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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢海蒂·乌尔里希斯对手稿的批判性阅读和编辑。
Materials
| 100 mm cell culture dishes | Falcon | 353003 | |
| 15 mL conical tissue culture tubes | VWR/Corning | 89039-664 | |
| 24-well tissue culture plates | Falcon | 353047 | |
| 24-well ultra-low-attachment plates | Corning | 07 200 601 and 07 200 602 | |
| 5% CO2/20% O2 tissue culture incubator | Thermo Fisher/Life Technologies | Heracell VIOS 160i | |
| 50 ml conical tissue culture tubes | VWR/Corning | 89039-656 | |
| 6-well tissue culture plates | Costar | 3516 | |
| Accutase | Innovation Cell Technologies | AT104500 | Cell dissociation solution |
| Anti-AP2a antibody | Abcam | ab108311 | Host: Rabbit; 1:400 dilution |
| Anti-Ascl1 antibody | BD Pharmingen | 556604 | Host: Mouse IgG1; 1:200 dilution |
| Anti-CD49D antibody | BioLegend | 304313 | Host: Mouse IgG1; 5 μl/million cells in 100 μl volume |
| Anti-CD49D antibody (isotype) | BioLegend | 400125 | Host: Mouse IgG1; 5 μl/million cells in 100 μl volume |
| Anti-DAPI antibody | Sigma | D9542 | 1:1000 dilution |
| Anti-DBH antibody | Immunostar | 22806 | Host: Rabbit; 1:500 dilution |
| Anti-GFP antibody | Abcam | ab13970 | Host: Chicken; 1:1000 dilution |
| Anti-HOXC9 antibody | Santa Cruz Biotechnology | sc-365692 | Host: Mouse IgG1; 1:100 dilution |
| Anti-NET1 antibody | Mab | NET17-1 | Host: Mouse; 1:1000 dilution |
| Anti-PRPH antibody | Santa Cruz Biotechnology | SC-377093/H0112 | Host: Mouse IgG2a; 1:200 dilution |
| Anti-SOX10 antibody | Abcam | ab50839 | Host: Mouse; 1:100 dilution |
| Anti-TH antibody | Pel-Freez | P40101- 150 | Host: Rabbit; 1:500 dilution |
| Ascorbic acid | Sigma | A8960-5G | Stock concentration: 100 mM |
| B27 supplement | Thermo Fisher/Life Technologies | 12587-010 | Stock concentration: 50x |
| BDNF | R&D Systems | 248-BD | Stock concentration: 10 μg/mL |
| BMP4 | R&D Systems | 314-BP | Stock concentration: 6 mM |
| Cell counter | Thermo Fisher/Life Technologies | Countess II | |
| Cell counting chamber slides | Invitrogen | C10312 | |
| Centrifuge | Eppendorf | 57021&5424R | |
| CHIR99021 | R&D Systems | 4423 | Stock concentration: 6 mM |
| Cryo-vial | Thermo Fisher/Life Technologies | 375353 | |
| dbcAMP | Sigma | D0627 | Stock concentration: 100 mM |
| DMEM | Thermo Fisher/Life Technologies | 10829-018 | Stock concentration: 1x |
| DMEM/F12 | Thermo Fisher/Life Technologies | 11330-057 | Stock concentration: 1x |
| DMSO | Thermo Fisher/Life Technologies | BP231-100 | |
| E6 medium | gibco | A15165-01 | |
| E8 medium | gibco | A15169-01 | Stock concentration: 1x |
| E8 supplement | gibco | A15171-01 | Stock concentration: 50x |
| EDTA | Sigma | ED2SS | Stock concentration: 0.5 M |
| Electrophysiology plates (AXION cytoview MEA96) | Axion BioSystems | M768-tMEA-96W | |
| FACS machine | Beckman Coulter | CytoFLEX (for FACS) | |
| FACS machine | Beckman Coulter | MoFlo Astrios EQ (for sorting) | |
| FACS tubes (blue filter cap) | Falcon | 352235 | |
| FACS tubes (white cap) | Falcon | 352063 | |
| Fetal bovine serum (FBS) | Atlanta Biologicals | S11150 | |
| GDNF | PeproTech | 450 | Stock concentration: 10 μg/mL |
| Geltrex | Invitrogen | A1413202 | Basement membrane matrix; Stock concentration: 100x |
| hPSCs | Thomson et al., (1998) | WA09 | |
| hPSCs | Oh et al. (2016) | H9-PHOX2B::eGFP | |
| Human fibronectin (FN) | VWR/Corning | 47743-654 | Stock concentration: 1 mg/mL |
| L-glutamine | Thermo Fisher/Gibco | 25030-081 | Stock concentration: 200 mM |
| LN tank | Custom Biogenic Systems | V-1500AB | |
| MEA reader | Axion BioSystems | Maestro Pro | |
| Mouse laminin I (LM) | R&D Systems | 3400-010-01 | Stock concentration: 1 mg/mL |
| N2 supplement | Thermo Fisher/Life Technologies | 17502-048 | Stock concentration: 100x |
| Neurobasal medium | gibco | 21103-049 | Stock concentration: 1x |
| NGF | PeproTech | 450-01 | Stock concentration: 25 μg/mL |
| Phosphate-buffered saline (PBS) | Gibco | 14190-136 | Stock concentration: 1x |
| Poly-L-ornithine hydrobromide (PO) | Sigma | P3655 | Stock concentration: 15 mg/mL |
| Primocin (antibiotics) | InvivoGen | ANTPM1 | Stock concentration: 50 mg/mL |
| qPCR machine | Bio-Rad Laboratories | C1000 Touch | |
| qPCR plates | Bio-Rad Laboratories | HSP9601 | |
| recombinant FGF2 | R&D Systems | 233-FB/CF | Stock concentration: 10 μg/mL |
| Retinoic acid | Sigma | R2625 | Stock concentration: 1 mM |
| SB431542 | Tocris/R&D Systems | 1614 | Stock concentration: 10 mM |
| Trypan blue | Corning | MT-25-900-CI | |
| Vitronectin (VTN) | Thermo Fisher/Life Technologies | A14700 | Stock concentration: 0.5 mg/mL |
| Water bath | VWR/Corning | 706308 | |
| Y27632 | R&D Systems | 1254 | Stock concentration: 10 mM |
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