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>

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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，我们已经成功地应用在这里描述的H1和H9 hESC细胞系，并从PD患者和未受影响的同时控制hiPSC线这个协议。 在我们的协议中，有三个主要步骤，以获得高的分化效率:首先，应该在单元之前定向分化没有区别HP​​SC菌落。作为自发分化为所有三种胚层通常被h中PSC培养，离解这些hPSCs成单个细胞之前去除这些分化的集落是很重要的。其次，MEF饲养应该从游离hPSCs被耗尽，因为以前的报告显示，MEF细胞促进自我更新和分化抑制21能分泌因素。为了这个目的，培养于明胶包被的板进行30分钟的HPSC和MEF细胞的混合物应足够以除去几乎所有的MEF细胞。第三，在单一hPSCs应在适当的细胞密度为分化被镀件。增加细胞密度将导致大多数细胞死亡在周围D11分化的，同时降低了细胞密度将导致脱离细胞的死亡，在板的短不少于7天的中心，虽然细胞上边缘将分化良好（数据未示出）。如果一个人观察所有这三个标准，它很容易从hPSCs与我们逐步协议有效地获得多巴胺神经元。 这里介绍的协议主要是基于由L.的Studer和他的同事14的一些修改以前的报告。首先，此处的分化开始单细胞铺板后48小时时，细胞达到大约只有70％汇合，而在他们的报告中，分化细胞时达到完全汇合（后单细胞电镀3天或更多天培养的）开始。根据我们的经验，开始从融合细胞分化无细胞传代至20天会导致严重的细胞死亡过程中的分化过程。第二，我们代替昂贵的SHH蛋白质在与凹陷含chlorobenzothiophene小分子激动剂Hh通路22的研究。因此，与先前公布的方案相比，这一次在这里描述的是饲养细胞和神经花环独立的，主要基于小分子的计划生育规范;它因此是较便宜和较少的时间和劳动消耗。当然，李该协议的mitation是利用KSR的分化的第一个工作日。 KSR从很多变化到很多，这可能会影响分化效率。因此，人们应该测试KSR不同批次，以获得一个诱导计划生育前体后第11天的分化效果最好。 总之，这里descried该分化方案应促进:从人类胚胎干细胞或正常人用于帕金森病的细胞替代疗法的人iPS细胞多巴胺神经元（1）的产生; （2）产生从PD患者的iPSC多巴胺神经元在体外的建模和测试潜在的治疗药物为PD; （3）基因的研究/信号转导通路调节人体多巴胺神经元的发展。

