, X., Huang, C. 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=Pax6+is+a+human+neuroectoderm+cell+fate+determinant.">Pax6 is a human neuroectoderm cell fate determinant.</a> Cell Stem Cell. 7, 90-100 (2010).</li><li>Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+patterning+of+the+central+nervous+system:+how+many+organizers.">Initial patterning of the central nervous system: how many organizers.</a> Nat. Rev. Neurosci. 2, 92-98 (2001).</li><li>Ma, L., Hu, B., 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+GABA+neurons+correct+locomotion+deficits+in+quinolinic+acid-lesioned+mice.">Human embryonic stem cell-derived GABA neurons correct locomotion deficits in quinolinic acid-lesioned mice.</a> Cell Stem Cell. 10, 455-464 (2012).</li><li>Li, W., Wei, W., 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=Generation+of+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors.">Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.</a> Cell Stem Cell. 4, 16-19 (2009).</li><li>Lowry, W. E., Richter, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+from+dermal+fibroblasts.">Generation of human induced pluripotent stem cells from dermal fibroblasts.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2883-2888 (2008).</li><li>Dimos, J. T., Rodolfa, K. 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=Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321, 1218-1221 (2008).</li><li>Ebert, A. D., Yu, 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=Induced+pluripotent+stem+cells+from+a+spinal+muscular+atrophy+patient.">Induced pluripotent stem cells from a spinal muscular atrophy patient.</a> Nature. 457, 277-280 (2009).</li><li>Lee, G., Papapetrou, E. P., 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=Modelling+pathogenesis+and+treatment+of+familial+dysautonomia+using+patient-specific+iPSCs.">Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.</a> Nature. 461, 402-406 (2009).</li><li>Park, I. H., Arora, 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=Disease-specific+induced+pluripotent+stem+cells.">Disease-specific induced pluripotent stem cells.</a> Cell. 134, 877-886 (2008).</li><li>Chamberlain, S. J., Chen, P. 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=Induced+pluripotent+stem+cell+models+of+the+genomic+imprinting+disorders+Angelman+and+Prader-Willi+syndromes.">Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader-Willi syndromes.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 17668-17673 (2010).</li><li>Kiskinis, E., Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+toward+the+clinical+application+of+patient-specific+pluripotent+stem+cells.">Progress toward the clinical application of patient-specific pluripotent stem cells.</a> J. Clin. Invest. 120, 51-59 (2010).</li><li>Koch, P., Tamboli, I. 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=Presenilin-1+L166P+mutant+human+pluripotent+stem+cell-derived+neurons+exhibit+partial+loss+of+gamma-secretase+activity+in+endogenous+amyloid-beta+generation.">Presenilin-1 L166P mutant human pluripotent stem cell-derived neurons exhibit partial loss of gamma-secretase activity in endogenous amyloid-beta generation.</a> Am. J. Pathol. 180, 2404-2416 (2012).</li><li>Walsh, R. M., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Rett+syndrome+with+stem+cells.">Modeling Rett syndrome with stem cells.</a> Cell. 143, 499-500 (2010).</li><li>Egawa, N., Kitaoka, 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=Drug+Screening+for+ALS+Using+Patient-Specific+Induced+Pluripotent+Stem+Cells.">Drug Screening for ALS Using Patient-Specific Induced Pluripotent Stem Cells.</a> Sci. Transl. Med. 4, (2012).</li><li>Kola, I., Landis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+the+pharmaceutical+industry+reduce+attrition+rates.">Can the pharmaceutical industry reduce attrition rates.</a> Nature Reviews Drug Discovery. 3, 711-716 (2004).</li></ol>

作者宣称，他们没有竞争的金融利益。

在神经细胞分化过程中有几个关键的步骤。重要的是要确保人的产品分成合同，因为否则的细胞是多能干细胞，可能已经偏向成为非神经元谱系。这可以通过以下的确认染色与多能性的标记物，如即Oct4，Sox2基因，Nanog基因，和的Tra-1-60 1-3的抗人类安全公司。如果人类安全公司不重视很好后传代，ROCK抑制剂（Y27632）可以被添加到帮助。对于那些有困难，保持其细胞多能性，一些潜在的问题...

