Center of Regenerative Medicine in Barcelona, Barcelona, Spain.
Nat Protoc. 2010 Feb;5(2):371-82. doi: 10.1038/nprot.2009.241. Epub 2010 Feb 4.
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.
诱导多能干细胞（iPS）的生成容易程度，以及它们在重编程后的特性，可能取决于起始体细胞群的来源。与成纤维细胞相比，角质细胞的重编程既更快又更有效，尽管在分离、培养和感染这些细胞时需要更加小心。在这项研究中，我们描述了详细的方案，使用有饲养细胞依赖和无血清及无饲养细胞条件，从包皮和皮内活检样本培养人角质细胞，以及如何从拔出的头发中分离角质细胞。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，从而生成 iPS 细胞。获得角质细胞的过程需要 10-14 天，而重编程和可分离并建立的 iPS 细胞集落的出现则需要另外 3-4 周。
