Human induced pluripotent stem cell (hiPSC)-derived neural cells provide valuable disease models for pathophysiological analysis and drug discovery for intractable neurodegenerative diseases. However, neural differentiation of hiPSCs requires a complex and long culture procedure, which has been a bottleneck for analysis. We previously demonstrated rapid, efficient and simple motor neuron differentiation from human pluripotent stem cells (hPSCs). Although optimization of the microenvironment for the differentiation of hPSCs has been considered to achieve more efficient differentiation, it has never been investigated in detail. Here, we demonstrated that three microenvironmental modifiers, oxygen (O2) tension, pH, and cell density, critically affect neural differentiation of hiPSCs. Hypoxia is known to be involved in neural development in vivo and to promote neural differentiation of PSCs. However, in this study, it caused significant cell death in aggregation culture of human embryoid bodies (hEBs) and negatively affected neural differentiation. Modulation of pH by optimized carbon dioxide (CO2) tension improved neural differentiation of hiPSCs, but mild acidosis caused by increased CO2 tension suppressed neural differentiation without cell death. Moreover, high-cell density culture resulted in prominent acidosis and cell death under hypoxic conditions, which synergistically suppressed neural differentiation of hiPSCs. These results suggest that optimization of the microenvironment via O2 tension, pH, and cell density enables more efficient neural differentiation of hiPSCs for the analysis of neurological diseases.

中文翻译：

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氧分压、酸中毒和细胞密度的调节对于人诱导多能干细胞的神经分化至关重要

人类诱导多能干细胞 (hiPSC) 衍生的神经细胞为难治性神经退行性疾病的病理生理学分析和药物发现提供了有价值的疾病模型。然而，hiPSCs 的神经分化需要复杂而漫长的培养过程，这一直是分析的瓶颈。我们之前证明了人类多能干细胞 (hPSC) 的快速、高效和简单的运动神经元分化。尽管优化用于 hPSC 分化的微环境已被认为可以实现更有效的分化，但从未对其进行详细研究。在这里，我们证明了三种微环境调节剂、氧 (O2) 张力、pH 值和细胞密度，严重影响了 hiPSC 的神经分化。已知缺氧参与体内神经发育并促进 PSC 的神经分化。然而，在这项研究中，它在人胚状体 (hEB) 的聚集培养中引起了显着的细胞死亡，并对神经分化产生了负面影响。通过优化二氧化碳 (CO2) 张力调节 pH 值改善了 hiPSC 的神经分化，但由二氧化碳张力增加引起的轻度酸中毒抑制了神经分化，而没有细胞死亡。此外，高细胞密度培养在缺氧条件下导致显着的酸中毒和细胞死亡，这协同抑制了 hiPSC 的神经分化。这些结果表明，通过 O2 张力、pH 值和细胞密度优化微环境能够更有效地对 hiPSC 进行神经分化，以分析神经系统疾病。