Unveiling the dynamic transcriptomic changes in and replicative senescence balance of hUC-MSCs: A comparative study...

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) hold significant potential for applications in regenerative medicine; however, the intrinsic heterogeneity associated with cell culture conditions requires further consideration. Culture medium supplements serve as critical modulators of the biological characteristics of MSCs; nevertheless, the transcriptomic changes and functional differences underlying supplement-induced heterogeneity remain poorly understood. This study compared the effects of two commercial supplements on hUC-MSC expansion, confirming that both maintained consistent immunophenotypes and trilineage differentiation potential. Serum-free (Helios, H) supplement culture maintained stable proliferation kinetics throughout passaging, whereas serum-containing (Begancell, B) supplement culture exhibited a significant increase in senescence-associated β-galactosidase activity in mid-to-late passages. RNA sequencing analysis of differentially expressed genes (DEGs) across passages revealed their primary enrichment in catabolic pathways, Wnt signaling, extracellular matrix organization, and protein degradation processes. Additionally, DEGs associated with growth factors and chemokines were identified, and the consistent expression patterns of ten growth-related genes, such as IL-6, CXCL12, and IGF2, were subsequently validated. Further PCA, covariate-adjusted DEG models, and multifactor transcriptomic analyses indicated that medium composition was the primary driver of transcriptional divergence, whereas senescence and passaging exert negligible confounding effects. LASSO regression analysis identified three candidate markers (ADAM12, MN1, LPIN1) to distinguish the two supplements. This investigation elucidates the complex effects of commercialized culture media on the functional heterogeneity of hUC-MSCs. Our findings contribute to a comprehensive understanding of the mechanisms by which culture conditions modulate MSC characteristics, highlighting the importance of optimizing culture conditions for controlling MSC expansion characteristics and transcriptomic heterogeneity.