Human embryonic stem cells (hESCs) are pluripotent cells, capable of differentiation into different cellular lineages given the opportunity. Derived from the inner cell mass of blastocysts in early embryonic development, the cell self-renewal ability makes them a great tool for regenerative medicine, and there are different protocols available for maintaining hESCs in their undifferentiated state. In addition, protocols for differentiation into functional human neural stem cells (hNSCs), which have the potential for further differentiation into various neural cell types, are available. However, many protocols are time-consuming and complex and do not always fit for purpose. In this study, we carefully combined, optimized, and developed protocols for differentiation of hESCs into adherent monolayer hNSCs over a short period of time, with the possibility of both expansion and freezing. Moreover, the method details further differentiation into neurons, cholinergic neurons, and glial cells in a simple, single step by step protocol. We performed immunocytochemistry, qPCR, and electrophysiology to examine the expression profile and characteristics of the cells to verify cell lineage. Using presented protocols, the creation of neuronal cultures, cholinergic neurons, and a mixed culture of astrocytes and oligodendrocytes can be completed within a three-week time period.

中文翻译：

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人胚干细胞向神经元，胆碱能和神经胶质细胞的分化

人胚胎干细胞（hESCs）是多能细胞，只要有机会，便能够分化为不同的细胞谱系。细胞的自我更新能力源自胚胎早期发育过程中的胚泡内部细胞团，使其成为再生医学的重要工具，并且有多种方案可将hESC维持在未分化状态。此外，还提供了用于分化为功能性人类神经干细胞（hNSC）的协议，这些协议具有进一步分化为各种神经细胞类型的潜力。但是，许多协议既耗时又复杂，并不总是适合目的。在这项研究中，我们精心组合，优化和开发了用于在短时间内将hESC分化为粘附性单层hNSC的方案，可能会膨胀和冻结。此外，该方法通过简单，一步一步的步骤详细介绍了进一步分化为神经元，胆碱能神经元和神经胶质细胞的方法。我们进行了免疫细胞化学，qPCR和电​​生理检查细胞的表达谱和特征，以验证细胞谱系。使用提出的协议，可以在三周的时间内完成神经元培养物，胆碱能神经元以及星形胶质细胞和少突胶质细胞混合培养的创建。电生理检查细胞的表达情况和特征以验证细胞谱系。使用提出的协议，可以在三周的时间内完成神经元培养物，胆碱能神经元以及星形胶质细胞和少突胶质细胞混合培养的创建。电生理检查细胞的表达情况和特征以验证细胞谱系。使用提出的协议，可以在三周的时间内完成神经元培养物，胆碱能神经元以及星形胶质细胞和少突胶质细胞混合培养的创建。