Name: directed dopaminergic neuron differentiation from human pluripotent stem cells
Uploaded: 2014-09-15T13:00:00.000+00:00
Duration: 6 min 40 s
Description: Watch this Scientific Journal Video about Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells at JoVE.com

The authors thank members of the Renee Reijo Pera laboratory for help during development of this protocol and during preparation of this manuscript. This work is supported by California Institute for Regenerative Medicine (CIRM) shared laboratory (CL-00518).

1，准备文化传媒<ol><li>通过结合下面的制备小鼠胚胎成纤维细胞（MEF）介质:445毫升的DMEM 50毫升胎牛血清（FBS）和5ml 100X青霉素/氨苄青霉素贮备液。保持过滤灭菌后的培养基在4℃不超过14天。 </li><li>制备HPSC培养基由下列组成含有血清的385毫升的DMEM / F 12，将100ml敲除血清替代品（KSR），将5ml 100×非必需氨基酸原液，将5ml 100×青霉素/氨苄青霉素贮备液，加入5ml 100倍巯基乙醇储备液和10ng / ml的bFGF。保持过滤灭菌后的培养基在4℃下不超过10天。 </li><li>制备mTeSR1无血清培养基中通过组合下列动作:400毫升mTeSR1基础培养基，加入100ml 5×补充和5ml 100X青霉素/氨苄青霉素贮备液。请媒体在4℃下不超过10天。 </li><li>准备10倍胶原酶储备液:称取0.5克胶原酶力量，用50ml的DMEM / F 12和过滤消毒溶解。请等份和股票它们在-20℃下几个月。 </li><li>制备0.1％的明胶溶液:称取0.5克明胶（由牛的皮肤，类型B）的功率，并与去离子水溶解。保持在室温下放置两周高压釜灭菌的溶液。 </li><li>制备KSR分化培养基中通过组合下列:410毫升的DMEM，75毫升KSR，将5ml 100倍的非必需氨基酸原液，将5ml 100×青霉素/氨苄青霉素贮备液，加入5ml 100X巯基乙醇储备液。保持过滤灭菌后的培养基在4℃下不超过10天。 注:KSR从很多很多，这可能会影响到效率的差异而不同。因此，最好进行测试KSR的几批找到最好的一个用于分化。 </li><li>准备N2分化培养基结合以下98毫升的DMEM，加入1ml 100X补充氮气和1毫升100X青霉素/氨苄西林原液。保持过滤灭菌后的培养基在4°下不超过10天。 </li><li>制备B27分化培养基中通过组合下列动作:480毫升Neurobasal培养基，将10毫升50×B27添加剂，加入5ml 100X Glutamax的储备溶液和5ml 100X青霉素/氨苄青霉素贮备液。保持过滤灭菌后的培养基在4℃下不超过10天。 </li></ol>在MEF饲养细胞的人类胚胎干细胞和iPS细胞的培养2。 该H9人类胚胎干细胞是从WiCell研究所获得，iPS细胞建立了通过山的逆转录病毒介导的转导Reijo佩拉实验室因子OCT3 / 4，SOX2，KLF4和c-MYC 18。 <ol><li>至少一天前孔通道，通过加入1ml 0.1％明胶为1井的6孔板中制备一些明胶板，孵育板在37℃下进行至少30分钟。 </li><li>温育后，从板抽吸明胶，和种子后照射在1.6×10 5个细胞的密度灭活的MEF细胞上的板/孔在MEF中使用血球计数细胞。 注:细胞传代之前，早在3天可选准备MEF饲养。 </li><li>传代细胞1天电镀MEF饲养后:从hPSCs吸出培养基，加入1毫克/毫升胶原酶细胞以1ml /孔，孵育细胞在37℃1小时。 </li><li>在此孵育后，用PBS洗一次MEF饲养，然后以1.5毫升/加温热（37℃）含血清HPSC培养基良好。 </li><li> 1小时后，挖掘培养板几次，使大部分的未分化克隆将分离的板块，而差异化的殖民地保持连接。仔细收集漂浮的殖民地，并把它们转移到15ml试管中。 </li><li>轻轻地用温水（37℃）的DMEM / F 12洗板一次和转移的DMEM / F12到相同的15ml试管中。 </li><li>离心管中，在179×g离心3分钟。 </li><li>从管，小心不要打扰颗粒吸出培养基。添加战争米HPSC培养基，吸取细胞上下数次打破成小群，并混合均匀。 </li><li>将细胞转移到新的MEF饲养在1:3-1:6的比率。 </li><li>每天与通路改变培养基中的细胞，每5-6天。 </li></ol> 3，制备细胞分化<ol><li>至少一天的准备细胞之前，准备一些基质胶板（通常为3孔6孔钢板）。稀释的Matrigel用冷的（4℃）的DMEM / F 12在1:40，添加的6孔板1毫升基底膜每孔。孵化基质胶板在4℃下过夜。注意:一个可以密封基底膜板用封口膜和股票他们在4℃下，只要一个星期。 </li><li>使用细胞（参见步骤2）通过后4天。取下所有板块分化的殖民地。从培养板吸去培养基1毫升的Accutase每加好，并孵育细胞，在37℃下加热5分钟。 </li><li>在培育期间，准备一些凝胶ATIN板加入1 ml明胶为1井的6孔板中。将这些板块在准备孵化前一天基质胶板材。 </li><li>之后的5分钟的孵育或当细胞已成为半浮动，吸管细胞上下数次，使它们成单个细胞。 </li><li>收集单细胞在15ml管中，离心分离机，在258×g离心3分钟。 </li><li>重悬细胞与含血清HPSC培养基的适当量，并Thiazovivin添加到2μM的最终浓度。 </li><li>转移细胞到明胶包被的板按1:1的比例，并孵育细胞，在37℃下进行30分钟以除去MEF饲养。 </li><li>将细胞转移到15 mL锥形管中，加入含HPSC中相应的血清，细胞计数与血球。 </li><li>离心将细胞在258×g离心3分钟。 </li><li>在离心分离机，加Thiazovivin到合适mTeSR1中做出2μm的浓度。 </li><li>经过离心机，spirate培养基并用Thiazovivin添加适当mTeSR1介质，以便有1.8×10 5个细胞/ ml培养基。 </li><li>取出基质胶板从培养箱中，吸出基质胶中加入2毫升上述混合细胞到1井的6孔板中。 注:细胞密度应为3.6×10 4个细胞/表面积厘米2。 </li><li>返回板返回孵化器，并让细胞附着24小时。 </li><li>电镀细胞24小时后，吸旧培养基并且每孔加入2ml新mTeSR1培养基，并让细胞生长另外24小时，在开始分化前。 </li></ol>细胞分化<ol><li> replating细胞基底膜上板后开始分化48小时。 </li><li>从平板上吸出培养基，用PBS洗一次细胞，并加入每孔下面的分化培养基中以2ml:KSR分化培养基中添加了10μMSB431542和100nM的LDN-193这一天，分化D0 189马克。 </li><li>每天从D0改变介质D20。 