Mesenchymal stem cell growth medium 2

For DNA extraction, we used three cell lines and lentiviral vectors. hiPSCs (HPS0002: 253G1), mesenchymal stem cells (hMSC), and undifferentiated type of hepatoma cells (HLF) were provided by the RIKEN BioResource Center Cell Bank [21 (link)] and the Cell Resource Center for Biomedical Research, Institute of Development, Takara Bio (Kusatsu, Shiga, Japan), and Aging and Cancer Tohoku University, respectively. HLF, MSC, and iPSC (253G1) were cultured in RPMI1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin, in Mesenchymal Stem Cell Growth Medium 2 (Takara Bio, Kusatsu, Shiga, Japan), and in ReproStem medium (ReproCell, Tokyo, Japan) with 10 ng/ml of bFGF-2, respectively. Additionally, for the transfection to HLF cells or hMSCs, the human mesangial cell line 293FT (Invitrogen Japan K.K., Tokyo, Japan) was used for producing 520d-5p expressing-lentiviral particles. 293FT cells were cultured in DMEM supplemented with 10% FBS, 0.1 mM MEM nonessential amino acid solution, 2 mM L-glutamine and 1% penicillin/streptomycin. Also, we induced hMSCs from iPSCs using STEMdiff Mesenchymal Progenitor kit (STEMCELL technologies, Seattle, WA, USA) and defined them as 520d/MSC after they were transfected by miR-520d-5p.

Primary cultured NHDFs, neonatal hASCs and HUVECs were purchased from Lonza (Walkersville, MD). Each cell type was derived from single donor. NHDFs were cultured in Dulbecco’s modified Eagle medium (DMEM, Wako Pure Chemical, Osaka, Japan) containing 10% fetal bovine serum (FBS, SAFC Biosciences; Lenexa, KS) under 5% CO₂ at 37 °C as described in our previous study^{4 (link)}. For cultivation of HUVECs, Endothelial Growth Medium-2 (Lonza; Walkersville, MD) was used as described previously^{3 (link)}. For cultivation of hASCs, Mesenchymal Stem Cell Growth Medium 2 (Takara Bio Inc., Kusatsu, Japan) was used. Transwell inserts with porous polyester bottom (pore size: 0.4 µm) for 24-well culture plate were purchased from CORNING Inc. (New York, NY).