Isolation and Characterization of HUCMSCs

HUCMSC were isolated and characterized as previously described (9 (link)). Briefly, umbilical cord tissues from healthy women were washed three times and cut into approximately 1 mm³ pieces. The pieces evenly spread on the bottom of a 75cm² flask with the serum-free MSC culture medium (TBD Science, China) for primary adherent culture and cultured in a 5% CO₂ incubator at 37°C. The medium was refreshed every 3-4 days. After achieving 80%–90% confluence, the cells were digested with 0.25% trypsin-EDTA (Gibco, Carlsbad, CA, USA) 2 minutes for passaging. The cells were seeded into 175cm² flasks at a density of 6000-8000 cells/cm². Until the cell confluence reached 80%-90%, the cells were digested and collected to obtain the passage one of HUCMSC. Repeat the above operations for subculture. HUCMSC at passage five were characterized by morphology, mesenchymal lineage differentiation and surface marker expression. The capacity of osteogenic, adipogenic, and chondrogenic mesenchymal lineage differentiation was detected using Alizarin Red, Oil Red O, and Alcian Blue staining (OriCell, Guangzhou, China), respectively. Surface marker expression was also characterized by flow cytometry with CD73, CD90, CD105, CD34, CD14 and HLA-DR antibodies (BD Bioscience, San Jose, CA, USA) as described by previous study (20 (link)). Then the cells were used for further experiments.