Small extracellular vesicles produced by homeostatic phenotype BMSC promote bone regeneration by transferring anti-senescent factor JMJD6

The therapeutic efficacy of extracellular vesicles (EVs) depends on the status of their donor cells. Cellular senescence of bone marrow mesenchymal stem cells (BMSCs), which induces significant changes to cellular secretome, greatly affects the osteogenesis of the donor BMSCs along with BMSC-derived EVs. Stem cells prefer a homeostatic self-renewing state to alleviate the senescence and loss of stemness caused by external disturbance, but which inevitable in EV produce. In this study, to avoid the senescence of BMSCs caused by in vitro culture and boost the osteogenesis efficacy of BMSC-derived small extracellular vesicles (sEVs), we established a niche-mimicking (NM) culturing system to maintain the cellular homeostasis that significantly delayed stem cell senescence in vitro. As a result, single cell transcriptome revealed that NM-culturing significantly enhanced the expression level of the homeostasis-related genes. Then we found that sEVs produced by homeostatic BMSCs exhibited superior osteogenic stimulation efficacy and bone defect repair compared to those from conventional monolayer-cultured BMSCs. Mechanically, by analyzing the "shuttling effect" of sEVs via multi-omic analysis, we found that the key homeostasis-related genes (including JMJD6, LIF, CYP19A1, and LAMA1) functioned throughout the entire process, by thus we defined the "homeostatic phenotype" of BMSC. Among the identified homeostasis-related genes in sEVs, JMJD6 emerges as a key gene, exhibiting anti-senescence characteristics and stimulating osteogenesis via Wnt pathway in BMSCs. This study optimizes the production part of stem cell-based EV therapy by maintaining the cellular homeostasis of donor cells, which delays cell senescence and boosts the osteogenesis stimulation of EVs, and JMJD6 is a key homeostasis-related gene that regulated cell senescence and osteogenesis of BMSC.