Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Human Pluripotent Cell Facility, Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Development. 2018 Jul 12;145(16):dev166215. doi: 10.1242/dev.166215.
Robust protocols for directed differentiation of human pluripotent cells are required to determine whether mechanisms operating in model organisms are relevant to our own development. Recent work in vertebrate embryos has identified neuromesodermal progenitors as a bipotent cell population that contributes to paraxial mesoderm and spinal cord. However, precise protocols for differentiation of human spinal cord progenitors are lacking. Informed by signalling in amniote embryos, we show here that transient dual-SMAD inhibition, together with retinoic acid (dSMADi-RA), provides rapid and reproducible induction of human spinal cord progenitors from neuromesodermal progenitor-like cells. Using CRISPR-Cas9 to engineer human embryonic stem cells with a GFP-reporter for neuromesodermal progenitor-associated gene we facilitate selection of this cell population. RNA-sequencing was then used to identify human and conserved neuromesodermal progenitor transcriptional signatures, to validate this differentiation protocol and to reveal new pathways/processes in human neural differentiation. This optimised protocol, novel reporter line and transcriptomic data are useful resources with which to dissect molecular mechanisms regulating human spinal cord generation and allow the scaling-up of distinct cell populations for global analyses, including proteomic, biochemical and chromatin interrogation.
需要稳健的定向分化人类多能细胞的方案来确定在模式生物中起作用的机制是否与我们自身的发育相关。最近在脊椎动物胚胎中的研究工作已经确定神经中胚层祖细胞是一种双能细胞群体，可分化为轴旁中胚层和脊髓。然而，缺乏用于分化人类脊髓祖细胞的精确方案。受羊膜动物胚胎中信号的启发，我们在这里表明，短暂的双重 SMAD 抑制，再加上视黄酸（dSMADi-RA），可快速且可重复地诱导神经中胚层祖细胞样细胞分化为人类脊髓祖细胞。我们使用 CRISPR-Cas9 工程技术对人类胚胎干细胞进行基因改造，使其带有 GFP 报告基因，用于神经中胚层祖细胞相关基因，从而有助于选择该细胞群体。然后进行 RNA 测序，以鉴定人类和保守的神经中胚层祖细胞转录特征，验证这种分化方案，并揭示人类神经分化中的新途径/过程。该优化方案、新型报告细胞系和转录组数据是有用的资源，可用于解析调节人类脊髓生成的分子机制，并允许对不同的细胞群体进行扩大规模，以进行包括蛋白质组学、生物化学和染色质探测在内的全面分析。
