P2 hUC-MSCs were purchased from Cyagen. The cell pellet was cultured in an expansion medium composed of the human umbilical cord mesenchymal stem cell basal medium (HUXUC-01001, Cyagen, China), supplemented with 10% human umbilical cord mesenchymal stem cell cell-qualified fetal bovine serum, 1% glutamine, and 1% penicillin-streptomycin (Cyagen), at 37°C, with 5% carbon dioxide in a fully humidified environment.
Native hUC-MSCs (at P5) were suspended at a concentration of 1 × 106 cells/mL, washed twice in phosphate-buffered saline (PBS), and then incubated for 30 min at 4°C in the dark with the following anti-human antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): anti-CD31-PE (303105), anti-CD45-PE (368509), anti-CD34-PE (343605), anti-CD44-FITC (338803), anti-CD90-FITC (328107), and anti-CD105-FITC (328107; all from BioLegend). After 30 min, cells were washed and resuspended in 300 mL Cell Fix (BD). PE-conjugated IgG1 and FITC-conjugated IgG1 were used as isotype controls (R&D Systems Inc. and Santa Cruz Biotechnology Inc.). Immunophenotyping of hUC-MSCs was performed by flow cytometry.
Native hUC-MSCs (at P5) were suspended at a concentration of 1 × 106 cells/mL, washed twice in phosphate-buffered saline (PBS), and then incubated for 30 min at 4°C in the dark with the following anti-human antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): anti-CD31-PE (303105), anti-CD45-PE (368509), anti-CD34-PE (343605), anti-CD44-FITC (338803), anti-CD90-FITC (328107), and anti-CD105-FITC (328107; all from BioLegend). After 30 min, cells were washed and resuspended in 300 mL Cell Fix (BD). PE-conjugated IgG1 and FITC-conjugated IgG1 were used as isotype controls (R&D Systems Inc. and Santa Cruz Biotechnology Inc.). Immunophenotyping of hUC-MSCs was performed by flow cytometry.