Quantitative proteomic analysis of Wharton’s Jelly Mesenchymal Stem Cell secretome: effects of culture time and passage

Wharton’s Jelly Mesenchymal Stem Cell (WJMSC) secretomes hold promise for regenerative medicine due to their rich protein composition and paracrine activity. Despite their promise, significant uncertainty remains regarding the conditions that yield the most therapeutically potent secretome. In particular, the effects of culture time and passage number on secretome composition are not well understood. This study addresses these gaps by characterizing time- and passage-dependent variability in WJMSC secretomes, intending to identify consistent patterns that support optimized and scalable secretome production. Quantitative proteomics was used to assess how culture passage (P3, P5, P10) and incubation time (24, 48, 72 h) shape WJMSC secretome profiles. Differentially expressed proteins (DEPs) were defined using a p-value < 0.05 and a false discovery rate (FDR) < 2%. Protein intensities were log2-transformed for normalisation. Pearson correlation was applied as the distance metric, and average linkage was used for clustering. A total of 646 proteins were identified via LC-MS/MS, with 62 differentially expressed proteins (DEPs) analyzed for secretory routes, protein-protein interactions (PPI), and functional enrichment. Most DEPs were predicted to be secreted through classical pathways, while a subset followed unconventional mechanisms. PPI analysis revealed three functional clusters related to protease regulation, extracellular matrix (ECM) organization, and cellular signalling. Gene Ontology and KEGG enrichment highlighted key roles in ECM remodelling, immune regulation, and coagulation. Gene Set Enrichment Analysis (GSEA) revealed time- and passage-dependent alterations, with early passages favouring ECM-associated processes and later passages enriched in immune and hemostatic functions. These findings suggest that WJMSC secretomes exhibit dynamic changes across different culture passages and incubation times, highlighting the potential importance of optimizing harvesting conditions for future cell-free therapeutic applications, including wound healing and immune modulation. Further validation in larger donor cohorts is required to confirm these observations.