Stemdiff mesoderm induction medium

Endothelial-cell (EC) differentiation of hiPSCs was performed in monolayers of cultured hiPSCs with the STEMdiff Endothelial Differentiation Kit (STEMCELL Technologies) as directed by the manufacturer’s instructions. Briefly, hiPSCs were seeded into mTeSR Plus (STEMCELL Technologies) in a 6 well plate at a density of 5.0 × 10⁴ per well and then on dD1 and dD2, the medium was changed to 3 ml STEMdiff Mesoderm Induction Medium (STEMCELL Technologies). The medium was replaced with 4 ml of STEMdiff Endothelial Induction Medium (STEMCELL Technologies) on dD3 and refreshed on dD5. On dD7, the cells were dissociated with ACCUTASE (Corning), transferred into a fibronectin-coated T75 flask, and cultured in EGM-2 MV (Lonza) supplemented with SB431542 (Fischer Scientific) until 100% confluent. Purification was performed by dissociating the cells, resuspending them in cold Dulbecco phosphate-buffered saline (DPBS) with 2% fetal bovine serum (FBS) at a density of 1 × 10⁶ cells/100 μL, and then collecting cells that expressed both CD31 and CD144 via flow cytometry on a BD FACS Aria II instrument; 100-µL samples were labeled by incubating them with 5 µL of AlexaFluor-conjugated CD31 and 20 µL of phycoerythrin-conjugated CD144 antibodies for 45 min on ice and then washed in DPBS.

iPSCs were seeded without feeder cells on Matrigel at 5000 cells/cm² and cultured in mTeSR Plus supplemented with ROCK pathway inhibitor Y-27632 (Enzo Life Sciences, Lörrach, Germany). After 24 h, the medium was changed to STEMdiff Mesoderm Induction Medium (STEMCELL Technologies) and replenished daily for four days. On day 5 of differentiation, medium was replaced with StemXVivo MSC Expansion Medium (R&D Systems, Minneapolis, MN, USA). After 48 h, cells were dissociated with 0.25% trypsin/EDTA (Thermo Fisher Scientific) and seeded onto gelatin-coated plates at 1  ×  10⁴ cells/cm² in MSC expansion medium. Medium was replenished every other day, and cells were propagated to 80% confluence in a humidified atmosphere at 37 °C and 5% CO₂. The differentiated cells derived from these culture conditions were termed iPSC-derived MSCs (iMSCs) and expanded. Cells of an early passage were used for characterization. For routine expansion, cells were plated at 5  ×  10⁴ cells/cm² onto Matrigel or uncoated culture dishes (starting with P3) and maintained in MSC growth medium. MSC growth medium consisted of Dulbecco’s modified Eagle’s medium-high glucose (DMEM-HG; Thermo Fisher Scientific), 10% FBS, and 1% PS.