注:可制备中使用不超过2天以上的时间。 </li><li>使用下面的培养基为D1和D2:添加了10μMSB431542，100nM的LDN-193189，0.25μM的凹陷，2μM的嘌吗啡胺和50毫微克/毫升FGF8b KSR分化培养基中。 </li><li>使用下面的培养基为D3和D4:添加了10μMSB431542，100nM的LDN-193189，0.25μM的凹陷，2μM的嘌吗啡胺，50毫微克/毫升FGF8b，和3μMCHIR99021 KSR分化培养基中。 </li><li>用于D5和D6以下介质:75％KSR分化培养基中加25％的N 2的分化培养基中添加100nM的LDN-193189，0.25μM的凹陷，2μM的嘌吗啡胺，50毫微克/毫升FGF8b，和3μMCHIR99021。 </li><li>使用的D7和D8以下介质:50％KSR分化培养基加上50％的氮气分化培养基中添加100纳米LDN-193189和3微米CHIR99021。</li><li>使用以下介质D9和D10:补充100纳米LDN-193189和3微米CHIR99021 25％KSR分化培养基加上75％的氮气分化培养基。 </li><li>使用下面的培养基为D11和D12:补充有3μMCHIR99021，10毫微克/毫升的BDNF，10毫微克/毫升的GDNF，1毫微克/毫升TGF3，0.2 mM的抗坏血酸和0.1mM腺苷B27分化培养基中。 </li><li>使用下面的培养基中分化的其余部分:B27分化培养基补充有10纳克/毫升的BDNF，10毫微克/毫升的GDNF，1毫微克/毫升TGF3，0.2 mM的抗坏血酸和0.1mM的cAMP。 </li><li>在大约D16分化，准备一些聚-L-鸟氨酸/层粘连蛋白/纤维连接蛋白片。稀聚-L-鸟氨酸原液用冷的（4℃）PBS中，以15微克/毫升，加入1毫升至1孔的6孔培养板，孵育所述板在37℃下过夜。 </li><li>吸出聚-L-鸟氨酸溶液，洗板3次，用无菌水，然后空气干燥该板。 </li><li>层粘连蛋白稀释溶胶股票ution用冷的（4℃）PBS中，以1微克/毫升，加入1毫升的6孔板一个孔中，并孵育所述板在37℃下过夜。 </li><li>吸出的层粘连蛋白溶液，洗板3次，用无菌水，然后空气干燥该板。 </li><li>稀释纤连蛋白原液用冷的（4℃）PBS中，以2微克/毫升，加入1毫升至1孔的6孔培养板，孵育所述板在37℃下过夜。 </li><li>密封板用封口膜，并将其放置在4℃下长达两个星期。 </li><li>经过20天的分化，replate的细胞在聚-L-鸟氨酸/层粘连蛋白/纤连蛋白的平板上。吸出物的分化培养基，加入1毫升温（37℃）的Accutase 1井的6孔板中，并培养细胞，在37℃下加热5分钟。 </li><li>在培育期间，将聚-L-鸟氨酸/层粘连蛋白/纤连蛋白板的孵化器。 </li><li>之后的5分钟的孵育或当细胞已成为半浮动，轻轻移液管细胞上下数次使他们成单个细胞。 </li><li>收集的单个细胞在15ml锥形管中，并加入适量的Neurobasal培养基用于细胞计数，这将取决于膨胀变化（11天后分化，细胞可以扩大15-20倍）。计数使用血球细胞。 </li><li>离心将细胞在258×g离心3分钟。吸出上清，重悬细胞与B27的分化培养基中，使细胞浓度为1×10 6个细胞/ ml培养基。 </li><li>从平板上吸出纤连蛋白，用PBS洗两次，然后加入2mL的细胞，以1个孔的6孔板中。 注:细胞密度为replating应为2×10 5个细胞/表面积厘米2。 </li><li>改变中24小时后，以消除任何独立的死细胞。 </li><li>改变培养基每隔一天，直到对于给定的实验所需要的时间点。 </li></ol>

