Insulin like growth factor igf 1

Human iPSCs (line 201B7; Riken Cell Bank, Tsukuba, Japan) were maintained with SNL76/7 feeder cells, clonally derived from a mouse fibroblast STO cell line transformed with neomycin resistance genes and murine LIF genes, in human ES medium (Dulbecco's modified Eagle's medium, nutrient mixture F-12 (DMEM/F-12, Invitrogen, Carlsbad, CA) with 20% knockout serum replacement (Invitrogen) supplemented with 1× non-essential amino acids solution (Chemicon, Temecula, CA), 2 mM l-glutamine (Chemicon), 1 mM 2-mercaptoethanol (Wako Pure Chemical Industries Ltd., Osaka, Japan), 1% penicillin/streptomycin (Invitrogen) and 5 ng ml⁻¹ human fibroblast growth factor (FGF)-2 (ReproCELL Incorporated, Yokohama, Japan)). hMSCs were purchased Lonza (Basel, Switzerland), cultured in BulletKit MSC growth medium (Lonza) and used at passage 3 to 5. The 1,25(OH)₂ vitamin D₃ was purchased from Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan). Insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-β were purchased from Wako Pure Chemical Industries Ltd.

Human CD34⁺, CD34⁻, or both CD34⁺ and CD34⁻ cells isolated from HUCB were cultured in methylcellulose-containing medium, H4236 (StemCell Technologies, Vancouver, Canada), supplemented with 20 ng/mL stem cell-derived factor (Kirin, Tokyo, Japan), 50 ng/mL vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, USA), 20 ng/mL interleukin (IL)-3 (Kirin), 50 ng/mL basic fibroblast growth factor (bFGF; Wako, Osaka, Japan), 50 ng/mL epidermal growth factor (EGF; Wako), 50 ng/mL insulin-like growth factor (IGF)-1 (Wako), 2 U/mL heparin (Ajinomoto, Tokyo, Japan), and 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA) on a 35-mm dish (Thermo SCIENTIFIC, Rockford, IL) for 8 d. The cell density of each sample was 5×10² cells per dish or was adjusted depending on the assay. The EPCs were identified as small EPC-CFUs or large EPC-CFUs by visual inspection using a light microscope (OLYMPUS, Tokyo, Japan) under 40x magnification. Small EPC-CFUs were composed of round adhesive cells, and large EPC-CFUs were composed of spindle-shaped cells. Nonattached cells were isolated as small EPCs by washing with PBS (WELGENE, Daegu, Korea), while attached cells were harvested as large EPCs by treatment with 5 mM EDTA (Sigma-Aldrich, St. Louis, MO) in PBS (5 mmol/L) for 5 min at 37°C.