Age-Associated Changes in Bone Marrow-Derived Extracellular Vesicles May Alter Their Effects on Murine Hematopoietic Stem Cell Function

Extracellular vesicles (EVs) are critical mediators of intercellular communication within the bone marrow niche and have been implicated in numerous features of aging. However, their role in natural hematopoietic stem cell (HSC) aging has not been fully elucidated. The goal of this work was to test the hypothesis that EVs from whole bone marrow (BM-EVs) can modulate the HSC aging phenotype in vivo. With respect to HSC aging, our prior work showed that, in contrast to the well-known reduced functional capacity and prominent myeloid skewing displayed by old immunophenotypically-defined HSCs, old unseparated whole bone marrow (WBM) (24-26-mo-old) had a 4-fold increase in functional HSCs compared to young (6-8-wk-old) WBM in limit dilution competitive bone marrow transplantation, and showed minimal to no myeloid skewing. In order to test the ability of BM-EVs derived from this total WBM population to alter HSC aging phenotype, we first isolated EVs from WBM flushed from old (24-26-month old) and young (6-8-week old) C57/BL6 (CD45.2) mice by differential centrifugation (2000 × g for 30 min, supernatant centrifuged 100,000 × g for 1 hour, BM-EV pellet collected). Utilizing nanoparticle tracking analysis, we found no difference in mean particle size between old and young BM-EVs, but there was an approximately 2-fold increase in the number of EVs from old WBM compared to young WBM. To test the ability of these BM-EVs to alter HSC function in vivo, we injected old CD45.2 mice with 2 x 10⁹ young BM-EVs and young CD45.2 mice with 2 x 10⁹ old BM-EVs mice via tail vein, daily x 3 days. Control mice were injected with age-matched BM-EVs or vehicle alone. At one-month post injection, we isolated total WBM and Lineage negative/c-Kit+/Sca-1+/CD150+ cells (LSK-SLAM) from the EV-exposed or vehicle control mice. We injected either 3 x 10⁵ WBM cells or 400 LSK-SLAM mixed with 3 x 10⁵ healthy WBM competitor cells from young B6.SJL (CD45.1) mice into lethally irradiated young CD45.1 hosts and measured peripheral blood chimerism and lineage contribution by flow cytometry up to 6 months post-transplant. For the young marrow, exposure to old BM-EVs had no appreciable effects on engraftment capacity or lineage distribution. However, old WBM exposed to young BM-EVs exhibited a significant decrease in engraftment (15% ± 5%) when compared to old WBM exposed to age-matched old BM-EVs (61% ± 14%) or vehicle control (47% ± 7%) (% average donor chimerism ± SEM, n=4-5 mice/group, p<0.04). Similarly, there was a trend toward decreased engraftment capacity by old LSK-SLAM after young BM-EV exposure (7% ± 4%) and increased engraftment capacity by the old LSK-SLAM after old BM-EV exposure (27% ± 10%) compared to vehicle control (15% ± 2%) (% average donor chimerism ± SEM, n=4-5 mice/group, p=not significant (ns)). These results are consistent with our prior data discussed above in which old un-separated WBM, the source of the old BM-EVs, had increased engraftment capacity compared to young WBM. Interestingly, there was also a trend toward reversal of classic myeloid skewing when old LSK-SLAM were exposed to young BM-EVs ( 36% ± 11%) compared to vehicle control (64% ± 3%) (average myeloid % of donor-derived peripheral blood ± SEM, n=4-5mice/group, p=ns). In addition, consistent with the known increase in LSK-SLAM with age, our preliminary data showed that old mice exposed to young BM-EVs had an approximately 7-fold decrease in the number of LSK-SLAM in marrow, indicating that BM-EVs within the young bone marrow microenvironment may modulate HSC population size. Finally, microRNA (miR) expression profiling (NanoString Technologies) indicated that a number of miRs known to be involved in hematopoiesis, proliferation, self-renewal, differentiation, senescence and inflammation were differentially represented in old and young BM-EVs, with miR-29a, miR-24, and miR-21 significantly increased and miR-105 significantly decreased in old BM-EVs compared to young BM-EVs. Together, these data indicate that young BM-EVs, via transfer of differentially age-related cargo, may alter engraftment capacity, modify the lineage commitment and may regulate LSK-SLAM population size during natural aging. Future studies more clearly delineating age-associated BM-EV effects on HSCs and defining the molecular mechanisms underlying these effects could yield key insights into the natural aging of HSCs and facilitate restoration of healthy hematopoiesis in the elderly.