<ol>
	<li>Freed, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Will+embryonic+stem+cells+be+a+useful+source+of+dopamine+neurons+for+transplant+into+patients+with+Parkinson's+disease.">Will embryonic stem cells be a useful source of dopamine neurons for transplant into patients with Parkinson's disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 1755-1757 (2002).</li><li>Perrier, A. 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=Derivation+of+midbrain+dopamine+neurons+from+human+embryonic+stem+cells.">Derivation of midbrain dopamine neurons from human embryonic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 12543-12548 (2004).</li><li>Park, C. 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=In+vitro+and+in+vivo+analyses+of+human+embryonic+stem+cell-derived+dopamine+neurons.">In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons.</a> Journal of Neurochemistry. 92, 1265-1276 (2005).</li><li>Buytaert-Hoefen, K. A., Alvarez, E., Freed, C. R. <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+tyrosine+hydroxylase+positive+neurons+from+human+embryonic+stem+cells+after+coculture+with+cellular+substrates+and+exposure+to+GDNF.">Generation of tyrosine hydroxylase positive neurons from human embryonic stem cells after coculture with cellular substrates and exposure to GDNF.</a> Stem Cells. 22, 669-674 (2004).</li><li>Zeng, 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=Dopaminergic+differentiation+of+human+embryonic+stem+cells.">Dopaminergic differentiation of human embryonic stem cells.</a> Stem Cells. 22, 925-940 (2004).</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=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>Schulz, T. 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=Differentiation+of+human+embryonic+stem+cells+to+dopaminergic+neurons+in+serum-free+suspension+culture.">Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.</a> Stem Cells. 22, 1218-1238 (2004).</li><li>Park, 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=Generation+of+dopaminergic+neurons+in+vitro+from+human+embryonic+stem+cells+treated+with+neurotrophic+factors.">Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors.</a> Neuroscience Letters. 359, 99-103 (2004).</li><li>Cho, 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=Highly+efficient+and+large-scale+generation+of+functional+dopamine+neurons+from+human+embryonic+stem+cells.">Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 3392-3397 (2008).</li><li>Cooper, O., 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+human+ES+and+Parkinson's+disease+iPS+cells+into+ventral+midbrain+dopaminergic+neurons+requires+a+high+activity+form+of+SHH,+FGF8a+and+specific+regionalization+by+retinoic+acid.">Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid.</a> Molecular and Cellular Neurosciences. 45, 258-266 (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, 275-280 (2009).</li><li>Sanchez-Danes, 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=Efficient+generation+of+A9+midbrain+dopaminergic+neurons+by+lentiviral+delivery+of+LMX1A+in+human+embryonic+stem+cells+and+induced+pluripotent+stem+cells.">Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem cells and induced pluripotent stem cells.</a> Human Gene Therapy. 23, 56-69 (2012).</li><li>Fasano, C. A., Chambers, S. M., Lee, G., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+derivation+of+functional+floor+plate+tissue+from+human+embryonic+stem+cells.">Efficient derivation of functional floor plate tissue from human embryonic stem cells.</a> Cell Stem Cell. 6, 336-347 (2010).</li><li>Kriks, 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=Dopamine+neurons+derived+from+human+ES+cells+efficiently+engraft+in+animal+models+of+Parkinson's+disease.">Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease.</a> Nature. 480, 547-551 (2011).</li><li>Xi, 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=Specification+of+midbrain+dopamine+neurons+from+primate+pluripotent+stem+cells.">Specification of midbrain dopamine neurons from primate pluripotent stem cells.</a> Stem Cells. 30, 1655-1663 (2012).</li><li>Kirkeby, 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=Generation+of+regionally+specified+neural+progenitors+and+functional+neurons+from+human+embryonic+stem+cells+under+defined+conditions.">Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions.</a> Cell Reports. 1, 703-714 (2012).</li><li>Mendez, I., 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=Cell+type+analysis+of+functional+fetal+dopamine+cell+suspension+transplants+in+the+striatum+and+substantia+nigra+of+patients+with+Parkinson's+disease.">Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson's disease.</a> Brain: a Journal of Neurology. 128, 1498-1510 (2005).</li><li>Takahashi, 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>Nishimura, K., Takahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+application+of+stem+cell+technology+toward+the+treatment+of+Parkinson's+disease.">Therapeutic application of stem cell technology toward the treatment of Parkinson's disease.</a> Biological &amp; Pharmaceutical Bulletin. 36, 171-175 (2013).</li><li>Smidt, M. P., Burbach, J. P. <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+a+mesodiencephalic+dopaminergic+neuron.">How to make a mesodiencephalic dopaminergic neuron.</a> Nature Reviews. Neuroscience. 8, 21-32 (2007).</li><li>Wang, 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=Noggin+and+bFGF+cooperate+to+maintain+the+pluripotency+of+human+embryonic+stem+cells+in+the+absence+of+feeder+layers.">Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers.</a> Biochemical and Biophysical Research Communications. 330, 934-942 (2005).</li><li>Chen, J. K., Taipale, J., Young, K. E., Maiti, T., 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=Small+molecule+modulation+of+Smoothened+activity.">Small molecule modulation of Smoothened activity.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 14071-14076 (2002).</li></ol>