人多能性干细胞（PSCs），包括人类胚胎干细胞（胚胎干细胞），诱导式多能性干细胞（iPS细胞），具有的能力，以产生在体内的每一个细胞类型，包括神经元1-3。定向分化为各种神经元亚型人分成合同持有这些细胞在再生医学中的应用的关键。在开发过程中的功能神经元亚型的产生是一个复杂的过程，涉及诱导的神经系的区域沿rostro尾鳍轴祖细胞，该规范，并从区域4,5祖细胞的有丝分裂后的神经元类型的分化。从2001年开始，一些系统已建立了从人类胚胎干细胞，它提供了一个平台，为后续神经元亚型6,7代产生神经系。根据发展原则，一些神经元类型，如8-12脊髓运动神经元，脑DOP13日至15日 ，胺类神经元和神经视网膜细胞16,17已有效地从人的产品分成合同中指定。这样做是通过施加临界形态发生素体内发育过程中的规格，这些神经元类型是重要的。其他协议也得到了发展，促进人类胚胎干细胞分化成神经元可以使用其他的因素，如小分子或与其他类型的细胞共培养，以18-20帮助促进花芽分化21。 人类的大脑新皮质是高度发达的，包含许多类型的细胞，包括谷氨酸能神经元在学习，记忆和认知功能的22,23，其中扮演了重要的角色。产生谷氨酸能神经元在培养的第一个步骤是指定端脑的祖细胞。笹井芳树的组首次报道了端脑前体定向分化的小鼠胚胎干细胞（mESCs）使用无血清悬索桥的N培养物的存在下，DKK1（抑制Wnt信号）以及LeftyA（抑制节点信令）24。随后，几个研究小组还报告了包括我们在内的人类在无血清培养基25日至27日的物业服务端脑前体的规范。并不需要外源的形态发生和效率产生这些前体的使用是远高于从mESCs 26,27的端脑前体从人类安全公司代。在这里，神经诱导，以及设立的张的基7的化学定义的系统已被描述。此协议没有此外外源性caudalizing因素，有效地产生端脑前体从人类安全公司27。通过调节Wnt信号和Sonic Hedgehog（SHH）的信令，这些祖细胞可以分化成背侧或腹侧祖细胞，，背祖细胞进一步分化成谷氨酸能神经元Ëfficiently 27。此外，该协议还可以很好地用于谷氨酸能神经元的产生，从人类iPSCs 28，这允许针对具体患者的神经元的产生，可以利用机制的探索行动，以及潜在的治疗疾病的大型阵列。此外，我们的系统还提供了一个平台，探索不同的前脑的神经元类型的发展和规范。

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Supplier</td>
			<td>Catalog #</td>
		</tr>
		<tr>
			<td>Dulbecco's modified eagle medium with F12 nutrient mixture (DMEM/F12)</td>
			<td>Gibco</td>
			<td>11330-032</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>Gibco</td>
			<td>10828-028</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030</td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>Gibco</td>
			<td>1140-050</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (14.3 M)</td>
			<td>Sigma</td>
			<td>M-7522</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>12587-010</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide (polyornithine)</td>
			<td>Sigma</td>
			<td>116K5103</td>
		</tr>
		<tr>
			<td>Laminin (human)</td>
			<td>Sigma</td>
			<td>L-6274</td>
		</tr>
		<tr>
			<td>Laminin (mouse)</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini</td>
			<td>100-106</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A-7906</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Invitrogen</td>
			<td>17104-019</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
		</tr>
		<tr>
			<td>ROCK Inhibitor</td>
			<td>Stemgent</td>
			<td>04-0012</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Stemgent</td>
			<td>04-0024</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor 2 (FGF2, bFGF)</td>
			<td>Invitrogen</td>
			<td>13256-029</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075</td>
		</tr>
		<tr>
			<td>0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
		</tr>
		<tr>
			<td>Foxg1 antibody</td>
			<td>Dr. Y. Sasai</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hoxb4 antibody (1:50)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>I12</td>
		</tr>
		<tr>
			<td>Pax6 antibody (1:5000)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>PAX6</td>
		</tr>
		<tr>
			<td>Nkx2.1 antibody (1:200)</td>
			<td>Chemicon</td>
			<td>MAB5460</td>
		</tr>
		<tr>
			<td>Tbr1 antibody (1:2000)</td>
			<td>Chemicon</td>
			<td>AB9616</td>
		</tr>
		<tr>
			<td>vGLUT1 antibody (1:100)</td>
			<td>Synaptic Systems</td>
			<td>135302</td>
		</tr>
		<tr>
			<td>Brain derived neurotrophic factor (BDNF)</td>
			<td>PrepoTech Inc.</td>
			<td>450-02</td>
		</tr>
		<tr>
			<td>Glial derived neurotrophic factor (GDNF)</td>
			<td>PrepoTech Inc.</td>
			<td>450-10</td>
		</tr>
		<tr>
			<td>Insulin growth factor 1 (IGF1)</td>
			<td>PrepoTech Inc.</td>
			<td>100-11</td>
		</tr>
		<tr>
			<td>Cyclic AMP (cAMP)</td>
			<td>Sigma</td>
			<td>D-0260</td>
		</tr>
		<tr>
			<td>Sonic hedgehog (SHH)</td>
			<td>R&amp;D</td>
			<td>1845-SH</td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Becton Dickinson (BD)</td>
			<td>352098</td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>BD</td>
			<td>352097</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>BD</td>
			<td>353046</td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>BD</td>
			<td>353047</td>
		</tr>
		<tr>
			<td>T25 flasks (untreated)</td>
			<td>BD</td>
			<td>353009</td>
		</tr>
		<tr>
			<td>T75 flasks (untreated)</td>
			<td>BD</td>
			<td>353133</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Chemiglass Life Sciences</td>
			<td>1760-012</td>
		</tr>
		<tr>
			<td>6 cm Petri dishes</td>
			<td>BD</td>
			<td>353004</td>
		</tr>
		<tr>
			<td>9'' glass pipetes</td>
			<td>Fisher</td>
			<td>13-678-20D</td>
		</tr>
		<tr>
			<td>Steriflip filters (0.22 &mu;M)</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
		</tr>
		<tr>
			<td>Stericup filters 1,000 ml (0.22 &mu;M)</td>
			<td>Millipore</td>
			<td>SCGPU10RE</td>
		</tr>
		<tr>
			<td>Phase contrast microscope (Observer A1)</td>
			<td>Zeiss</td>
			<td>R2625</td>
		</tr>
		<tr>
			<td>Carbon dioxide incubator (Hera Cell 150)</td>
			<td>Thermo Electron Corporation</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biosafety hood (Sterilgard III Advance)</td>
			<td>The Baker Company</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge (5702 R)</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

