Multi‐Omics Assessment of Primed Tissue‐Specific MSCs‐sEV to Unveil Their Regenerative Potential

ABSTRACT Small extracellular vesicles (sEV) play a pivotal role in intercellular communication and hold immense therapeutic potential. In this study, we aimed to characterize sEV derived from bone marrow mesenchymal stromal cells (BM‐MSCs) and Wharton's jelly mesenchymal stromal cells (WJ‐MSCs), cultured under normoxic and hypoxic conditions for elucidating their functional inclinations. Using high‐throughput miRNA sequencing, we identified distinct miRNA profiles across cell types and oxygenation states, revealing unique signatures associated with hypoxia‐driven cellular adaptations. Concurrently, mass spectrometry‐based proteomic analysis of sEV provided a comprehensive catalog of proteins, highlighting the molecular cargo influenced by both cell source and environmental conditions. Comparative analyses showed overlapping and unique miRNA‐protein networks between BM‐MSC and WJ‐MSC sEV, shedding light on their differential regulatory roles. Conclusively, hypoxia was found to enhance the enrichment of specific miRNAs and proteins implicated in angiogenesis, immunomodulation, and tissue regeneration. These findings underscore the influence of the cellular microenvironment on sEV composition and provide insights into their potential application in regenerative medicine and therapeutic development. Summary This study provides a comprehensive comparative analysis of sEV derived from BM and WJ‐MSCs, highlighting how both cell source and oxygenation state influence their molecular composition and functional potential. By integrating high‐throughput miRNA sequencing with proteomic profiling, we demonstrate that hypoxic preconditioning induces distinct shifts in sEV cargo, enriching their regenerative and immunomodulatory profiles. The findings suggest that the therapeutic efficacy of MSC‐sEV can be significantly modulated by microenvironmental conditions, particularly oxygen availability. Bioinformatic analyses reveal unique molecular interaction networks and clustering patterns reflective of each MSC source, supporting the idea that different MSC‐sEV may be suited to specific therapeutic contexts. The work advances the understanding of MSC‐sEV heterogeneity and lays the groundwork for the rational design of sEV‐based therapies. By identifying key environmental and cellular determinants of sEV composition, the study offers valuable insights for optimizing their use in regenerative medicine, with potential applications in targeting inflammation, ischemia, and tissue repair. Ultimately, the findings contribute to the development of more effective, tailored, and cell‐free therapeutic strategies.