A real-time pluripotency reporter for the long-term and real-time monitoring of pluripotency changes in induced pluripotent stem cells.
A real-time pluripotency reporter for the long-term and real-time monitoring of pluripotency changes in induced pluripotent stem cells.
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实时多能性报告仪，用于长期实时监测诱导多能干细胞的多能性变化
DOI：
10.18632/aging.204083
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发表时间：
2022-05-15
期刊：
影响因子：
5.2
通讯作者：
Xiao, Dong
中科院分区：
文献类型：
--
作者：
Shen, Hong-Fen;Li, Yong-Long;Huang, Shi-Hao;Xia, Jia-Wei;Yao, Zhi-Fang;Xiao, Gao-Fang;Zhou, Ying;Li, Ying-Chun;Shi, Jun-Wen;Lin, Xiao-Lin;Zhao, Wen-Tao;Sun, Yan;Tian, Yu-Guang;Jia, Jun-Shuang;Xiao, Dong
关键词：
掌握将小鼠体细胞重编程为诱导多能干细胞（induced pluripotent stem cells，iPSCs）的技术，为建立将人类癌细胞重编程为iPSCs的技术平台奠定良好的基础。小鼠iPSC（即，通过在小鼠胚胎干细胞（mESC）培养条件下引入四种因子Oct 4、Sox 2、c-Myc和Klf 4，从携带Oct 4-EGFP转基因的小鼠胚胎成纤维细胞（MEF）成功地产生Oct 4-GFP miPSC。Oct 4-GFP miPSC在形态、增殖、mESC特异性表面抗原和基因表达方面与mESC相似。此外，Oct 4-GFP miPSC可以悬浮培养以形成胚状体（EB）并在体外分化为三个胚层的细胞类型。Oct 4-GFP miPSC在体内可发育为畸胎瘤和嵌合体。与MEFs的细胞周期分布不同，Oct 4-GFP miPSC在细胞周期结构上与mESC相似，由较高的S期和较低的G1期组成。更重要的是，我们的数据表明，携带Oct 4-EGFP转基因的MEF不表达GFP，直到它们被重编程到多能阶段（iPSC），而当这些多能Oct 4-GFP miPSC暴露于EB介导的分化条件时，GFP表达逐渐丧失，这表明Oct 4-GFP miPSC的多能性可以通过GFP测定在长时间内实时监测。综上所述，我们的研究结果表明Oct 4-GFP miPSC系成功建立，这将为建立将癌细胞重编程为iPSCs的技术平台奠定坚实的基础。此外，该多能性报告系统允许长期实时监测活单细胞及其后代中的多能性变化。
To master the technology of reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs), which will lay a good foundation for setting up a technology platform on reprogramming human cancer cells into iPSCs. Mouse iPSCs (i.e., Oct4-GFP miPSCs) was successfully generated from mouse embryonic fibroblasts (MEFs) harboring Oct4-EGFP transgene by introducing four factors, Oct4, Sox2, c-Myc and Klf4, under mESC (Murine embryonic stem cells) culture conditions. Oct4-GFP miPSCs were similar to mESCs in morphology, proliferation, mESC-specific surface antigens and gene expression. Additionally, Oct4-GFP miPSCs could be cultured in suspension to form embryoid bodies (EBs) and differentiate into cell types of the three germ layers in vitro. Moreover, Oct4-GFP miPSCs could develop to teratoma and chimera in vivo. Unlike cell cycle distribution of MEFs, Oct4-GFP miPSCs are similar to mESCs in the cell cycle structure which consists of higher S phase and lower G1 phase. More importantly, our data demonstrated that MEFs harboring Oct4-EGFP transgene did not express GFP, until they were reprogrammed to the pluripotent stage (iPSCs), while the GFP expression was progressively lost when these pluripotent Oct4-GFP miPSCs exposed to EB-mediated differentiation conditions, suggesting the pluripotency of Oct4-GFP miPSCs can be real-time monitored over long periods of time via GFP assay. Altogether, our findings demonstrate that Oct4-GFP miPSC line is successfully established, which will lay a solid foundation for setting up a technology platform on reprogramming cancer cells into iPSCs. Furthermore, this pluripotency reporter system permits the long-term real-time monitoring of pluripotency changes in a live single-cell, and its progeny.
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