Name: efficient differentiation of postganglionic sympathetic neurons using human pluripotent stem cells under feeder-free and chemically defined culture conditions
Uploaded: 2020-05-24T13:30:00
Description: Watch this Scientific Journal Video about Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

我们要感谢海蒂·乌尔里希斯对手稿的批判性阅读和编辑。

注：H9 PHOX2B：GFP记者专线由Oh等人提供。本文中使用的一些qPCR引物来自OriGene技术公司，而一些序列则从Frith等人20、30.,30
1. 设置用于盘涂、介质制备和 hPSC 维护
<ol><li>碟涂层<ol>
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体外内他 （VTN） 涂层
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<li>将 VTN 小瓶放入 37 °C 水浴中，直到完全解冻，然后彻底混合。</li>
			<li>对于 100 mm 的 Petr...

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我们最近发表了两篇评论，一篇讨论使用hPSC衍生的symN进行疾病建模31，以及深入比较现有的分化协议22。因此，在这里，我们重点对当前协议进行故障排除，以帮助感兴趣的研究人员成功创建 symN。在整个分化过程中，为了获得一致的数据以及健康的分化细胞，应仔细控制各个阶段的污染。在常规 hPSC 维护中，支原体测试应每两周或每月执行一次。如果在第10天...

后刚体交感神经元（symNs）属于自主神经系统（ANS），在独立于意识的个体的反应和调节平衡方面具有多重重要作用。例如，压力刺激symN，并唤起战斗或逃跑的反应，导致心率，血压和出汗的增加。SymNs 因遗传、毒性/伤害或其他疾病的同伴而在多种人类疾病中受到影响。遗传神经病变的一个例子是儿童疾病家庭性肌瘤（FD），其中，严重的调节调节的共生导致反乌托邦危机，明显的出汗，皮肤斑点，呕吐发作，高血压和焦虑1。毒性的一个例子是化疗治疗，据报道，它对自主神经元有毒副作用2。众所周知，自主变性和超内侧病既可导致或伴随，如帕金森病或高血压肾病33，4。4因此，能够进行研究，了解symN生物学的机制和疾病背景下的缺陷，有利于寻找新颖有效的治疗方法。
解剖 外围神经系统分支成感官和自主的分裂。感觉神经系统的发泡神经负责疼痛和触觉的感觉，而ANS负责将信息从所有器官中继到大脑。ANS分为肠神经系统，内化胃肠道，寄生神经系统，这是放松的重要，和寄分神经系统（SNS），这是重要的激活/调节器官。SNS 调整双神经元系统5。前刚利感神经斧头在脊髓第一个项目到交感性神经神经神经斧子，其中后刚体symN细胞体的位置。然后，这些神经元发送长投影，以内化身体中每个器官的目标组织。由前江柱神经元传输的信号是胆碱化的，而后江脂符号是可德雷纳的，因此表达去甲肾上腺素（NE）作为其主要神经递质。很少有明显的例外，后血管，同情神经元是胆碱化，包括那些内化血管。腺素后神经神经元表达酶酪氨酸羟基酶（TH），芳香L-氨基酸脱氧血脂酶（AAAD），多巴胺β-羟基酶（DBH），和单胺氧化酶（MAO-A），所有负责产生和代谢NE。此外，它们还表达NE回收运输机和/或受体β-肾上腺素受体（ADRA2）、β-肾上腺素受体（ADR2B）、去甲肾上腺素输送器（NET1）和光体单胺输送器（VMAT1/2）。
