Papel del estroma medular en la mejora de la función hematopoyéticadesde la irradiación hasta las vesículas extracelulares

Resumen

Role of Bone Marrow Stroma in the Improvement of Hematopoetic Function: From Irradiation to Extracellular Vesicles Background Bone marrow (BM) microenvironment regulates growth and differentiation of hematopoietic stem cells (HSC) and is composed of several cell types, including osteoblasts (that have a critical role in the regulation of hematopoiesis and in the maintenance of the clonogenic potential of HSC) and adipocytes (which exert an inhibitory effect on hematopoiesis), both derived from mesenchymal stromal cells (MSC). Allogeneic hematopoietic cell transplantation (allo-SCT) remains as the only curative therapeutic approach for a variety of hematopoietic diseases. An adequate hematopoietic function after an allo-SCT is not only dependent on the number of hematopoietic stem cells infused, that is a key factor for engraftment. In the last few years, the focus has also been oriented to the BM microenvironment, especially to MSC. In the current work, we have hypothesized that inducing some modifications in the BM microenvironment (as the ones induced by low-dose irradiation, that is increasingly used in the conditioning regimens used in elderly patients) or the release of MSC-derived extracellular vesicles (MSC-EV) (one of the most important mechanism by which MSC exert their therapeutic effects) could increase the hematopoietic function, with its potential application to improve the outcome of allo-SCT, especially in those cases with poor graft function. Objectives Thus, the main objective of our work was to evaluate multiparametrically the effects of a reduced dose (2.5Gy) of irradiation on MSC and the effects of the incorporation of MSC-EV into HSC and their potential role in the improvement of the hematopoietic function. The specific aims were: 1) To evaluate the effects of low-dose irradiation on MSC and their impact on hematopoietic function, 2) To study the effects on MSC-EV incorporation into CD34+ cells and their impact on hematopoietic function, 3) To study the effects of previous-irradiation of MSC on their VE (IRR-MSC-EV) capacity on hematopoietic function improvement, and 4) To evaluate the effects of different doses of MSC-EV incorporation into HSC and their capacity on hematopoietic function improvement. Methods In the current work, a total of 70 bone marrow samples from healthy donors, 45 leukapheresis samples and 33 cord blood units were used, after proper informed consent was obtained and with the approval of the local Ethics Committee. To evaluate the effects of low-dose irradiation on MSC and their impact on hematopoietic function, MSC at third passage were irradiated with 2.5 Gy or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Adipogenic differentiation was assessed by Oil-Red staining and reverse transcriptase (RT)-PCR of CEBPA and PPARG, osteogenic differentiation was evaluated by alkaline phosphatase staining and RT-PCR of RUNX2 and ALP and mineralization was analyzed RT-PCR of SPP1 and quantified by Alizarin Red staining. Apoptosis was evaluated by flow cytometry with annexin V/7-AAD staining. Gene expression profile was studied by Chip Human Gene ST Arrays and the most relevant genes involved in hematopoiesis maintenance (SDF-1, ANGPT-1, COL1A1, THPO, CXCR4, ITGA-4, CD44 and NGF) were analyzed by RT-PCR, SDF-1 expression was confirmed by ELISA. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Clonal growth of progenitor cell population was assayed weekly culturing CD34+ cells in methylcellulose Media. Differentiation status of stroma was evaluated during culture. To study the effects on MSC-EV incorporation into CD34+ cells and their impact on hematopoietic function, MSC-EV were characterized by flow cytometry, Western blot, electron microscopy (TEM), and nano-particle tracking analysis (NTA). Micro-RNA content of MSC-EV and IRR-MSC-EV was analyzed by TaqMan Arrays. 1x105 CD34+ cells were co-cultured with EV isolated from 3x106 MSC and EV incorporation into CD34+ cells was confirmed by flow cytometry and confocal microscopy after staining EV with Vybrant Dil cell labeling solution. Then Gene expression profile was studied by Chip Human Gene ST Arrays. Apoptosis and cell cycle were evaluated by flow cytometry and Caspase 3/7 and Caspase 9 activity was measured by luminescence. RT-PCR were performed in modified CD34+ cells in order to analyze expression of some genes (SDF-1, COL1A1, CD44, CXCR4, ITGA-4 y cKIT) and micro-RNAs (150, 155, 181a, 17, 363, 494 and Let7g). Protein expression of CD44, CXCR4, ITGA-4 and cKIT was evaluated by flow cytometry and CXCR4 and cKIT expression was confirmed by Western blot (Wes Simple). Phosphorylation of STAT5 was also analyzed by WES Simple. Finally, clonal growth of CD34+ cells in the different experimental conditions (after MSC-EV incorporation or not) was assessed by clonogenic assays and their capacity of engraftment was analyzed 4 weeks after CD34+ cell transplantation in non-obese diabetic/severe combined immunodeficient mice by flow cytometry in bone marrow and spleen. Similar experiments were done to evaluate either the effect of different doses of MSC-EV (isolated from 3x106MSC or 9x106MSC), or the effect of EV released by low-dose irradiated MSC on CD34+ cells. Results After low-dose irradiation of MSC, the immunophenotypic characterization and viability of irradiated MSC was comparable to that of control cells. Gene expression profiling showed a significant differential expression in 50 genes. Of them, 5 genes were overexpressed and 45 were down-regulated in irradiated compared with non-irradiated MSC. The most downregulated gene was pyruvate dehydrogenase kinase 1 (PDK1), which is involved in the regulation of adipogenesis. By RT-PCR, we observed that SDF-1 and ANGPT were overexpressed, whereas COL1A1 was down-regulated in irradiated cells (p=0.015, p=0.007, and p=0.031, respectively). The over-expression of SDF-1 in irradiated cells was confirmed