Maintaining Undifferentiated H9-hESCs and Differentiating into M-MSCs

Maintenance of undifferentiated H9-hESCs and differentiation into M-MSCs (Fig. 1a) were performed as previously described^{23 (link), 24 (link)}. The established M-MSCs were cultured with EGM2-MV medium (Lonza, San Diego, CA, USA) on plates coated with rat tail collagen type I (Sigma-Aldrich, St. Louis, MO, USA) in a humidified atmosphere with 5% CO₂ at 37 °C. All M-MSCs used in experiments were expanded less than ten passages to ensure multipotency. Characterization of basic features such as surface protein expression, cell proliferation, multipotency (in vitro differentiation into osteogenic, chondrogenic, or adipogenic lineages), in vitro angiogenesis assays, and karyotyping were performed as previously described^{23 (link), 24 (link)}. The M-MSC line stably expressing GFP was established by infection of GFP-expressing lentivirus produced as previously described^{14 (link)}. Human BM-MSCs purchased from Lonza (Basel, Switzerland) were cultured following the manufacturer’s instructions. Cells expanded for fewer than seven passages were used for experiments to ensure multipotency. All cells were tested for mycoplasm content each month (Mycoplasma Hoechst Stain Kit; 3030000; MP Biomedicals, LLC, Santa Ana, CA, USA).