the specification of telencephalic glutamatergic neurons from human pluripotent stem cells

这里，已经描述了用于高效地生成从人多能性干细胞（PSCs）端脑谷氨酸能神经元的一个逐步的过程。的分化过程开始向上舍入到时形成聚集体的悬浮培养细胞被放置在成团块通过打破人类安全公司。聚集在人类胚胎干细胞培养基中生长1-4天，以允许自发分化。在这段时间内，将细胞有能力成为任何三种胚层。从5-8天，将细胞放置在一个神经诱导培养基中，推入神经谱系。第8天左右，细胞被允许连接到6孔板上，并分化期间的时间的神经上皮细胞形式。这些神经上皮细胞，可以隔离17天。这些细胞可以保持神经球，直到他们准备好盖玻片的培养皿上。使用碱性介质中没有任何caudalizing因素，神经上皮细胞specifieD插入端脑前体，然后可以进一步区分有效地背端脑祖细胞和谷氨酸能神经元的。总的来说，我们的系统提供了一个工具来生成人体谷氨酸能神经元的研究人员研究这些神经元疾病，影响到他们的发展。

在这里，一个协议来区分人类安全公司到端脑谷氨酸能神经元通过几个关键步骤：PSC的聚集体的形成，神经上皮细胞的诱导，在前脑祖细胞的生成，这些祖细胞的终末分化的成端脑的神经元（ 图1）具有被描述。此系统是稳健的和有效率的，在端脑祖细胞和谷氨酸能神经元的生成。作为一个例子（ 图2），未经此外caudalizing因素，胚胎干细胞分化为神经系27。在24天...

Watch this Scientific Journal Video about The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells at JoVE.com

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

此过程产生在前脑的​​神经元通过检查站，这是人类发展过程中所观察到的类似。细胞可自发地分化，暴露因素推动对神经系，是孤立的，并镀上盖玻片，以便终末分化和成熟。

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 ...

the-specification-telencephalic-glutamatergic-neurons-from-human

Research

JoVE Journal

Neuroscience

10.8K 次观看.  The University of Connecticut Health Center. 此过程产生在前脑的​​神经元通过检查站，这是人类发展过程中所观察到的类似。细胞可自发地分化，暴露因素推动对神经系，是孤立的，并镀上盖玻片，以便终末分化和成熟。

视频: The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

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 ...

科研

神经科学

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

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

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

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 ...

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

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

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

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

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

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 ...

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

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

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 ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

人小胶质细胞样细胞：使用人突触体从诱导多能干细胞和 体外 活细胞吞噬测定中分化

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...