The authors declare that they have no competing financial interests.

人多能干细胞（包括胚胎干细胞和人iPS细胞）可以在体外分化而产生大部分，如果不是全部，我们的身体，包括多巴胺神经元，这已被证明在以前的研究中19的细胞类型。这里，基于从胚胎多巴胺能神经元的发展20知识和公布的协议从其他实验室14,15，我们优化了培养条件对从胚胎干细胞和iPS细胞多巴胺神经元的产生。这个协议是有效的，可重复的，我们已经成功地?...

多巴胺（DA）神经元可以在几个脑区域，包括脑，下丘脑，视网膜，和嗅球中找到。在A9多巴胺神经元在黑质致密部（黑质）控制由突出到前脑，纹状体和形成锥体外运动系统的行为和动作。 A9多巴胺神经元变性导致帕金森氏病（PD），这是第二个最常见的人类神经变性疾病，目前无法治愈的。细胞替代疗法是最有前途的策略为PD 1的处理中的一个，因此，出现了在由人多能干细胞（hPSCs）导出A9多巴胺神经元的极大兴趣，包括人类胚胎干细胞（胚胎干细胞）和最近的人类诱导多能干细胞（iPS细胞）。 许多研究都试图通过各种方法hPSCs得到A9的多巴胺神经元。通过神经网络的最早报道都产生多巴胺的神经元莲座祖阶段。通过合作，培养与MS5 2或3-5尼龙6间质细胞有或没有外部生长因子2-4周，L的Studer和同事成功地诱导人类胚胎干细胞产生神经花环其他三个组。然后，他们丰富了这些玫瑰花机械清扫或酶消化进一步分化。在其他报告中，研究人员通过生成的胚体悬浮培养分化6-9神经花环（EB）。后来，研究人员建立了基于单层分化方法10,11，在那里它们镀人类胚胎干细胞和人iPS细胞的细胞外基质，添加于不同生长因子的培养以诱导hPSCs分化为多巴胺神经元，其模拟了体内胚胎DA神经元的发展。虽然所有这些研究中得到的酪氨酸羟化酶（TH）表达细胞与多巴胺神经元的某些特征，整个分化过程中的时间和劳动消耗，通常效率低，而且更重要的是，这些神经元的A9身份，除了一个与LMX1a异位表达12多数研究都没有表现出来。最近，一种新的地板板（FP）的协议被开发13-16，其中，首先由活化的音猬和经典Wnt信号转导途径的过程中分化的早期阶段产生的FP的前体与多巴胺神经元的电势，然后这些计划生育细胞进一步指明DA能神经元。虽然这个协议是更有效的，但仍存在一些问题;例如，在整个分化过程需要很长的时间（至少35天），是饲养细胞依赖的15，或者是EB依赖性16或A9身份没有被证实14。 在此基础上，从体内胚胎多巴胺神经元发育等研究人员公布业绩的知识，我们优化培养条件为EFFicient代来自人类胚胎干细胞和iPS细胞多巴胺神经元。我们首先生成的FP的前体细胞通过激活Wnt信号传导的小分子CHIR99021和音猬蛋白与小分子SAG和嘌吗啡胺的信令。这些计划生育细胞表达FOXA2，LMX1a，CORIN，OTX2和巢。然后，我们指定了这些计划生育细胞对多巴胺神经元的生长因子，包括BDNF，GDNF 等 。生成的多巴胺神经元的A9细胞类型，因为它们是阳性GIRK2而负钙结合蛋白17。该协议是饲养细胞和EB独立，高效和可重复性。通过此协议，一个也可以，或者从PD患者的体外模型的PD或测试潜在的治疗药物为PD iPS细胞在不到4周的胚胎干细胞或正常人的细胞移植研究iPS细胞获得多巴胺神经元。