by ELISA. Analyzing their differentiation capacity, we observed that, differentiation of irradiated MSC was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of SPP1 (p=0.039), involved in mineralization, and lower expression of genes involved in adipogenesis, CEBPA and PPARG (p=0.003 and p=0.019), was observed in irradiated cells. Moreover, an increase in the mineralization capacity quantified by Alizarin Red staining and a decrease in adipocyte counts were observed in irradiated cells at days 7, 14, and 21 after culture in specific differentiation media (p=0.018 p=0.046, and p=0.018, respectively). Finally, colony-forming unit granulocyte macrophage (CFU-GM) numbers in long-term bone marrow cultures were higher in the irradiated cells during the five weeks of the culture, with significant differences after 4 and 5 weeks (p=0.046 and p=0.007). In summary, the irradiation of MSC with 2.5 Gy improved their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis. Regarding the effects on MSC-EV incorporation into CD34+ cells and their impact on hematopoietic function, the isolation and characterization of the EV showed that EV size evaluated by NTA was homogeneous among samples with a mean of 131.93 nm (124.4–143.6 nm) and a mean particle concentration of 9.09E+10 particles/milliliter (5.16E+10–1.21E+11) in preparations of EV isolated from 3x106MSC. In addition, by TEM we observed the characteristic rounded morphology of EV. By flow cytometry, we confirmed that EV were smaller than 1 μm, negative for hematopoietic markers and positive for MSC and exosome markers (CD81 and CD63). CD63 expression was also confirmed by Western blot. MSC-EV incorporation was visualized by confocal microscopy and quantified by flow cytometry. Upon incorporation into CD34+ cells, MSC-EV induced a down-regulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the JAK-STAT pathway. A significant decrease in apoptosis and Caspases 3/7 and Caspase 9 activation was observed in CD34+ cells after the incorporation of MSC-EV. Increased CD44 and CXCR4 expression and decreased cKIT expression upon the incorporation of MSC-EV were confirmed by FC. Increased levels of phospho-STAT5 were detected by WES Simple in CD34+ cells with MSC-EV. In addition, these cells displayed a higher colony-forming unit granulocyte/macrophage clonogenic potential and the in vivo bone marrow lodging ability of human CD34+ cells with MSC-EV was significantly increased in the injected femurs. In summary, the incorporation of MSC-EV induces genomic and functional changes in CD34+ cells, increasing their clonogenic capacity and their bone marrow lodging ability. When comparing the effects of EV from pre-irradiated MSC (IRR-MSC-EV) we observed that were similar in size, morphology, concentration and immunophenotype to non irradiated MSC-EV and they had similar capacity of incorporation into CD34+ cells. Regarding micro-RNA content, we found 19 micro-RNAs significantly downregulated in IRR-MSC-EV compared to MSC-EV. Some of them were analyzed in CD34+ cells that had incorporated either MSC-EV or IRR-MSC-EV but we did not found differences in its expression in these cells. However, upon the incorporation of IRR-MSC-EV, 1330 genes were modified in CD34+ cells inducing a downregulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the JAK-STAT pathway as MSC-EV did. Moreover, we detected an over-expression of many HLA molecules resulting in the overexpression of antigen processing and presentation, graft versus host disease or allograft rejection pathways. Also proteasome, ribosome biogenesis and other pathway were altered. Despite these differences in gene expression, we did not find differences in viability, cell cycle, expression of molecules involved in hematopoiesis or colony formation capacity between CD34+ cells that had incorporated IRR-MSC-EV or MSC-EV. In addition, IRR-MSC-EV and MSC-EV significantly increase BM lodging ability of CD34+ cells without significant differences among both experimental groups. Finally we evaluated the potential impact of the MSC-EV dose in the observed effects in CD34+ cells. As expected, particle concentration assessed by NTA of EV preparations isolated from 9x106 MSC (3.02E+12 particles/ml) was higher than that of EV isolated from 3x106MSC (2,88E+11 particles/ml). Besides, we observed by flow cytometry a significant increase of CD34+ cells that had incorporated EV when they were co-cultured with the higher dose of EV. However, despite the fact that some effects (increase of viability, CD44 expression and clonogenic capacity) were more pronounced after the incorporation of higher dose of EV in CD34+ cells, we did not found significant differences between different doses, neither in their bone marrow lodging ability. So we can conclude that the lower dose (VE isolated from 3x106 MSC) could be optimal for inducing changes in CD34+ cells in the experiments performed and that higher doses do not contribute to improve these beneficial effects. Conclusions 1) Low-dose γ-irradiation of MSC causes an alteration in their gene expression profile increasing the expression levels of SDF-1 and ANGPT, favoring osteogenic differentiation and decreasing adipogenesis. These modifications lead to an improvement of MSC hematopoietic-supporting ability. 2) Human MSC-EV are able to incorporate into human CD34+ cells, modifying their gene expression and increasing their viability, clonogenic capacity in vitro, and their 4-week BM lodging ability in vivo. 3) IRR-MSC-EV induce similar genomic and functional changes as MSC-EV when incorporate into CD34+ cells without significant differences in the viability, cell cycle, colony forming capacity and BM lodging ability of CD34+ cells. These results confirms that the beneficial effect of low-irradiation on MSC hematopoietic-supporting ability is not related to their exchange of molecules through EV. 4) EV isolated from 9x106 MSC do not improve the beneficial effects in the hematopoietic function induced by EV isolated from 3x106 when they incorporate into CD34+ cells.