Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells.
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.
Aasen
Trond
T
Center of Regenerative Medicine in Barcelona, Barcelona, Spain.
Izpisúa Belmonte
Juan Carlos
JC
D001706:Biopsy; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D003470:Culture Media; D016895:Culture Media, Serum-Free; D005347:Fibroblasts / Q000166:cytology; D052816:Foreskin / Q000166:cytology; D006197:Hair / Q000166:cytology*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D008297:Male; D009432:Neural Crest / Q000166:cytology; D039904:Pluripotent Stem Cells / Q000166:cytology*; D012867:Skin / Q000166:cytology*
D003470:Culture Media; D016895:Culture Media, Serum-Free