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，形成在所述板约70％汇合。然后分化后，这些细胞达到完全汇合在短至3-4天，并从周围D16-D17开始，这些融合细胞开始，使在板的一些空间，一些轴突/神经突可能已经在这些空间和细胞层下进行观察。而再接种20日龄后，分化的细胞附着和生长的小团块。然后，在接下来的5天，更密集，形态完好的轴突/神经突起的团块出来，轴突/从不同的团块突起形成一些连接。在长期的文化，在一丛的神经元产生更多的扩展功能，获得更成熟，并与邻近神经细胞团块更多的连接。 11天后分化，hPSCs可以有效地转化成FP的前体，并作为图3所示，几乎所有的细胞表达的FP的前体细胞标志物巢蛋白和FOXA2，并将细胞在90％以上表示另一三个标记CORIN，OTX2和LMX1a。然后后分化的多天里，在FP的前体细胞被指定为多巴胺神经元，因为它们表达TUJ1和TH（ 图4A）。这些多巴胺神经元是A9的身份，因为他们EXPRESŠGIRK2同时为负的钙结合蛋白（ 图4B）。免疫荧光实验还表明，没有GFAP +胶质细胞和很少GABA能神经元（ 图4C），表明分化专门向DA神经元谱系指定。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51737/51737fig1highres.jpg" width="500" /> 图1概述了分化方案，用生长因子/小分子和用于每一天的培养基。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51737/51737fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51737/51737fig2highres.jpg" width="500" /> 图2形态在分化期间人的变化。如所述的H9胚胎干细胞铺板为单细胞在适当的细胞密度，此后48小时，这些细胞中的板达到约70％汇合的菌落（D0）。在分化的第几天，这些细胞迅速扩大，分化24小时（D）后达到90％汇合，再经过48多个小时（D3），成为融合。然而，大多数这些细胞仍表现出典型的胚胎干细胞形态和高细胞核向细胞质比（D3）。随着分化的推移，细胞扩展更多的逐渐丧失人类胚胎干细胞的形态。在分化的D11的一端，有在该板的许多份以上的电池层，如由细胞在某些方面比其它（D11）较暗的颜色。大约D17到D20开始，部分细胞死亡，留下一些空间的板块，以及一些神经元的预测已经可以在这些空间被观察到第二有时细胞层（D20）下。细胞replating在D20，使更多的空间用于细胞生长和分化结束后，细胞聚集为小群，多了很多轴突/轴突从这些集群的出现，和轴突/从不同的集群突起形成短一些连接4-5天（D25）。比例尺，200微米。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51737/51737fig3highres.jpg" width="500" /> 图3:免疫染色为FP标志物的差异。H9胚胎干细胞的早期阶段分化为使用此处描述的协议11天。然后将细胞用4％多聚甲醛固定，透化和曲拉通X-100，阻断与驴血清，然后染色以FP前体细胞标志物。如这里所示，几乎所有的细胞表达FOXA2和巢蛋白，并且所述细胞的90％以上的表达LMX1a，CORIN，并OTX2，表示由hESCs到FP单元非常高的效率转换。比例尺，200微米。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51737/51737fig4highres.jpg" width="500" /> 图4为免疫染色的DA神经元标志物在分化后期 。 （ 一）H9人类胚胎干细胞分化为25天，而细胞受到免疫染色泛神经元标记TUJ1和常用的DA神经元标志位。如这里所示的，虽然有更多TUJ1阳性细胞比TH阳性细胞，所有的TH-表达细胞残留在TUJ1表达细胞，以及TH-表达细胞代表至少一半的所有单元格。 （B）。该H9人类胚胎干细胞进一步分化为另外25天数（共50天），然后细胞受到免疫。结果表明，存在的TH-表达细胞的次高百分比一，在D25。几乎所有这些TH-表达细胞也表达GIRK2而其中大部分是负的钙结合蛋白，表明所生成的多巴胺神经元的A9亚型的身份，这是失去了在PD的细胞类型。 （c）在分化D25免疫染色的细胞显示没有细胞表达GFAP，只有极少数细胞表达GABA的影响。比例尺，200微米。

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

Nanyang Technological University

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

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 ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

血源性内皮细胞与人多能干细胞的定向分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

人类多能干细胞的神经元在一个Multititre的板格式的快速和高效地生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

生物学

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 ...

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Source: Cosset, E. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/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 ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

Differentiating Non-Transgenic and Transgenic Human Pluripotent Stem Cells into Neural Progenitor Cells

Source: Cvetkovic, C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates the generation of neural progenitor cells from non-transgenic and transgenic human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural progenitor cells using a neuronal differentiation medium. While differentiation is induced in non-transgenic cells using small molecules, the transgenic hPSCs are differentiated by adding an antibiotic that induces the promoter for a neuronal differentiation-specific transcription factor.

将非转基因和转基因人多能干细胞分化为神经祖细胞

Ethic approvals were obtained when performing the experiments.
1. Cell Culture and Reagent Preparation

	Prepare coated plates for cell culture.
	
		...

Neuronal Culture Techniques

Advanced Neuronal Models

Human Pluripotent Stem Cell Differentiation in Neurons Using Engineered Lentiviruses

Source: Assetta, B., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61778">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates the process of inducing human pluripotent stem cells to differentiate into neurons by transfecting them with engineered lentiviruses carrying a neurogenic transcription factor gene under the control of&#160; tetracycline-responsive element, followed by treatment with a tetracycline derivative and maturation medium.

使用工程慢病毒在神经元中分化人类多能干细胞

1. Human neuron induction from human pluripotent stem cells

	Lentivirus preparation (~5 days, detailed protocol as described previously)
	
		Plate ~1 ...