Centre for Gene Therapy and Regenerative Medicine, Centre for Developmental Neurobiology, MRC Centre for Neurodevelopmental Disorders, King's College London, 28th Floor Tower Wing, Guy's Campus, Great Maze Pond, London, SE1 9RT, UK.
The Wolfson Centre for Age-Related Diseases, King's College London, London, UK.
Sci Rep. 2023 Feb 3;13(1):2008. doi: 10.1038/s41598-023-29165-z.
V3 spinal interneurons are a key element of the spinal circuits, which control motor function. However, to date, there are no effective ways of deriving a pure V3 population from human pluripotent stem cells. Here, we report a method for differentiation and isolation of spinal V3 interneurons, combining extrinsic factor-mediated differentiation and magnetic activated cell sorting. We found that differentiation of V3 progenitors can be enhanced with a higher concentration of Sonic Hedgehog agonist, as well as culturing cells in 3D format. To enable V3 progenitor purification from mixed differentiation cultures, we developed a transgene reporter, with a part of the regulatory region of V3-specific gene Nkx2-2 driving the expression of a membrane marker CD14. We found that in human cells, NKX2-2 initially exhibited co-labelling with motor neuron progenitor marker, but V3 specificity emerged as the differentiation culture progressed. At these later differentiation timepoints, we were able to enrich V3 progenitors labelled with CD14 to ~ 95% purity, and mature them to postmitotic V3 interneurons. This purification tool for V3 interneurons will be useful for in vitro disease modeling, studies of normal human neural development and potential cell therapies for disorders of the spinal cord.
V3 脊髓中间神经元是控制运动功能的脊髓回路的关键组成部分。然而，迄今为止，尚无有效的方法从人类多能干细胞中获得纯 V3 群体。在这里，我们报告了一种结合外源性因子介导的分化和磁激活细胞分选从人多能干细胞中分离 V3 中间神经元的方法。我们发现，V3 前体细胞的分化可以通过提高 Sonic Hedgehog 激动剂的浓度以及在 3D 培养格式中培养细胞来增强。为了能够从混合分化培养物中纯化 V3 前体细胞，我们开发了一种转基因报告基因，其 V3 特异性基因 Nkx2-2 的一部分调节区驱动膜标记物 CD14 的表达。我们发现，在人细胞中，NKX2-2 最初与运动神经元祖细胞标记物共标记，但随着分化培养的进行，出现了 V3 特异性。在这些较晚的分化时间点，我们能够将 CD14 标记的 V3 前体细胞富集到~95%的纯度，并将其成熟为有丝分裂后 V3 中间神经元。这种 V3 中间神经元的纯化工具将有助于体外疾病建模、正常人类神经发育的研究以及脊髓疾病的潜在细胞治疗。
