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Abstract：
Rationale: Demyelination is a major component of white matter injury, characterized by oligodendrocyte (OL) death and myelin sheath loss, which result in memory loss and cognitive impairment in the context of ischemic stroke. Accumulating evidence has shown that OLs can be generated by the direct activation of defined transcription factors (TFs) in human induced pluripotent stem cells (hiPSCs); however, the rapid acquisition of single TF-induced OL progenitor cells (OPCs) as cell therapy for ischemic stroke remains to be thoroughly explored. Methods: A stable, chemically defined protocol was used to generate a substantial number of transplantable and functional OLs through the partial inhibition of sonic hedgehog (Shh) activity by GANT61 during neural induction from hiPSCs and sequential induction of TF Olig2 overexpression. Transcriptome and metabolome analyses further revealed a novel molecular event in which Olig2 regulates OL differentiation from hiPSC-derived neural progenitor cells (NPCs). Olig2-induced NG2+ OPCs (Olig2-OPCs) were then evaluated for their therapeutic potential in cell-based therapy for ischemic stroke. Results: GANT61 treatment resulted in a motor neuron (MN)-OL fate switch during neural induction, and induced overexpression of Olig2 accelerated oligodendroglial lineage cell specification. Olig2-OPCs expressed typical oligodendroglial lineage marker genes, including NKX2.2, CSPG4, and ST8SIA1, and displayed superior ability to differentiate into mature OLs in vitro. Mechanistically, Olig2-OPCs showed increased gene expression of the peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway, and activated CEPT1-mediated phospholipogenesis. Functionally, inhibiting PPARγ and knocking down CEPT1 further compromised the terminal differentiation of Olig2-OPCs. Most importantly, when transplanted into a rat model of transient middle cerebral artery occlusion (tMCAO), Olig2-OPCs efficiently promoted neurological functional recovery by reducing neuronal death, promoting remyelination, and rescuing spatial memory decline. Conclusions: We developed a stable, chemically defined protocol to generate OPCs/OLs with partial inhibition of Shh activity by GANT61 from hiPSCs and sequentially induced the expression of the single TF Olig2. Olig2-OPC transplantation may be an ideal alternative approach for ischemic stroke rehabilitation therapy.
摘要：
原理：脱髓鞘是白质损伤的主要组成部分，以少突胶质细胞（OL）死亡和髓鞘丢失为特征，导致缺血性卒中的记忆丧失和认知障碍。越来越多的证据表明，人诱导多能干细胞（hiPSC）中确定的转录因子（TFs）的直接激活可以产生OLs；然而，快速获取单个TF诱导的OL祖细胞(OPCs)作为缺血性卒中的细胞疗法仍有待深入探索.方法：稳定，化学定义的方案用于通过GANT61在从hiPSC的神经诱导和TFOlig2过表达的顺序诱导过程中部分抑制Sonichedgehog（Shh）活性来产生大量可移植和功能性OLs。转录组和代谢组分析进一步揭示了一个新的分子事件，其中Olig2调节从hiPSC衍生的神经祖细胞（NPC）的OL分化。然后评估Olig2诱导的NG2+OPC(Olig2-OPC)在缺血性卒中的基于细胞的治疗中的治疗潜力。结果：GANT61治疗在神经诱导过程中导致运动神经元（MN）-OL命运转换，并诱导Olig2的过表达加速了少突胶质细胞谱系的规范。Olig2-OPCs表达典型的少突胶质谱系标记基因，包括NKX2.2，CSPG4和ST8SIA1，并且在体外表现出优异的分化为成熟OLs的能力。机械上,Olig2-OPCs显示过氧化物酶体增殖物激活受体γ（PPARγ）信号通路的基因表达增加，和激活CEPT1介导的磷脂生成。功能上,抑制PPARγ和敲低CEPT1进一步损害了Olig2-OPCs的终末分化。最重要的是,当移植到短暂性大脑中动脉闭塞（tMCAO）的大鼠模型中时，Olig2-OPCs通过减少神经元死亡有效促进神经功能恢复，促进髓鞘再生，拯救空间记忆衰退。结论：我们开发了一种稳定的，化学定义的方案，以从hiPSC产生GANT61部分抑制Shh活性的OPCs/OLs，并依次诱导单个TFOlig2的表达。Olig2-OPC移植可能是缺血性中风康复治疗的理想替代方法。
