Generation of Human iPSCs from Hair Keratinocytes and Fibroblasts

Human iPSCs were generated from plucked human hair keratinocytes and from human foreskin fibroblasts (System Biosciences). Keratinocytes were cultured and infected as described in^{32 (link)}. Fibroblasts were cultured in DMEM, 10% FBS, 1% antibiotic-antimycotic, 1% NEAA, and 1% GlutaMAX. For reprogramming 1*10⁵ fibroblasts were plated on coated 6-well plates and were infected with 5*10⁸ viral copies of STEM CCA³⁹ OKSM lentivirus on two subsequent days in culture medium supplemented with 10 µM Rock inhibitor/Y-27632 (Selleckchem), 8 µg/ml polybrene (Sigma Aldrich). On the third day infected keratinocytes and fibroblasts were distributed equally into 6-well plates on mitomycin-inactivated rat embryonic fibroblast (REF) feeder cells. 1,5*10⁴ REFs were mitotically inactivated with 7,5 µg/ml mitomycin C for 2,5 h. During reprogramming cells were cultured in KO-DMEM, 20% KOSR, 1% antibiotic-antimycotic, 100 μM NEAA, 1% GlutaMAX, 50 mM β-mercaptoethanol, 50 μg/ml L-Ascorbic acid (Carl Roth), 10ng/ml FGF2 (Cell Guidance Systems), 10 µM Rock inhibitor/Y-27632 (Selleckchem) at 5% CO2, 5% O2, and 37 °C, and medium was changed every second day. IPSC colonies were mechanically transferred onto Matrigel coated (Corning) 6-well plates after three weeks.