Highly Pure and Expandable PSA-NCAM-Positive Neural Precursors from Human ESC and iPSC-Derived Neural Rosettes
Highly Pure and Expandable PSA-NCAM-Positive Neural Precursors from Human ESC and iPSC-Derived Neural Rosettes
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DOI：
10.1371/journal.pone.0039715
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发表时间：
2012-07-20
期刊：
影响因子：
3.7
通讯作者：
Kim, Dong-Wook
中科院分区：
文献类型：
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作者：
Kim, Dae-Sung;Lee, Dongjin R.;Kim, Dong-Wook
源自人多能干细胞（hPSCs）的神经前体细胞（NPCs）的同质培养将为生物医学应用提供有力工具。然而，先前为培养NPCs而对机械分离的神经花环进行扩增的尝试，仍然存在非神经细胞污染的问题。此外，一些利用细胞表面标志物纯化NPCs的尝试尚未证明分选细胞的扩增能力。在本研究中，我们发现多聚唾液酸 - 神经细胞黏附分子（PSA - NCAM）在源自hPSCs的神经花环细胞中可被检测到，并将PSA - NCAM用作从神经花环中纯化可扩增的原始NPCs的标志物。使用基于磁性的细胞分选技术，从机械收获的神经花环的异质细胞群中分离出PSA - NCAM阳性的NPCs（称为hNPC(PSA - NCAM+)）。hNPC(PSA - NCAM+)广泛表达神经标志物，如Sox1、Sox2、巢蛋白和Musashi - 1（占总细胞的80% - 98%），并且在我们的培养条件下能够传代多次，同时保留其原始特性。有趣的是，PSA - NCAM阴性细胞在很大程度上表现出神经嵴细胞的特征。hNPC(PSA - NCAM+)在体外显示出多能性以及对区域特异性神经元亚型的指导性信号的响应能力。当移植到大鼠纹状体中时，hNPC(PSA - NCAM+)分化为神经元、星形胶质细胞和少突胶质细胞，没有明显的肿瘤发生迹象。此外，与神经花环细胞的移植物相比，在hNPC(PSA - NCAM+)的移植物中很少检测到Ki67阳性的增殖细胞和非神经谱系细胞。我们的结果表明，PSA - NCAM介导的细胞分离从hPSCs中提供了一种高度可扩增的纯原始NPCs群体，这将成为神经退行性疾病药物筛选和细胞治疗的一种有前景的策略。
Homogeneous culture of neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) would provide a powerful tool for biomedical applications. However, previous efforts to expand mechanically dissected neural rosettes for cultivation of NPCs remain concerns regarding non-neural cell contamination. In addition, several attempts to purify NPCs using cell surface markers have not demonstrated the expansion capability of the sorted cells. In the present study, we show that polysialic acid-neural cell adhesion molecule (PSA-NCAM) is detected in neural rosette cells derived from hPSCs, and employ PSA-NCAM as a marker for purifying expandable primitive NPCs from the neural rosettes. PSA-NCAM-positive NPCs (termed hNPC(PSA-NCAM+)) were isolated from the heterogeneous cell population of mechanically harvested neural rosettes using magnetic-based cell sorting. The hNPC(PSA-NCAM+) extensively expressed neural markers such as Sox1, Sox2, Nestin, and Musashi-1 (80 similar to 98% of the total cells) and were propagated for multiple passages while retaining their primitive characteristics in our culture condition. Interestingly, PSA-NCAM-negative cells largely exhibited characteristics of neural crest cells. The hNPC(PSA-NCAM+) showed multipotency and responsiveness to instructive cues towards region-specific neuronal subtypes in vitro. When transplanted into the rat striatum, hNPC(PSA-NCAM+) differentiated into neurons, astrocytes, and oligodendrocytes without particular signs of tumorigenesis. Furthermore, Ki67-positive proliferating cells and non-neural lineage cells were rarely detected in the grafts of hNPC(PSA-NCAM+) compared to those of neural rosette cells. Our results suggest that PSA-NCAM-mediated cell isolation provides a highly expandable population of pure primitive NPCs from hPSCs that will lend themselves as a promising strategy for drug screening and cell therapy for neurodegenerative disorders.