Generation of oligodendrocytes and establishment of an all-human myelinating platform from human pluripotent stem cells
Generation of oligodendrocytes and establishment of an all-human myelinating platform from human pluripotent stem cells
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DOI：
10.1038/s41596-020-0395-4
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发表时间：
2020-10-23
期刊：
影响因子：
14.8
通讯作者：
Verfaillie, Catherine M.
中科院分区：
文献类型：
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作者：
Garcia-Leon, Juan Antonio;Garcia-Diaz, Beatriz;Verfaillie, Catherine M.
少突胶质细胞（OL）负责髓磷脂的产生和神经元的代谢支持。 OL 的缺陷在多种神经退行性疾病中至关重要，包括多发性硬化症 (MS) 和肌萎缩侧索硬化症 (ALS)。该协议描述了一种在大约 20 d 内从人类多能干细胞 (hPSC) 生成少突胶质细胞前体细胞 (OPC) 的方法，随后可以在体外和体内使神经元有髓鞘化。迄今为止，OPC 已源自八种不同的 hPSC 系，包括分别源自自发性和家族性 MS 和 ALS 患者的 hPSC 系。使用编码 SOX10 的诱导型慢病毒载体转导 hPSC，在 8 天后将形成神经祖细胞。添加多西环素 10 天后，>60% 的细胞成为表达 O4 的 OPC，其中 20% 共表达成熟 OL 标记髓磷脂碱性蛋白 (MBP)。该协议还描述了病毒转导的替代方案，通过在安全港基因座 AAVS1 中掺入诱导型 SOX10，产生类似于 100% 纯 OPC 的结果。 O4(+) OPC 可以纯化并冷冻保存或用于功能研究。作为可以使用衍生细胞进行功能研究类型的一个例子，O4(+) 细胞可以与成熟的 hPSC 衍生神经元在 96/384 孔板中共培养，从而可以筛选促髓鞘形成化合物。
Oligodendrocytes (OLs) are responsible for myelin production and metabolic support of neurons. Defects in OLs are crucial in several neurodegenerative diseases including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). This protocol describes a method to generate oligodendrocyte precursor cells (OPCs) from human pluripotent stem cells (hPSCs) in only similar to 20 d, which can subsequently myelinate neurons, both in vitro and in vivo. To date, OPCs have been derived from eight different hPSC lines including those derived from patients with spontaneous and familial forms of MS and ALS, respectively. hPSCs, fated for 8 d toward neural progenitors, are transduced with an inducible lentiviral vector encoding for SOX10. The addition of doxycycline for 10 d results in >60% of cells being O4-expressing OPCs, of which 20% co-express the mature OL marker myelin basic protein (MBP). The protocol also describes an alternative for viral transduction, by incorporating an inducible SOX10 in the safe harbor locus AAVS1, yielding similar to 100% pure OPCs. O4(+) OPCs can be purified and either cryopreserved or used for functional studies. As an example of the type of functional study for which the derived cells could be used, O4(+) cells can be co-cultured with maturing hPSC-derived neurons in 96/384-well-format plates, allowing the screening of pro-myelinating compounds.