Rock inhibitor y 27632

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.

P6T and P26T human colorectal cancer organoids were established in a previous study (Van De Wetering et al, 2015 (link)) and obtained a following material transfer agreement with Hubrecht Organoid Technology. Organoids were cultured in advanced DMEM/F12 medium (Thermo Fisher Scientific), supplemented with penicillin–streptomycin (Sigma-Aldrich), 10 mM HEPES (Thermo Fisher Scientific), 1X GlutaMAX (Thermo Fisher Scientific), B27 (Thermo Fisher Scientific), 10 mM nicotinamide (Sigma-Aldrich), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 10% vol/vol Noggin-conditioned medium, 50 ng/ml human EGF (Peprotech), 500 nM A83-01 TGF-ß type 1 receptor inhibitor (Tocris), and 10 μM SB202190 P38 MAPK inhibitor (Sigma-Aldrich). Organoids were maintained in Cultrex BME (R&D systems) and dissociated using TrypLE (Thermo Fisher Scientific) during passaging. Medium was supplemented with 10 μM Y-27632 ROCK inhibitor (Selleck Chemicals) after splitting.