Alternative Splicing Constitutes a Transcription-independent Regulatory Layer in Replicative Senescence of Human Umbilical Cord Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) inevitably undergo replicative senescence during in vitro expansion, leading to progressive loss of therapeutic efficacy. However, the global landscape of alternative splicing (AS) during this process and its relationship with transcriptional regulation remain uncharacterized. Here, we performed splicing-aware transcriptomic profiling of human umbilical cord derived MSCs across four passage points (P2, P8, P10, P12; n = 11). Using stringent AND criteria (FDR < 0.05, |ΔPSI| ≥ 0.05), we identified 112 differential splicing events (DSEs) affecting 96 genes. Skipped exon (SE) events predominated (82/112, 73.2%), with DSEs peaking at P8 (83 events), indicating that splicing reprogramming is an early senescence event. Only four genes overlapped between the 699 differentially expressed genes (DEGs) and 96 DSE genes (COL12A1, ITGA7, MYOCD, TNS3), indicating that splicing and transcriptional changes affect largely distinct gene sets. Functional enrichment revealed that DEGs converged on ECM remodeling and immune/inflammatory pathways, whereas DSE genes were significantly enriched in DNA repair pathways (Fanconi anemia, homologous recombination), and splicing factor genes showed a 5.6-fold enrichment trend among DSEs (p = 0.054). PTC-50nt rule based classification showed that in-frame changes constituted the predominant category (58.5%), with NMD_risk at only 14.6% and NMD_rescue at 26.8%. The core spliceosome component SF3B1 harbored a predicted NMD-targeting event (745 bp exon inclusion, dPSI = + 0.122), while the SR protein SRSF4 experienced a predicted NMD-rescue event (dPSI = - 0.098). Splicing factor expression profiling revealed that PTBP1 exhibited an early sharp decline at P8 (- 20.2%) followed by partial recovery, with divergent regulation of SRSF and HNRNP families. Multi-layer convergence analysis resolved the senescent MSC output into two separable molecular programs, ECM remodeling and immune modulation, driven predominantly at the transcriptional level. This study provides the first systematic characterization of the alternative splicing landscape during human MSC replicative senescence, revealing that splicing changes are largely gene-level distinct from transcriptional changes, that in-frame changes rather than NMD dominate the predicted functional consequences, and that splicing factors and DNA repair genes are preferentially affected. These findings integrate alternative splicing into the regulatory landscape of MSC senescence and establish a molecular foundation for developing splicing-aware quality control strategies in ATMP manufacturing.