Shenzhen Children's Hospital, Shenzhen, 518026, China,
Protein Cell. 2014 Jan;5(1):69-79. doi: 10.1007/s13238-013-0007-y. Epub 2014 Jan 29.
With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry(+) NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.
人类胚胎干细胞 (hESC) 具有无限自我更新和分化为体内所有细胞类型的能力，在人类细胞治疗中具有巨大的应用前景。然而，目前用于轻易识别和分离活的 hESC 来源细胞的工具十分有限。为了追踪 hESC 来源的神经祖细胞 (NPC)，我们应用同源重组将 mCherry 基因敲入 hESC 的 Nestin 基因座。这使得 Nestin 阳性神经祖细胞的遗传标记成为可能。我们的报告基因系统能够可视化 hESC 的神经诱导，无论是在体外（类胚体）还是在体内（畸胎瘤）。该系统还可以根据我们荧光报告基因的强度来识别不同的神经亚群。在这种情况下，高 mCherry 表达水平显示了神经祖细胞的富集，而低 mCherry 则对应于更成熟的神经状态。mCherry 高表达与细胞表面抗原染色相结合，可进一步富集 hESC 来源的 NPC。这些 mCherry(+) NPC 可以在培养中扩增，其分化导致 mCherry 的下调与 Nestin 表达的丧失一致。因此，我们开发了一种荧光报告基因系统，可用于追踪 hESC 的神经分化事件。
