Human fgf 2

hiPSCs (line 201B7, Riken Cell Bank, Tsukuba, Japan) [9] (link) were maintained with SNL76/7 feeder cells [clonally derived from a mouse fibroblast Sandoz inbred mouse-derived thioguanine-resistant and ouabain-resistant (STO) cell line transformed with neomycin resistance and murine LIF genes; American Type Culture Collection, Manassas, VA, USA] in human ES medium [Dulbecco's modified Eagle's medium: nutrient mixture F-12 (DMEM/F-12) (Invitrogen, Carlsbad, CA, USA) with 20% knockout serum replacement (Invitrogen) supplemented with 1× nonessential amino acid solution (Chemicon, Temecula, CA, USA), 2 mM l-glutamine (Chemicon), 1 mM 2-mercaptoethanol (Wako Pure Chemical Industries Ltd., Osaka, Japan), 1% penicillin/streptomycin (Invitrogen), and 5 ng/ml human FGF-2 (ReproCELL Inc., Yokohama, Japan)].

An LGR5-positive colon cancer stem cell line PLR123 was cultured in cell culture flasks (Corning) in SCM which consisted of DMEM/F-12 (Cat# 11330-057) supplemented with 1× Antibiotic-Antimycotic, 1× N-2 Supplement, 4 mg/mL AlbuMAX I Lipid-Rich BSA, 20 ng/mL human EGF, 20 μg/mL human insulin (all from Thermo Fisher Scientific), 2.9 mg/mL glucose (Merck, total 6.0 mg/mL glucose), 4 μg/mL heparin (Merck), and 10 ng/mL human FGF2 (Reprocell) at 37 °C under 5% CO₂ as described previously^{8 (link)}. The colorectal cancer cell lines LoVo and LS174T (both from ATCC) were grown in F-12K (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS) or Eagle’s MEM supplemented with 1% Non-Essential Amino Acids Solution, 1 mM Sodium Pyruvate (all from Thermo Fisher Scientific), and 10% FBS, respectively, at 37 °C under 5% CO₂. The cell lines were adapted to SCM before used in 3D cultures.