发展 在胚胎发育过程中，符号来自神经峰（NC），神经管和覆盖的ectoderm6之间出现，可以分化成多个细胞谱系，包括黑色素细胞、骨细胞、副细胞、胶质、肠神经元、感觉神经元和自主神经元7。神经峰细胞（NCCs）是高度迁移的细胞，通过胚胎走几条途径。在NC发育的早期阶段，细胞表示标记SNAIL1/2、FOXD3和SOX1088、9、10、11。9,10,11迁移路线以及它们采用的轴向位置决定了它们将发展到的 NC 子类型。这些NC亚型可以通过其特定的HOX基因表达来区分：颅内NCC不表达HOX基因，vagal NCC表达HOX1+5，中继NCCs表达HOX6+9，以及食质NCC表达HOX10~1112。其中，中继NcC被公认为是符号的主要来源。SymN 前体表示转录因子 MASH1/ASCL113，它促进 PHOX2B14和 INSM115的表达。《国家与加体刑部》的转录因子家族在晚期同情发展期间表达。GATA2 和 GATA3 表示在符号中，然后激活 DBH16。转录因子HAND2对DBH和TH17的表达和维护也很重要。
HPSC（例如胚胎和诱导多能干细胞）是一个强大的工具18，用于概括发展范式并生成符号，然后可用于各种人类疾病的疾病建模。因此，在从 hPSC 生成符号的同时，必须遵循发展准则，并评估在差别化过程中表达适当的标记。
以前的 symN 协议 很少有研究小组以前曾报告过hPSC19、20、21,20,21的共生生成。最近22年，我们审查了这些协议与彼此的直接比较。2016年23月，我们发布了一种分化协议，用于生成具有symN字符的自主神经元（图1A）。该协议使用基于KSR的介质，用于维持无差别的hPSCs和细胞分化。此外，在小鼠胚胎成纤维细胞（MEF馈送细胞）上维持hPSCs。我们使用这个协议和PSC从FD患者建模紊乱23。在2019年，我们描述了这个旧协议24的更详细的版本。综上所述，神经命运是由双SMAD抑制25在前2天阻止TGF-β和BMP信号诱导的。使用CHIR99021进行WNT激活促进神经祖细胞成为NC细胞。第11天，通过FACS对CD49D+或+SOX10=种群26，23,23的细胞进行排序，产生了约40%的NC生成效率。+因此，需要分拣以确保下一步差异化的效率和纯度。通过FGF2和CHIR的组合处理，将NCC作为球体进行维护和放大。4天后，对维护的NC球体进行镀层，并给予BDNF、GDNF和NGF，以完成symN成熟。虽然这些符号表示强烈的symN标记，如ASCL1，TH，DBH和PHOX2A，更成熟的符号，包括尼古丁乙酰胆碱受体（CHRNA3/CHRNB4）和囊泡运输器（VMAT1/2）的表达，即使在70天的分化后，也很低。该协议中的HOX基因没有经过正式测试，成熟的神经特性，包括细胞的电生理活性，未经验证。
在这里，我们提出了一个优化的协议来生成符号（图1B）。HPSC 在无馈送条件下保持，在体外内他丁 （VTN） 涂层的菜肴上，使用基本 8 （E8） 介质27。分化介质的公式在每个阶段都进行了修改，从而增加了NC人口28的百分比。symN 成熟可以在 CD49D+/SOX10=排序或未排序的散装 NCC 种群上完成。两者都显示第 30 天的高级别 symN 标记表达式。此外，使用该协议生成的符号对电生理记录和使用symN活化剂和抑制剂化合物的处理有反应。

