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?...

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。

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			<td style="width:167px;">1/200 dilution</td>
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			<td style="width:167px;">1/1000 dilution</td>
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			<td style="width:184px;">Pel Freez</td>
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			<td style="width:167px;">1/500 dilution</td>
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			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9702</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Mouse anti human TUJ1 antibody</td>
			<td style="width:184px;">Covance</td>
			<td style="width:119px;">MMS-435P</td>
			<td style="width:167px;">1/2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human GIRK2 antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab30738</td>
			<td style="width:167px;">1/300 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human Calbindin antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab25085</td>
			<td style="width:167px;">1/400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Centrifuge</td>
			<td style="width:184px;">Eppendorf</td>
			<td style="width:119px;">5804</td>
			<td style="width:167px;"></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

Research

JoVE Journal

Neuroscience

16.4K 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

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

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

从人类多能干细胞的规范在前脑谷氨酸能神经元

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

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 ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

分化后再蛋白的人类多能干细胞衍生神经元，用于高含量的内尿成生和突合性成熟筛查

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

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 ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

成人皮层的器官型培养作为人类干细胞移植和验证的 离体 模型

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...