• • Mid-passage UC-MSCs (passage 5-7) demonstrated superior functional performance compared to early-passage (passage 2-3) cells, as evidenced by differential gene expression and functional enrichment analysis, indicating optimal timing for clinical cell manufacturing.
• • Single-cell RNA sequencing of 78,178 cells identified 14 subpopulations; subpopulation C8 exhibited the highest stemness, hematopoietic support, and immunomodulatory potential, providing a target for quality control.
• • NOTCH signaling, mediated predominantly by NOTCH2, was identified as the key pathway maintaining C8 stemness; this was confirmed by cell-cell communication network analysis, suggesting a mechanistic target for enhancing MSC potency.
• • MLPH and LPXN were validated as surface markers; MLPHhighLPXNhigh UC-MSCs showed significantly higher hematopoietic support (e.g., increased CD34+ cell output in co-culture) and immunosuppression (e.g., reduced T-cell proliferation by 40%) compared to MLPHlowLPXNlow cells, enabling prospective isolation of functional cells.