<table>
	<tbody>
		<tr>
			<td>100 mm cell culture dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tissue culture tubes</td>
			<td>VWR/Corning</td>
			<td>89039-664</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plates</td>
			<td>Falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well ultra-low-attachment plates</td>
			<td>Corning</td>
			<td>07 200 601 and 07 200 602</td>
			<td></td>
		</tr>
		<tr>
			<td>5% CO2/20% O2 tissue culture incubator</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>Heracell VIOS 160i</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tissue culture tubes</td>
			<td>VWR/Corning</td>
			<td>89039-656</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovation Cell Technologies</td>
			<td>AT104500</td>
			<td>Cell dissociation solution</td>
		</tr>
		<tr>
			<td>Anti-AP2a antibody</td>
			<td>Abcam</td>
			<td>ab108311</td>
			<td>Host: Rabbit; 1:400 dilution</td>
		</tr>
		<tr>
			<td>Anti-Ascl1 antibody</td>
			<td>BD Pharmingen</td>
			<td>556604</td>
			<td>Host: Mouse IgG1; 1:200 dilution</td>
		</tr>
		<tr>
			<td>Anti-CD49D antibody</td>
			<td>BioLegend</td>
			<td>304313</td>
			<td>Host: Mouse IgG1; 5 &#956;l/million cells in 100 &#956;l volume</td>
		</tr>
		<tr>
			<td>Anti-CD49D antibody (isotype)</td>
			<td>BioLegend</td>
			<td>400125</td>
			<td>Host: Mouse IgG1; 5 &#956;l/million cells in 100 &#956;l volume</td>
		</tr>
		<tr>
			<td>Anti-DAPI antibody</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Anti-DBH antibody</td>
			<td>Immunostar</td>
			<td>22806</td>
			<td>Host: Rabbit; 1:500 dilution</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td>Host: Chicken; 1:1000 dilution</td>
		</tr>
		<tr>
			<td>Anti-HOXC9 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-365692</td>
			<td>Host: Mouse IgG1; 1:100 dilution</td>
		</tr>
		<tr>
			<td>Anti-NET1 antibody</td>
			<td>Mab</td>
			<td>NET17-1</td>
			<td>Host: Mouse; 1:1000 dilution</td>
		</tr>
		<tr>
			<td>Anti-PRPH antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-377093/H0112</td>
			<td>Host: Mouse IgG2a; 1:200 dilution</td>
		</tr>
		<tr>
			<td>Anti-SOX10 antibody</td>
			<td>Abcam</td>
			<td>ab50839</td>
			<td>Host: Mouse; 1:100 dilution</td>
		</tr>
		<tr>
			<td>Anti-TH antibody</td>
			<td>Pel-Freez</td>
			<td>P40101- 150</td>
			<td>Host: Rabbit; 1:500 dilution</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A8960-5G</td>
			<td>Stock concentration: 100 mM</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>12587-010</td>
			<td>Stock concentration: 50x</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>R&#38;D Systems</td>
			<td>248-BD</td>
			<td>Stock concentration: 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-BP</td>
			<td>Stock concentration: 6 mM</td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>Countess II</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting chamber slides</td>
			<td>Invitrogen</td>
			<td>C10312</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>57021&#38;5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>R&#38;D Systems</td>
			<td>4423</td>
			<td>Stock concentration: 6 mM</td>
		</tr>
		<tr>
			<td>Cryo-vial</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>375353</td>
			<td></td>
		</tr>
		<tr>
			<td>dbcAMP</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>Stock concentration: 100 mM</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>10829-018</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>11330-057</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>E6 medium</td>
			<td>gibco</td>
			<td>A15165-01</td>
			<td></td>
		</tr>
		<tr>
			<td>E8 medium</td>
			<td>gibco</td>
			<td>A15169-01</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>E8 supplement</td>
			<td>gibco</td>
			<td>A15171-01</td>
			<td>Stock concentration: 50x</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>ED2SS</td>
			<td>Stock concentration: 0.5 M</td>
		</tr>
		<tr>
			<td>Electrophysiology plates (AXION cytoview MEA96)</td>
			<td>Axion BioSystems</td>
			<td>M768-tMEA-96W</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS machine</td>
			<td>Beckman Coulter</td>
			<td>CytoFLEX (for FACS)</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS machine</td>
			<td>Beckman Coulter</td>
			<td>MoFlo Astrios EQ (for sorting)</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes (blue filter cap)</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes (white cap)</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>PeproTech</td>
			<td>450</td>
			<td>Stock concentration: 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Geltrex</td>
			<td>Invitrogen</td>
			<td>A1413202</td>
			<td>Basement membrane matrix; Stock concentration: 100x</td>
		</tr>
		<tr>
			<td>hPSCs</td>
			<td>Thomson et al., (1998)</td>
			<td>WA09</td>
			<td></td>
		</tr>
		<tr>
			<td>hPSCs</td>
			<td>Oh et al. (2016)</td>
			<td>H9-PHOX2B::eGFP</td>
			<td></td>
		</tr>
		<tr>
			<td>Human fibronectin (FN)</td>
			<td>VWR/Corning</td>
			<td>47743-654</td>
			<td>Stock concentration: 1 mg/mL</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher/Gibco</td>
			<td>25030-081</td>
			<td>Stock concentration: 200 mM</td>
		</tr>
		<tr>
			<td>LN tank</td>
			<td>Custom Biogenic Systems</td>
			<td>V-1500AB</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA reader</td>
			<td>Axion BioSystems</td>
			<td>Maestro Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse laminin I (LM)</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td>Stock concentration: 1 mg/mL</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>17502-048</td>
			<td>Stock concentration: 100x</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>gibco</td>
			<td>21103-049</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>NGF</td>
			<td>PeproTech</td>
			<td>450-01</td>
			<td>Stock concentration: 25 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide (PO)</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td>Stock concentration: 15 mg/mL</td>
		</tr>
		<tr>
			<td>Primocin (antibiotics)</td>
			<td>InvivoGen</td>
			<td>ANTPM1</td>
			<td>Stock concentration: 50 mg/mL</td>
		</tr>
		<tr>
			<td>qPCR machine</td>
			<td>Bio-Rad Laboratories</td>
			<td>C1000 Touch</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR plates</td>
			<td>Bio-Rad Laboratories</td>
			<td>HSP9601</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant FGF2</td>
			<td>R&#38;D Systems</td>
			<td>233-FB/CF</td>
			<td>Stock concentration: 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Stock concentration: 1 mM</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris/R&#38;D Systems</td>
			<td>1614</td>
			<td>Stock concentration: 10 mM</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Corning</td>
			<td>MT-25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitronectin (VTN)</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>A14700</td>
			<td>Stock concentration: 0.5 mg/mL</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>VWR/Corning</td>
			<td>706308</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>R&#38;D Systems</td>
			<td>1254</td>
			<td>Stock concentration: 10 mM</td>
		</tr>
	</tbody>
</table>

