Aplastic anemia is a potentially life-threatening bone marrow failure in which the body stops producing enough new blood cells. Marrow transplantation, antithymocyte globulin (ATG) treatment and other approaches can vary in effectiveness in approaching marrow aplasia. Extracellular vesicles are small particles released from cells that were initially thought to present cell “junk”, and later evaluated to be involved in cell-to-cell communication and affect the phenotype of target cells. In this study, we will evaluate the capacity of mesenchymal stem cell derived-extracellular vesicles (MSC-EV) to reverse aplasia in a murine model of aplastic anemia and identify the MSC-EV associated miRNA responsible for the reversal of aplastic anemia. This AA&MDSIF research grant will give us an opportunity to address novel basic mechanisms of MSC-EV healing effects and open exciting possibilities for therapy of various marrow disorders, and by extension, potentially many other disease states.
Aplastic anemia is a blood disorder where the body doesn't make enough blood cells. Marrow transplantation, antithymocyte globulin treatment and other approaches can be variably in effectiveness in approaching marrow aplasia. Extracellular vesicles (EV) are membrane surrounded small particles released by cells. Microvesicles and exosomes are the two major types of EV, and having been found to deliver both mRNA and transcriptional modulators to target cells and affect their phenotype. We have recently demonstrated the capacity of EV from mesenchymal stem cells (MSC) reverse radiation toxicity in marrow stem cells. Therefore, in this study, we plan to investigate the capacity of MSC-derived vesicles to reverse aplasia in a murine model of aplastic anemia.
In the first year of our research, we have been able to evaluate the stimulation of hematopoiesis in normal murine marrow by in vitro and in vivo exposure of hematopoietic cells to EVs. We found that the combined exosome and microvesicle fractions were superior to the exosome and microvesicle fractions in the stimulation of murine hematopoietic cell line, FDC-P1 cells. We demonstrated that the MSC-EV exposure could partially reverse aplasia in the myleran treated mouse model of aplastic anemia. To identify the MSC-EV associated miRNA responsible for the reversal aplastic anemia, we have identified miRNAs profile of three fractions vesicles by deep sequencing and are evaluating the effect of MSC-EV associated miRNA candidates on reversal of aplastic anemia.
Reversal of aplastic anemia by mesenchymal stem cell-derived extracellular vesicles
Principal Investigator: Sicheng Wen
Aplastic anemia is one of the most common diseases with 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 barrow 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 induced marrow aplasia by administered 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 final BU dose. We observed that the human MSC EVs treatment significantly prolonged survival in these mice. 50% of BU treated mince died by 30 days after BU injection, whereas only 30% of EVs-injected mice did so by this time. The mice were sacrificed to harvest WBM cells and peripheral blood for cell count at 10 days after final BU dose. There were significant decreases in WBM cell counts from tibia and femur 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±0.46 x106 cells /tibia and femur for the vesicle treated mice compared to 2.05±0.46 x 106 cells for the non-vesicle treated mice. The restoration growth of bone marrow stem cells was further confirmed by WBM cell culture. These studies suggested that the MSC-EVs could partially reverse of bone marrow failure in acute aplastic an anemia mouse model. We also established a chronic aplastic anemia mouse model up to 388 days. However, the treatment with MSC-EVs couldn’t reverse chronic aplastic anemia in our current protocol, we need further optimize the dose of MSC-EVs. 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 treated, 115 showed partial or complete reversal of these alterations after EV treatment. We also identified several micoRNA candidates associated with recovery the inhibition of bone marrow induced by Busulfan. We found that over expressed microRNAs candidates in FDC-P1 cells could enhance recovery from Busulfan–induced inhibition of cell growth. In summary, our data suggested that MSC-EV treatment could partially reverse Busulfan induced marrow failure at early time points. The reversal of the Busultan induced chronic bone marrow failure by MSC-EVs need further investigate.