Extracellular vesicles from antler blastema progenitor cells reverse bone loss and improve aging-related phenotypes in mice and macaques
Antler blastema progenitor cell (ABPC) is a novel type of skeletal mesenchymal stem cell with strong stemness and renewal ability. Here, we discovered that ABPC-derived extracellular vesicles (EVsABPCs) can strongly rejuvenate aging tissues in aged mice and rhesus macaques (Macaca mulattas). We identified a variety of rejuvenating molecules in EVsABPCs that differed from EVs of mouse bone marrow stromal stem cells (EVsBMSCs). EVsABPCs exhibited a robust in vitro ability to rejuvenate senescence BMSCs of aged mice and shift their molecular signature into a youthful state. In vivo, EVsABPCs increased bone mineral density in the femur by 2.4-fold in mice, and 1.53-fold in rhesus macaques. Besides, intravenous EVsABPCs significantly outperformed mouse fetal-EVsBMSCs in improving physical performance, reducing systemic inflammation, and rejuvenating major tissues of elderly mice, with no observed immunological side effects. Furthermore, EVsABPCs showed similar rejuvenating effects in old rhesus macaques as in elderly mice. Particularly, EVsABPCs resulted in remarkable improvements in brain structure and behaviors of elderly rhesus macaques, providing the first batch of evidence of EVs' beneficial effects on aging brains in primates. These findings suggest that the ABPC is a novel and practical source of EVs containing a wealth of systemic rejuvenating factors and has considerable translational value for anti-aging interventions, particularly the rejuvenation of aging bones. Overall design: Antler blastema progenitor cells (ABPCs) were separated using a method described in our previous study 20. Single-cell suspensions of ABPCs comprising Dulbecco's modified Eagle's medium (DMEM; Gibco, 11965092), 1% penicillin-streptomycin (Gibco, 15140122), 0.1% mycoplasma removal agent and fetal bovine serum (FBS; Gibco, 10099141C) were seeded in 10 cm culture dishes at a density of 2 × 105 cells/cm2 and incubated at 37 °C in 5% CO2. After 24 h, non-adherent cells were removed, whereas attached cells were incubated further. When cellular growth reached 80–90% confluence, adherent cells were harvested and reseeded at a density of 2 × 105 cells/cm2 for further amplification. ABPCs of passages 6 to 10 (P6 to P10) were used for the experiments. Bone marrow stromal cells (BMSCs) were isolated from 18-month-old (A-BMSCs) and 10-day-old (F-BMSCs) male Sprague-Dawley rats. The proximal and distal metaphyses of femurs and tibias were cut and the bone marrow was flushed from the diaphyses with 10% FBS in phosphate buffer solution (PBS). A single-cell suspension comprising a-minimum essential medium (MEM a; Gibco, 12571063), 1% penicillin-streptomycin (Gibco, 15140122), 0.1% mycoplasma removal agent and FBS (Gibco, 10099141C) was seeded on a 10 cm culture dish at a density of 2 × 105 cells/cm2 and left to incubate at 37 °C in 5% CO2. After 24 h, non-adherent cells were removed, whereas attached BMSCs were incubated further. When cell growth reached 80–90% confluence, adherent cells were harvested and seeded at a density of 2 × 105 cells/cm2 for amplification. F-BMSCs and A-BMSCs of passages 6 to 10 (P6 to P10) were used for the experiments. The culture conditions for the two types of BMSCs (F-BMSCs and A-BMSCs) were identical.