Fibroblast growth factor 2 (fgf2)

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.

ESCs were maintained on laminin in Falcon® plates and cultured in Chemically Defined Medium (CDM) supplemented with 0.7 µM PD0325901 (AxonMedCHem), 2.5 µM CHIR99201 (AxonMedChem), and 700 U/ml LIF (Cell Guidance Systems). Passage of ESCs is performed every 3 days after Trypsin treatment. The conversion of ESCs into cEpiSCs was performed by switching medium to CDM supplemented with 20 ng/ml Activin A (Cell Guidance Systems) and 12 ng/ml FGF2 (Cell Guidance Systems)^{53 (link)}. cEpiSCs were seeded on serum-coated Falcon® plates and passed every 3 or 4 days after collagenase treatment. TSCs were cultured on serum-coated Falcon® plates according to Ohinata and Tsukiyama^{30 (link)} with some modifications: We used a serum-free medium i.e. CDM supplemented with 12 ng/ml FGF2, 20 ng/ml Activin A, 10 nM XAV939 (Sigma) and 5 nM Y27632 (Cell Guidance Systems) called FAXY medium and passed every 3 days. Immunostaining and RNA-FISH experiments were performed on cells cultured at least for 14 days or 10 days for cEpiSCs.

EpiSCs were routinely grown in FAX medium on fibronectin-coated tissue culture plastic. Coating was performed with 20 µg/ml human fibronectin (Merck) in PBS for at least 30 min. FAX medium is N2B27 supplemented with 12 ng/ml FGF2 (Cell Guidance Systems), 25 ng/ml ActivinA (Cell Guidance Systems) and 20 µM XAV939 (Cell Guidance Systems). N2B27 was prepared as a 1:1 mixture of Dulbecco's modified Eagle medium/F12 (PAN Biotech) and Neuropan basal medium (PAN Biotech) with 0.0025% bovine serum albumin (BSA; Gibco), 1× N2 and 1× B27 supplements (Thermo Fisher Scientific), 1× GlutaMAX (Gibco), and 50 μM 2-mercaptoethanol. Cells were split with Accutase (Merck) every 2 days or upon reaching confluency, and replated at a density of 8000-10,000 cells/cm². For mesoderm differentiation, EpiSCs were seeded at a density of 1300-2000 cells/cm². The day after seeding, medium was changed to N2B27 supplemented with growth factors and small-molecule inhibitors: CHIR99201 (Merck), murine BMP4 (PeproTech), FGF2 (Cell Guidance System), FGF4 (PeproTech) or AZD4547 (Selleckchem). During differentiation, medium was changed daily.