Data from: Comparison and optimization of hiPSC forebrain cortical differentiation protocols

Several protocols have been developed for human induced pluripotent stem cell neuronal differentiation. We compare several methods for forebrain cortical neuronal differentiation by assessing cell morphology, immunostaining and gene expression. We evaluate embryoid aggregate vs. monolayer with dual SMAD inhibition differentiation protocols, manual vs. AggreWell aggregate formation, plating substrates, neural progenitor cell (NPC) isolation methods, NPC maintenance and expansion, and astrocyte co-culture. The embryoid aggregate protocol, using a Matrigel substrate, consistently generates a high yield and purity of neurons. NPC isolation by manual selection, enzymatic rosette selection, or FACS all are efficient, but exhibit some differences in resulting cell populations. Expansion of NPCs as neural aggregates yields higher cell purity than expansion in a monolayer. Finally, co-culture of iPSC-derived neurons with astrocytes increases neuronal maturity by day 40. This study directly compares commonly employed methods for neuronal differentiation of iPSCs, and can be used as a resource for choosing between various differentiation protocols.

目前已开发出多种用于人类诱导多能干细胞（induced pluripotent stem cell, iPSC）神经元分化的实验方案。本研究通过评估细胞形态、免疫染色与基因表达水平，对多种前脑皮层神经元分化方法展开对比。本次评估涵盖多组对比项：采用双SMAD抑制策略的拟胚体聚集培养与单层培养分化方案、手工聚集与AggreWell聚集形成方式、细胞接种底物、神经前体细胞（neural progenitor cell, NPC）分离方法、NPC的维持与扩增策略，以及星形胶质细胞共培养体系。采用基质胶（Matrigel）作为底物的拟胚体聚集培养方案，可稳定获得高产率、高纯度的神经元。通过手工挑选、酶法玫瑰簇筛选或荧光激活细胞分选（fluorescence-activated cell sorting, FACS）开展NPC分离，均具备较高分离效率，但所得细胞群体存在一定差异。以神经聚集形式扩增NPC，所得细胞纯度高于单层培养扩增方式。最后，将iPSC来源的神经元与星形胶质细胞共培养，可在培养至第40天时提升神经元的成熟程度。本研究直接对比了当前常用的iPSC神经元分化方法，可为研究者在各类分化方案中进行选择提供参考依据。