<table><tbody><tr><td>DMEM</td><td>Life Technologies</td><td>10569-010</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>26140</td><td></td></tr><tr><td>penicillin/ampicillin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>10565-018</td><td></td></tr><tr><td>KSR</td><td>Life Technologies</td><td>10828-028</td><td></td></tr><tr><td>Non-essential amino acid</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>b-mercaptoethanol</td><td>Millipore</td><td>ES-007-E</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D Systems</td><td>233-FB-025</td><td></td></tr><tr><td>mTesR1</td><td>STEMCELL Technologies</td><td>5850</td><td></td></tr><tr><td>Collagenase IV</td><td>Life Technologies</td><td>17104-019</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G9391</td><td></td></tr><tr><td>N2 supplement</td><td>Life Technologies</td><td>17502-048</td><td></td></tr><tr><td>B27 supplement</td><td>Life Technologies</td><td>17504-044</td><td></td></tr><tr><td>Neurobasal</td><td>Life Technologies</td><td>21103-049</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Growth factor reduced matrigel</td><td>BD Biosciences</td><td>354230</td><td></td></tr><tr><td>Accutase</td><td>MP Biomedicals</td><td>1000449</td><td></td></tr><tr><td>Thiazovivin</td><td>Santa Cruz Biotechnology</td><td>sc-361380</td><td></td></tr><tr><td>SB431542</td><td>Tocris Bioscience</td><td>1614</td><td></td></tr><tr><td>LDN-193189</td><td>Stemgent</td><td>04-0074</td><td></td></tr><tr><td>SAG</td><td>EMD Millipore</td><td>566660-1MG</td><td></td></tr><tr><td>Purmorphamine</td><td>Santa Cruz Biotechnology</td><td>sc-202785</td><td></td></tr><tr><td>FGF8b</td><td>R&amp;amp;D Systems</td><td>423-F8-025</td><td></td></tr><tr><td>CHIR99021</td><td>Cellagen Technology</td><td>C2447-2s</td><td></td></tr><tr><td>BDNF</td><td>R&amp;amp;D Systems</td><td>248-BD-025</td><td></td></tr><tr><td>GDNF</td><td>R&amp;amp;D Systems</td><td>212-GD-010</td><td></td></tr><tr><td>TGF-beta3</td><td>R&amp;amp;D Systems</td><td>243-B3-002</td><td></td></tr><tr><td>Ascorbic acid</td><td>Sigma-Aldrich</td><td>A4034</td><td></td></tr><tr><td>cAMP</td><td>Sigma-Aldrich</td><td>D0627</td><td></td></tr><tr><td>Mouse anti human NESTIN antibody</td><td>Santa Cruz Biotechnology</td><td>sc-23927</td><td>1/1,000 dilution</td></tr><tr><td>Rabbit anti human OTX2 antibody</td><td>Millipore</td><td>AB9566</td><td>1/2,000 dilutiion</td></tr><tr><td>Goat anti human FOXA2 antibody</td><td>R&amp;amp;D Systems</td><td>AF2400</td><td>1/200 dilution</td></tr><tr><td>rabbit anti human LMX1a antibody</td><td>Millipore</td><td>AB10533</td><td>1/1,000 dilution</td></tr><tr><td>Rabbit anti human TH antibody</td><td>Pel Freez</td><td>P40101</td><td>1/500 dilution</td></tr><tr><td>Chicken anti human TH antibody</td><td>Millipore</td><td>AB9702</td><td>1/500 dilution</td></tr><tr><td>Mouse anti human TUJ1 antibody</td><td>Covance</td><td>MMS-435P</td><td>1/,2000 dilution</td></tr><tr><td>Rabbit anti human GIRK2 antibody</td><td>Abcam</td><td>ab30738</td><td>1/300 dilution</td></tr><tr><td>Rabbit anti human Calbindin antibody</td><td>Abcam</td><td>ab25085</td><td>1/400 dilution</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804</td><td></td></tr></tbody></table>

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.

