Generation, culture, and identification of iMSC

The use of human iPSC in this study was approved by the local ethics committee of the Shanghai Sixth People’s Hospital affiliated with Shanghai Jiao Tong University. The generation of mesenchymal stem cells from human induced pluripotent stem cells was previously described [16] with a few modifications. The human iPSC line (iPS-S-01) used in this study was from Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences in agreement with Liao and Xiao [31]. Six-well plate was pre-coated with vitronectin (Nuwacell™ VTN, Nuwacel Biotechnology, RP01002) in a concentration of 1 μg/cm² in DMEM/F12 at room temperature for at least an hour. Next, iPSC were cultured in vitronectin-coated 6-well plate with iPSC culture medium containing basal medium (Nuwacell™ Nova Basal Medium, Nuwacell Biotechnology, RP01001-1) and supplement (Nuwacell™ Nova Supplement, Nuwacell Biotechnology, RP01001-2). When cells reached to 90% confluency, the culture medium was changed to MSC culture medium containing basal medium (Nuwacell™ Nova Missoin Basal Medium, Nuwacell Biotechnology, RP020101) and supplement (Nuwacell™ Nova Missoin Supplement, Nuwacell Biotechnology, RP02010-2). Culture medium was changed every 2 days for 14 days. The cells were then trypsinized with 0.25% trypsin/1 mM EDTA (Gibco) and seeded to 25- and 75-cm² cell culture flasks (Corning) at a density of 1 × 10⁵/mL in MSC culture medium mentioned above. The cells were sub-cultured every 2–3 days when cell reached to 85% confluency. The morphology of cells was changed to fibroblast-like cells at passage 4, and the cells were utilized to identify iMSC phenotypical characteristics and tri-lineage differentiation ability [16]. Passages 5 to 10 were used for the following experiments.