Temporal and partial inhibition of GLI1 in neural stem cells (NSCs) results in the early maturation of NSC derived oligodendrocytes in vitro
Temporal and partial inhibition of GLI1 in neural stem cells (NSCs) results in the early maturation of NSC derived oligodendrocytes in vitro
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DOI：
10.1186/s13287-019-1374-y
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发表时间：
2019-08-27
影响因子：
7.5
通讯作者：
Deng, Wenbin
中科院分区：
文献类型：
--
作者：
Namchaiw, Poommaree;Wen, Han;Deng, Wenbin
背景少突胶质细胞是一种合成轴突周围髓鞘的胶质细胞，对中枢神经系统的神经传导至关重要。少突胶质细胞死亡和缺陷是几种髓鞘疾病的主要原因，如多发性硬化症、进行性多灶性白质脑病、脑室周围白质软化症和几种白质营养不良。在哺乳动物胚胎发育过程中，Sonic Hedgehog （SHH）通路的时间激活对于少突胶质细胞祖细胞的产生及其在脑和脊髓中的分化和成熟至关重要。方法在SOX2/ pax6阳性神经干细胞（NSCs）形成过程中，利用人诱导多能干细胞（iPSC）和人胚胎干细胞（hESCs）的粘附培养，将绿色荧光蛋白（GFP）报告基因敲入OLIG2基因位点的一个等位基因，双SMAD抑制和小分子GANT61对胶质瘤相关癌基因1 （GLI1）的短暂部分抑制。SHH通路后来被一种平滑的激动剂氨基吗啡重新刺激，以诱导OLIG2胶质前体的产生。采用单细胞RNA测序（RNA- seq）对GANT61和对照组192个少突胶质前体细胞（OPCs）进行分析。我们在这里证明，小分子抑制剂GANT61对NSCs中SHH通路转录因子GLI1的短暂和部分抑制可以产生更具迁移性的OPCs，并且可以更早地向产生髓磷脂的少突胶质细胞分化。单细胞转录组学分析（RNA-Seq）显示，gant61 - nsc来源的少突胶质前体细胞（OPCs）在细胞骨架重排途径中具有一些基因的差异激活，这些基因参与了OPC的运动和诱导成熟。在蛋白质水平上，与对照组相比，GANT61组的髓磷脂特异性基因水平也较高。gant61 - nsc来源的OPCs在髓磷脂缺乏的震颤小鼠体内和体外移植后均能产生致密髓磷脂。结论这是一种基于小分子的体外培养方案，可以更快地生成功能性少突胶质细胞。少突胶质细胞从祖细胞中高效、快速分化的方案的发展，为利用人类多能干细胞开发基于自体神经干细胞的治疗方法提供了重要进展。
BackgroundOligodendrocytes are a type of glial cells that synthesize the myelin sheath around the axons and are critical for the nerve conduction in the CNS. Oligodendrocyte death and defects are the leading causes of several myelin disorders such as multiple sclerosis, progressive multifocal leukoencephalopathy, periventricular leukomalacia, and several leukodystrophies. Temporal activation of the Sonic Hedgehog (SHH) pathway is critical for the generation of oligodendrocyte progenitors, and their differentiation and maturation in the brain and spinal cord during embryonic development in mammals.MethodsOur protocol utilized adherent cultures of human induced pluripotent stem cells (iPSC) and human embryonic stem cells (hESCs) with a green fluorescent protein (GFP) reporter knocked into one allele of the OLIG2 gene locus, dual SMAD inhibition, and transient partial inhibition of glioma-associated oncogene 1 (GLI1) by the small molecule GANT61 during the formation of the SOX2/PAX6-positive neural stem cells (NSCs). The SHH pathway was later restimulated by a Smoothened agonist purmorphamine to induce the generation of OLIG2 glial precursors. One hundred ninety-two individual oligodendrocyte precursor cells (OPCs) from GANT61 and control group were analyzed by single-cell RNA sequencing (RNA-Seq).ResultsWe demonstrate here that transient and partial inhibition of the SHH pathway transcription factor GLI1 in NSCs by a small molecule inhibitor GANT61 was found to generate OPCs that were more migratory and could differentiate earlier toward myelin-producing oligodendrocytes. Single-cell transcriptomic analysis (RNA-Seq) showed that GANT61-NSC-derived oligodendrocyte precursor cells (OPCs) had differential activation of some of the genes in the cytoskeleton rearrangement pathways that are involved in OPC motility and induction of maturation. At the protein level, this was also associated with higher levels of myelin-specific genes in the GANT61 group compared to controls. GANT61-NSC-derived OPCs were functional and could generate compact myelin in vitro and in vivo after transplantation in myelin-deficient shiverer mice.ConclusionsThis is a small molecule-based in vitro protocol that leads to the faster generation of functional oligodendrocytes. The development of protocols that lead to efficient and faster differentiation of oligodendrocytes from progenitors provides important advances toward the development of autologous neural stem cell-based therapies using human iPSCs.