efficient differentiation of postganglionic sympathetic neurons using human pluripotent stem cells under feeder-free and chemically defined culture conditions

人类多能干细胞（hPSCs）已成为疾病建模和体外人类胚胎发育研究的有力工具。我们之前提出了一种分化协议，用于具有交感特性的自主神经元的衍生，该特性已应用于自主神经病变患者。然而，该协议建立在敲出血清替代（KSR）和基于饲料的培养条件之上，为了确保高分化效率，细胞分拣是必要的。这些因素导致高可变性、高成本和低可重复性。此外，成熟的寄感特性，包括电活动，尚未得到证实。在这里，我们提出了一个优化的协议，其中PSC培养和分化是在无馈料和化学定义的培养条件下进行的。识别躯干神经峰的基因标记被识别。在20天后实现进一步分化成后刚体交感神经元，无需细胞分拣。电生理记录进一步显示功能神经元身份。从我们分化的神经元检测到的激发可以通过尼古丁增强，并抑制由肾上腺素受体拮抗剂蛋白醇。该协议中的中间交感神经祖生可以作为神经球体维持长达2周，这允许培养物的扩展。总之，与以前的版本相比，我们更新的交感神经元分化协议表现出高分化效率、更好的可重复性、更大的灵活性和更好的神经成熟度。该协议将为研究人员提供研究影响自主神经系统的人类疾病所需的细胞。

在此协议中，我们将给出有关如何从 hPSC 生成符号的说明。这里演示的培养条件从先前公布的协议23、24（图1A）到无馈送和化学定义的条件（图1B）都得到了改善。23,提供了两个选项，一个在 20 天内生成符号，另一个选项可以扩展 NcC 2 周以生成更多的单元格，然后可以分化为符号（图 1B、</st...

Watch this Scientific Journal Video about Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

