Kim Jong Soo, Choi Hyun Woo, Hong Yean Ju, Do Jeong Tae
Department of Animal Biotechnology, Konkuk University, Seoul, South Korea.
Department of Animal Biotechnology, College of Animal Bioscience and Technology, 120 Neungdong-Ro, Gwangjin-gu, Seoul, 143-701, South Korea.
Methods Mol Biol. 2016;1357:85-95. doi: 10.1007/7651_2014_161.
Induced pluripotent stem (iPS) cells can be directly generated from somatic cells by overexpression of defined transcription factors. iPS cells can perpetually self-renew and differentiate into all cell types of an organism. iPS cells were first generated through infection with retroviruses that contain reprogramming factors. However, development of an exogene-free iPS cell generation method is crucial for future therapeutic applications, because integrated exogenes result in the formation of tumors in chimeras and regain pluripotency after differentiation in vitro. Here, we describe a method to generate iPS cells by transfection of plasmid vectors and to convert partially reprogrammed cells into fully reprogrammed iPS cells by switching from mouse ESC culture conditions to KOSR-based media with bFGF. We also describe basic methods used to characterize fully reprogrammed iPS cells.
通过过表达特定转录因子，可直接从体细胞生成诱导多能干细胞（iPS细胞）。iPS细胞能够持续自我更新并分化为生物体的所有细胞类型。iPS细胞最初是通过感染携带重编程因子的逆转录病毒产生的。然而，开发无外源基因的iPS细胞生成方法对于未来的治疗应用至关重要，因为整合的外源基因会导致嵌合体中形成肿瘤，并在体外分化后恢复多能性。在此，我们描述了一种通过转染质粒载体生成iPS细胞的方法，以及通过从小鼠胚胎干细胞（ESC）培养条件转换为含碱性成纤维细胞生长因子（bFGF）的基于 KnockOut血清替代物（KOSR）的培养基，将部分重编程细胞转化为完全重编程的iPS细胞的方法。我们还描述了用于鉴定完全重编程的iPS细胞的基本方法。
