利用人类胚胎干细胞的单细胞培养进行高效神经分化
Summary
这里介绍的是人类胚胎干细胞单细胞培养及其随后分化为神经祖细胞的一个方案。该协议简单、可靠、可扩展，适用于药物筛选和再生医学应用。
Abstract
人类胚胎干细胞（hESCs）的体外分化改变了研究人体在生物学和分子水平上发育的能力，并为再生应用提供了用于再生的细胞。hESC 培养的标准方法使用菌落类型培养来保持未分化的 hESC 和胚胎体 （EB） 和玫瑰形形成，以分化成不同的胚层，效率低且耗时。此处介绍的是使用 hESC 而不是殖民地类型的区域性的单细胞培养方法。此方法允许维护未分化 hESC 的特征，包括与菌群类型 hESC 相媲美的 hESC 标记的表达。此外，该协议还提出了一种从单细胞型hESC生成神经祖细胞（NPC）的有效方法，在1周内产生NPC。这些细胞高度表达几个NPC标记基因，并可以分化成各种神经细胞类型，包括多巴胺能神经元和星形细胞。这种用于hESC的单细胞培养系统将有助于研究这些过程的分子机制、某些疾病的研究以及药物发现屏幕。
Introduction
人类胚胎干细胞（hESCs）有可能分化成三种主要生殖层，然后分化成各种多能的祖细胞系。这些血统随后引起人体的所有细胞类型。体外hESC培养系统已经改变了研究人类胚胎发育的能力，并已成为获得关于如何在生物和分子水平上对这些过程进行调控的新见解的宝贵工具。同样，从人类患者中分离出来的诱导多能干细胞（iPSCs）的研究提供了对各种疾病的新见解。此外，从hESCs衍生的祖细胞和分化细胞可用于干细胞治疗和药物筛选1、2、3、4的研究。
hESCs 可以诱导分化成神经祖细胞 （NPC），这是具有广泛自我更新能力的多电位细胞。随后，这些细胞可以分化成神经元、星形细胞和寡核苷酸细胞5，6。NPC还为神经发育生物学和各种神经系统疾病的体外研究提供了一个细胞系统。然而，目前涉及hESC及其分化为NPC的菌群培养方法效率不高，往往涉及共栽以及胚胎体（EB）和玫瑰花形成5、7、8、9。这些协议表现出较低的存活率和自发分化，并且更耗时。
这里介绍的是一个改进和强大的培养系统，易于扩展，并使用高密度单细胞类型培养hESCs10。在hESC10、11、12、13、14的单细胞类型培养中，加入Roh-激酶（ROCK）抑制剂有助于显著提高生存效率。在此文化系统中，hESC 可以轻松维护和扩展。此外，该议定书还提出了一种从hESCs的单细胞类型培养物生成NPC的有效方法，它允许产生高度纯的NPC。 用ALK抑制剂抑制BMP/TGF+/activin信号通路，有效地诱导单细胞型hESC分化为NPC15、16，然后可以诱导分化成功能神经谱系，如多巴胺能神经元和星形。
总之，使用hESCs的单细胞类型培养协议为研究这些细胞分化为包括NPC在内的各种谱系提供了一个有吸引力的模型。该协议易于扩展，因此适合生成细胞，用于涉及再生治疗和药物筛选的研究。
Protocol
1. 制备符合hESC资格的地下室膜基质涂层板 在4°C下缓慢解冻hESC认证的基底膜基质（见材料表）溶液至少2~3小时或过夜，以避免形成凝胶。 要制备基底膜基质涂层板，请将冷 DMEM/F12 中的基质稀释至 2% 的最终浓度。混合好，用1 mL的稀释基质溶液涂覆6孔板。 在室温 （RT） 下孵育基质涂层板至少 3 小时或 4°C 过夜。注：在去除基质溶液和使用板之前，具有基底?…
Representative Results
这里介绍的是一个改进的协议，用于维护和扩展hESC的单细胞类型培养及其有效分化为神经祖细胞，后者随后分化成各种下游神经谱系，包括多巴胺能神经元和星形细胞。 代表性相位对比图像显示细胞形态在适应单细胞类型培养过程中不同步骤的细胞形态（图1A）。通过单细胞培养条件，发现适应的hESC能够维持在高密度，然后在达到汇合时?…
Discussion
将hESCs分化成各种谱系和产生足够数量的分化细胞的可扩展和有效方法是药物筛选和干细胞治疗的重要标准。已经发表了各种单细胞传递方法，其中细胞在ROCK抑制剂或其他小分子的存在下培养，以提高存活率，但这些培养方法的最终产物是菌群型hESCs17，18，19，20，21。单细?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢卡尔·博尔特纳博士（NIEHS）协助FACS分析。这项研究得到了国家环境卫生科学研究所、国家卫生研究院、Z01-ES-101585至AMJ的校内研究计划的支持。
Materials
| 35 mm m-dishes | ibidi | 81156 | Cell culture dish |
| 6-well plates | Corning | 3516 | |
| Accutase | Innovative Cell Technologies | AT104-500 | Cell detachment solution |
| Activin A | R&D system | 338-AC-050 | |
| Ascorbic Acid | Sigma Aldrich | A4403 | |
| B27 supplement | Thermo Fisher | 17504044 | |
| B27 supplement (-Vit A) | Thermo Fisher | 12587010 | |
| BDNF | Applied Biological Materials | Z100065 | |
| bFGF | Peprotech | 100-18C | |
| Centrifuge | DAMON/ICE | 428-6759 | |
| CO2 incubator | Thermo Fisher | 4110 | |
| Corning hESC-qulified Matrix (Magrigel) | Corning | 354277 | Basement membrane matrix (used for most of the protocol here) |
| Cryostor CS 10 | Stemcell Technologies | 7930 | Cell freezing solution |
| Dispase | Stemcell Technologies | 7923 | |
| DMEM | Thermo Fisher | 10569-010 | |
| DMEM/F12 | Thermo Fisher | 10565-018 | |
| Dorsomorphin | Tocris | 3093 | |
| EGF | Peprotech | AF-100-16A | |
| Fetal Bovine Serum | Fisher Scientific | SH3007003HI | |
| FGF8 | Applied Biological Materials | Z101705 | |
| GDNF | Applied Biological Materials | Z101057 | |
| Geltrex matrix | Thermo Fisher | A1569601 | Basement membrane matrix |
| GlutaMax | Thermo Fisher | 35050061 | Glutamine supplement, 100X |
| H9 (WA09) human embryonic stem cell line | WiCell | WA09 | |
| Heregulin b-1 | Peprotech | 100-3 | |
| IGF | Peprotech | 100-11 | |
| Knockout DMEM | Thermo Fisher | 10829018 | |
| Knockout Serum Replacement | Thermo Fisher | 10828028 | |
| Laminin | Sigma Aldrich | L2020 | |
| mTeSR1 | Stemcell Technologies | 85850 | hESC culture medium |
| N2 supplement | Thermo Fisher | 17502001 | |
| NEAA | Thermo Fisher | 11140050 | |
| Neurobasal | Thermo Fisher | 21103049 | |
| Poly-L-ornithine | Sigma Aldrich | P3655 | |
| ROCK inhibitor | Tocris | 1254 | |
| SB431542 | Tocris | 1614 | |
| SHH | Applied Biological Materials | Z200617 | |
| Stemdiff Neural Progenitor medium | Stemcell Technologies | 5833 | NPC expansion medium |
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Cite This Article
Jeon, K., Park, K., Jetten, A. M. Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells. J. Vis. Exp. (155), e60571, doi:10.3791/60571 (2020).