Developmental Biology Program, Sloan-Kettering Institute, New York, NY, USA.
Nat Protoc. 2010 Apr;5(4):688-701. doi: 10.1038/nprot.2010.35. Epub 2010 Mar 18.
Human pluripotent stem cell (hPSC)-derived neural crest (NC) cells present a valuable tool for modeling aspects of human NC development, including cell fate specification, multipotency and cell migration. hPSC-derived NC cells are also suitable for modeling human disease and as a renewable cell source for applications in regenerative medicine. Here we provide protocols for the step-wise differentiation of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into neuroectodermal and NC cells using either the MS5 coculture system or a novel defined culture method based on pharmacological inhibition of bone morphogenetic protein and transforming growth factor-beta signaling pathways. Furthermore, we present protocols for the purification and propagation of hPSC-NC cells using flow cytometry and defined in vitro culture conditions. Our protocol has been validated in multiple independent hESC and hiPSC lines. The average time required for generating purified hPSC-NC precursors using this protocol is 2-5 weeks.
人多能干细胞（hPSC）衍生的神经嵴（NC）细胞为模拟人类 NC 发育的各个方面提供了有价值的工具，包括细胞命运特化、多能性和细胞迁移。hPSC 衍生的 NC 细胞也适合用于模拟人类疾病，并作为可再生细胞来源，用于再生医学应用。在这里，我们提供了使用 MS5 共培养系统或基于骨形态发生蛋白和转化生长因子-β信号通路的药理学抑制的新型定义培养方法，逐步将人胚胎干细胞（hESC）或人诱导多能干细胞（hiPSC）分化为神经外胚层和 NC 细胞的方案。此外，我们还提供了使用流式细胞术和定义的体外培养条件纯化和扩增 hPSC-NC 细胞的方案。我们的方案已经在多个独立的 hESC 和 hiPSC 系中得到验证。使用此方案生成纯化的 hPSC-NC 前体所需的平均时间为 2-5 周。
