Apoptosis linked gene-2 interacting protein X as a target to regulate the yield of small extracellular vesicles derived from tissue specific mesenchymal stem cells for translational applications.

Small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) have emerged as promising therapeutic agents in regenerative medicine. However, their clinical translation remains limited by low production yields. The prime objective of this study was aimed at dissecting the regulatory mechanisms of sEV biogenesis in MSCs by investigating the roles of key proteins involved in both ESCRT-dependent (HRS, STAM, TSG101, ALIX) and ESCRT-independent (RAB27A, RAB27B) pathways. A sequential siRNA-mediated knockdown approach was employed to assess the contribution of each protein to sEV formation and secretion. Comparative analyses were performed using bone marrow-derived MSCs (BM-MSCs) and Wharton's jelly-derived MSCs (WJ-MSCs) under normoxic and hypoxia-preconditioned culture conditions to evaluate their sEV production efficiency and responsiveness to physiological modulation. Our results identified ALIX as a central regulatory protein that significantly enhances sEV yield-by approximately 4-fold and 3-fold in BM-MSCs, and 5-fold and 7-fold in WJ-MSCs under normoxic and hypoxic conditions, respectively. WJ-MSCs emerged as a superior source for scalable sEV production due to their dual engagement of ESCRT pathways and higher basal yield, in addition to being a readily available, non-invasive, and ethically non-contentious tissue source. Hypoxia was further validated as a minimally manipulative and effective strategy to augment sEV yield. Together, this study provides a mechanistic framework to improve sEV biogenesis through targeted modulation of ALIX and supports the clinical potential of WJ-MSC-derived sEVs for regenerative therapeutics.