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

在此协议中，我们描述了一种稳定、高效的分化策略，用于从人类多能干细胞生成后强感致感神经元。此模型将使神经元可用于多个自主障碍的研究。

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

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

该协议允许从人类多能干细胞中衍生出交感神经元。这些细胞将使科学家能够研究影响交感神经系统的人类疾病。该技术允许在分裂的无馈线满足条件下以高可扩展效率推导交感神经元。它在20天内产生电活性神经元。该技术可应用于由功能失调的交感神经系统引起的疾病，也可用于疾病建模、再生医学和药物筛查。这些细胞可以在体外用于创新联合培养系统内的任何组织，例如，用于心脏组织调节的研究。当人类多能干细胞培养达到80%至90%的汇合时，应观察具有光滑明亮边缘的大菌落。对于分裂，在用4毫升0.25摩尔EDTA在37摄氏度和5%的二氧化碳下处理细胞之前，用PBS清洗培养基。两分钟后，用 10 毫升 E8 介质冲洗板，将分离的电池悬浮液转移到 15 毫升锥形管中。然后将500微升细胞转移到9.5毫升的新鲜E8介质中。将干细胞板到涂有100毫米的新的玻璃素上。在分化感应前一天，将适当数量的六孔板涂上每孔两毫升的基底膜基质。一夜之间把盘子储存在摄氏四度。第二天早上，将盘子加热到室温，用每次洗涤的新鲜PBS洗涤两次，以分离人类多能干细胞培养。第二次洗涤后，在细胞培养培养箱中用7毫升0.5毫升EDTA治疗细胞15分钟，然后吸上漂浮的人类多能干细胞，并将收获的细胞转移到50毫升的管中。向管中加入等量的PBS，稀释EDTA，通过离心收集细胞。将颗粒重新在01天1毫升的分型介质中，然后加入适当的体积的介质进行核算。将细胞稀释到每平方厘米浓度的1.25倍至第五细胞，形成每井分型介质的毫升。从先前准备的六孔板的每个井中吸气所有基底膜基质溶液。然后将两毫升细胞放入每个井中，将盘子放入细胞培养箱中。第二天早上，每井用三毫升01日增殖中等的细胞。在分化的第二天，每井用三毫升2至10个分化介质喂养细胞，然后每隔一天喂一次细胞直到第10天。要将神经峰细胞聚合到球体中，第10天，用PBS清洗细胞。并添加两毫升的分离介质到每一个井。在细胞培养培养箱中20分钟后，将漂浮细胞转移到50毫升锥形管中，用新鲜的PBS将管中的悬浮量达到50毫升。通过离心收集细胞。并在足够的体积 10 至 14 球体介质中重新暂停颗粒以进行计数。计数后，使用第10至14天球体介质将细胞稀释至每500微升浓度的5倍至第5细胞。并板500微升的细胞进入每一个井的超低附件24井板。然后将细胞返回到细胞培养箱。为了诱导最小的球体培养，在培养的第11天，每孔每孔10至14个球体介质中，再为神经波峰球体提供500微升。第 12 天，倾斜板以在板的一侧积聚球体。并仔细吸气尽可能多的介质从每一个井，而不吸球体。然后每井用一毫升的10至14球体介质给球体喂食。为了诱导膨胀的球类培养，第15天，用第10天至14号球体介质的1.5毫升喂球体，每井补充0.5微摩尔视网膜酸。然后将球体返回到细胞培养箱。对于交感神经元分化，第 14 或 28 天，倾斜板以在板的一侧积聚球体，并仔细吸气尽可能多的介质。用一毫升的交感神经元介质喂养细胞。要将大球体聚合分解成较小的球体，每井添加不超过一毫升的介质，并在分裂前移液球体 5 到 10 次。并从先前准备的24孔板中去除多余的层压纤维素。将板状球体从 24 孔球形培养板上的每口孔中分离到新涂层 24 孔板的四个独立孔之间。在每个井中加入250微升新鲜交感神经元介质。将盘子放在细胞培养箱中。第二天早上，用1毫升的交感神经元介质代替培养剂，补充0.125微摩尔视网膜酸，将板回细胞培养箱。第35天之后，通过小心地用新鲜介质替换每个井中现有介质的一半来喂养神经元。在分化之前，人类多能干细胞菌落应该圆，边缘有光泽，光滑，很少或没有分化。细胞从第四天到第10天表达神经峰标记Sox10。神经峰细胞出现在密集的暗脊，从第六天开始可见。这些山脊也表达索克斯10。在神经波峰细胞阶段，Sox10 与细胞表面标记 CD49D 关联，该标记可用于对神经波峰细胞进行排序。正排序和未排序的种群以类似的方式产生神经波峰球状体。虽然负分细胞不能正确聚合，但不会产生圆形、平滑的健康球体，并在三到四天内死亡。此外，在第14天比较神经波峰球状体时，神经峰和交感神经祖细胞标记的定量逆转录酶PCR，无法检测到排序细胞和未分类细胞之间的显著差异。在第20天或第35天，各自的最小或扩展协议，神经ID可以观察到生长在径向模式从附加的球体。与去甲肾上腺素合成和运输有关的标记在这个阶段表达， 以及 Hox6 通过九个树干神经波峰基因。电生理记录还检测从第20天开始的神经活动。这种活性可以通过针对交感神经元的自感知器的药物增强或抑制，这些药物调整分化神经元的功能。人类的支持者自己必须健康地从零天开始分化，否则，实验可能会失败。药理抑制剂或活化剂可以添加到神经元中，以了解其正常或病理行为。这种方法为研究同情生物学和病理学开辟了一条新途径，因为现在可以很容易地从人类获得交感神经元的活检。

Research

JoVE Journal

Developmental Biology

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

从人外周T细胞无饲养条件下，使用仙台病毒诱导多能干细胞的生成

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

科研

发育生物学

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

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

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

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

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

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

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

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

高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化

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

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...