Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells
Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells
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DOI：
10.1038/nprot.2009.241
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发表时间：
2010-01-01
期刊：
影响因子：
14.8
通讯作者：
Izpisua Belmonte, Juan Carlos
中科院分区：
文献类型：
--
作者：
Aasen, Trond;Izpisua Belmonte, Juan Carlos
产生诱导多能干（iPS）细胞的难易程度以及它们在重编程后的可能性质取决于体细胞起始群体的来源。与成纤维细胞相比，角质形成细胞的重编程既快又有效，尽管在分离、培养和感染这些细胞时需要更多的注意。在这项研究中，我们描述了详细的协议，使用饲养层依赖和定义的血清和饲养层的条件下培养人角质形成细胞从包皮样本和打孔活检，以及如何分离角质形成细胞从拔出的头发。我们进一步描述了培养技术和方法，以有效地感染和重编程这些细胞，以产生iPS细胞的目的。衍生角质形成细胞的过程需要10-14天，而重编程和可分离和建立的iPS细胞集落的出现需要另外3-4周。
the ease of generating induced pluripotent stem (iPS) cells, and possibly their properties after reprogramming, depends on the origin of the somatic cell starting population. reprogramming of keratinocytes is both faster and more efficient compared with fibroblasts, although more care is required when isolating, culturing and infecting these cells. In this study, we describe detailed protocols using both feeder-dependent and defined serum-and feeder-free conditions for culturing human keratinocytes from foreskin samples and punch biopsies, as well as how to isolate keratinocytes from plucked hair. We further describe culture techniques and approaches to efficiently infect and reprogram these cells for the purpose of generating iPS cells. the procedure of deriving keratinocytes takes 10-14 d, whereas reprogramming and the appearance of iPS cell colonies that can be isolated and established requires another 3-4 weeks.