质粒重编程诱导多潜能干细胞及定向分化神经干细胞

林成楷; 戎利民; 刘斌

doi:10.13418/j.issn.1001-165x.2016.06.011

中国临床解剖学杂志 ›› 2016, Vol. 34 ›› Issue (6) : 647-654. DOI: 10.13418/j.issn.1001-165x.2016.06.011

实验研究

质粒重编程诱导多潜能干细胞及定向分化神经干细胞

林成楷，　戎利民，　刘斌

作者信息 +

Generation of non-integrating induced pluripotent stem cells with episomal plasmids and differentiation into neural stem cells

LIN Cheng-kai, RONG Li-ming, LIU Bin

Author information +

文章历史 +

摘要

目的　探讨游离质粒载体重编程诱导小鼠胚胎成纤维细胞(mouse embryonic fibroblasts, MEFs)为非整合型诱导多潜能干细胞,并在体外定向分化为神经干细胞(neural stem cells, NSCs), 为神经干细胞移植治疗神经损伤提供稳定、安全的细胞来源。方法　使用电转仪将质粒pEP4-EO2S-ET2K转入小鼠MEFs, 经诱导培养重编程为诱导多潜能干细胞(induced pluripotent stem cells, iPSCs), iPSCs在不同诱导培养基中经2次悬浮及贴壁培养分化为NSCs,在体内及体外实验鉴定iPSCs多向分化潜能特性及NSCs特性。结果　体内外实验显示iPSCs具有与胚胎干细胞(embryonic stem cells, ESCs)相似的多向分化潜能, 且不整合外源性基因。iPSCs进一步分化的NSCs其相关标志基因表达与野生型NSCs相近,且较iPSCs显著增加,免疫荧光显示NSCs高表达NSC标志物 NESTIN及PAX6,在体外存活能分化为神经元、少突胶质细胞及星形胶质细胞。结论　游离质粒能重编程诱导非整合型iPSCs, 并定向分化为神经干细胞及神经元, 是神经损伤修复的理想种子细胞。

Abstract

Objective　To study the induction of non-intergrated induced pluripotent stem cells (iPSCs) with episomal plasmid vectors, and differentiating into neural stem cells in vitro.　Methods　Non-integrating mouse iPSCs were induced from mouse embryonic fibroblasts (MEFs) with plasmid vectors pCEP4-EO2S-ET2K. For the differentiation of neural stem cells (NSCs), iPSCs were cultured in suspension and adherent cultural plate for 2 times with different mediums. The characterization of iPSCs and NSCs were then investigated.　Results　Our results demonstrated that mouse iPSC could be effectively generated and the pluripotency of iPSC was similar to that of mESCs. In addition, no vector integration was found in iPSCs of 10th passages. Quantitative real-time PCR showed that the neural stem cell marker gene expression was significantly improved in iPSC-derived NSCs, which was similar to that of wild-type NSCs. Immunofluorescence demonstrated that iPSC-derived NSCs expressed NESTIN and PAX6 and could differentiated into neurons, oligodendrocytes and astrocytes in vitro.　Conclusions　 Non-integrated iPSCs could be generated with episomal plasmid vectors and differentiated into NSCs which could be the ideal seeding cells for the treatment of spinal cord injury.

导出引用

林成楷，　戎利民，　刘斌. 质粒重编程诱导多潜能干细胞及定向分化神经干细胞[J]. 中国临床解剖学杂志. 2016, 34(6): 647-654 https://doi.org/10.13418/j.issn.1001-165x.2016.06.011

LIN Cheng-kai, RONG Li-ming, LIU Bin. Generation of non-integrating induced pluripotent stem cells with episomal plasmids and differentiation into neural stem cells[J]. Chinese Journal of Clinical Anatomy. 2016, 34(6): 647-654 https://doi.org/10.13418/j.issn.1001-165x.2016.06.011

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基金

国家自然科学基金资助项目(31170947, 31470949, 81472122); 广东省自然科学基金资助项目(S2012020011099, S2013 010016413) ; 广东省科技计划(2012B060300008) ; 教育部博士点新教师基金资助项目(20100171120088); 广州市科技计划(2013J4100062)