该分化方案的概要示于图1。这里给出的分化方案的效率依赖于起始细胞的状态。因此，重要的是要确保，第一个删除所有分化集落解离hPSCs成单个细胞的分化之前，第二，1耗尽大部分，如果不是全部，在MEF饲养细胞在明胶包被的平板孵育细胞30分钟后，和第三，1板的HPSC单个细胞在适当的密度到基质胶盘。如示于图2中 ，再接种HPSC单个细胞将在分化之前的48小时为簇扩大?...

Watch this Scientific Journal Video about Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells at JoVE.com

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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 ...

directed-dopaminergic-neuron-differentiation-from-human-pluripotent

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

16.5K 次观看.  Stanford University School of Medicine. 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’s disease.

视频: Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

科研

发育生物学

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 ...

神经科学

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

在无馈体和化学定义的培养条件下，使用人类多能干细胞的后多能感感神经元的有效分化

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...

从人多能干细胞衍生肠道神经元

Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls bowel motility, enzyme secretion, and nutrient absorption and interacts with the immune system and the gut microbiome. Consequently, developmental and acquired defects of the ENS are responsible for many human diseases and may contribute to symptoms of Parkinson's disease. Limitations in animal model systems and access to primary tissue pose significant experimental challenges in studies of the human ENS. Here, a detailed protocol is presented for effective in vitro derivation of the ENS lineages from human pluripotent stem cells, hPSC, using defined culture conditions. Our protocol begins with directed differentiation of hPSCs to enteric neural crest cells within 15 days and yields diverse subtypes of functional enteric neurons within 30 days. This platform provides a scalable resource for developmental studies, disease modeling, drug discovery, and regenerative applications.

作者聚焦：来自人多能干细胞的人肠道神经元的稳健培养

Deriving enteric nervous system (ENS) lineages from human pluripotent stem cells (hPSC) provides a scalable source of cells to study ENS development and disease, and to use in regenerative medicine. Here, a detailed in vitro protocol to derive enteric neurons from hPSCs using chemically defined culture conditions is presented.

肠神经系统谱系与人多能干细胞的分化

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

通过集成活细胞监测，自动生产人类诱导多能干细胞衍生皮质神经元和多巴胺神经元

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

生物学

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Source: Cosset, E. et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59682">Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases.</a> J. Vis. Exp. (2019)
This video demonstrates the differentiation of human embryonic stem cells (hESCs) into dopaminergic neurons, to provide a model for studying Parkinson&#39;s disease. The hESCs are cultured in a neural induction medium in a microwell plate to form 3D neurospheres. These are then cultured in a neural induction medium to promote the differentiation into dopaminergic progenitor neurons. The final maturation of the neurons occurs in a culture insert under air-liquid interface conditions, resulting in 3D organoids consisting of mature dopaminergic neurons.

从人胚胎干细胞产生多巴胺能神经元类器官

1. hESC-derived dopaminergic organoids for Parkinson&#39;s Disease (PD) studies

	Day 0: Amplify hESCs in 2D culture up to 60% confluency (day 0), then ...

从人多能干细胞定向多巴胺能神经元分化

摘要

探索更多视频

此视频中的章节

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

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

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究

在无馈体和化学定义的培养条件下，使用人类多能干细胞的后多能感感神经元的有效分化

肠神经系统谱系与人多能干细胞的分化

肠神经系统谱系与人多能干细胞的分化

肠神经系统谱系与人多能干细胞的分化

通过集成活细胞监测，自动生产人类诱导多能干细胞衍生皮质神经元和多巴胺神经元

从人胚胎干细胞产生多巴胺能神经元类器官