Reversal of Aplastic Anemia By Mesenchymal Stem Cell-Derived Extracellular Vesicles

Aplastic anemia is a disease of bone marrow failure. Aplastic anemia remains a challenging disease as far as etiology and therapeutic approaches. Extracellular vesicles (EVs) have been studied for many years. These were initially felt to be cell junk largely from platelets and erythrocytes. But, more recently, intense interest has been focused on their potential for altering cell fate and reversing tissue injury. Vesicles derived from mesenchymal stem cells (MSC) have been shown to affect the phenotype and induce healing of many different cell types. Our recent published work has shown that human or murine MSC-EV treatment dramatically reversed radiation damage to bone marrow both in vitro and in vivo by inhibition of DNA damage and apoptosis. In this study, we evaluated whether human MSC-EVs could reverse marrow failure in a Busulfan induced aplastic anemia mouse model. We have carried out studies on Busulfan (BU) induced marrow aplasia by administering Busulfan in doses of 9.25 mg/kg intraperitoneally (9 times over 20 days) to induce bone marrow aplasia in female Balb/cByJ mice. Mice were then infused with 4x10⁸ human MSC-EVs at 1, 2 and 3 days after the final BU dose. We observed that the human MSC EV treatment significantly prolonged survival in these mice. 50% of the BU treated mice died by 30 days after BU injection, whereas only 30% of EV-injected mice died by this time. The mice were sacrificed to harvest WBM cells and peripheral blood for cell count at 10 days after the final BU dose. There were significant decreases in marrow cellularity from tibiae and femurs and complete blood count (CBC) including RBC, WBC, HBG and PLT in BU treated mice compared to vehicle control mice. The vesicle treatment led to a statistically significant (p<0.05) increase in bone marrow cell count with the average of 3.30 x10⁶ ±0.46 x10⁶ cells /tibia and femur for the vesicle treated mice compared to 2.05 x10⁶ ± 0.46 x 10⁶ cells for the non-vesicle treated mice. The restoration growth of bone marrow stem cells was further confirmed by WBM proliferation assessment in cell culture. We further investigated the effect of MSC-EVs on the microRNA expression in bone marrow cells from Busulfan induced aplastic anemia mice by microRNA realtime PCR array. 151 microRNAs were altered by Busulfan treatment. 88 showed partial or complete reversal of these alterations after EV treatment. In summary, our data suggest that MSC-EV treatment could partially reverse Busulfan induced marrow failure at early time points. The reversal of the Busulfan induced chronic bone marrow failure by MSC-EVs needs further investigation.