| Background:Currently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative modality for patients with certain hematological malignancies. Acute graft-versus-host disease (aGVHD) remains a major cause of mortality and morbidity for allo-HSCT recipients. To date, no optimal aGVHD prophylaxis strategy has been proposed, and more effective prophylaxis for aGVHD is urgently needed. Mesenchymal stem cells (MSCs) are ideal candidates for use as cellular therapy during allo-HSCT due to their unique immunoregulatory and regenerative properties. Numerous clinical trials have indicated that the adoptive transfer of MSCs prevents life-threatening aGVHD, and accelerates hematopoietic reconstruction. However, safety concerns remain about the clinical application of MSCs, mainly due to their uncontrolled differentiation and unwanted long-term side effects. Emerging evidence indicates that paracrine effects, which are mediated by extracellular vesicles (EVs), play a key role in MSC-based therapy, and an increasing number of preclinical and clinical studies have indicated that MSC-derived exosomes and microvesicles are attractive alternative to MSCs in the fields of tissue regeneration and immunomodulation.Objective:In this study, we aim to explore efficacy and mechanism of EVs released from human umbilical cord derived MSCs (hUC-MSC-EVs) in allo-HSCT.Methods and Results:① Recipient BALB/c mice received lethal TBI of 7.5 Gy by an X-ray source; then, 6×106 donor C57BL/6 mice bone marrow cells, together with 3×106 splenocytes, were injected within 4 to 6 h after irradiation. The recipient mice developed severe aGVHD clinical manifestations approximately 21 days post allo-HSCT. Consistently, the histology of target organs of aGVHD changed profoundly. All recipient mice died from severe aGVHD approximately two months after allo-HSCT.② Culture supernatants were collected after hUC-MSCs were cultured for 48 h in fetal bovine serum-free medium, and were centrifuged at 2000 g for 30 min to remove cells and debris. The supernatants were then submitted to two cycles of ultra-centrifugation, and the final EV pellets were suspended in PBS. The protein concentration of the hUC-MSC-EVs was quantified via the BCA method. Using transmission electron microscopy, hUC-MSC-EVs appeared as a group of heterogeneous spheroids with sizes ranging from 30 to 100 nm.③ To investigate the prophylactic effects of hUC-MSC-EVs on aGVHD,200 μg of hUC-MSC-EVs were injected on days 0 and 7 after transplantation. hUC-MSC-EVs alleviated the clinical manifestations and histologic changes of aGVHD and significantly reduced the mortality of the recipient mice. Recipients treated with hUC-MSC-EVs had significantly lower frequencies and absolute numbers of CD3+CD8+T cells and serum levels of IL-2, TNF-α and IFN-γ, a higher ratio of CD3+CD4+and CD3+CD8+T cells, and higher serum levels of IL-10. An in vitro experiment indicated that hUC-MSC-EVs inhibited mitogen-induced proliferation of splenocytes, and cytokine changes were similar to the in vivo results.④To investigate the effect of hUC-MSC-EVs on hematopoietic reconstruction,200 μg of hUC-MSC-EVs were injected on day 0 after transplantation. hUC-MSC-EVs accelerated the hematopoietic reconstruction of recipient mice. An in vitro experiment indicated that hUC-MSC-EVs promoted BM cells to form more CFU.Conclusions:① A mouse model of allogeneic HSCT is successfully established.② Sequential centrifugation is an effective method for obtaining hUC-MSC-EVs.③ hUC-MSC-EVs can prevent life-threatening aGVHD by modulating immune responses.④ hUC-MSC-EVs can acclerate hematopoietic reconstitution post allo-HSCT. |
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