Papel del Estroma Medular en la Mejora de la Función Hematopoyética: Desde la Irradiación hasta las Vesículas Extracelulares

[EN] 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

Experiencia piloto de “micro flipped teaching” (clase invertida) en la asignatura “Células madre de la Médula Ósea: características biológicas y su posible papel en el desarrollo de neoplasias”

Memoria ID2019/067. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2019-2020

Mesenchymal stromal cells combined with elastin-like recombinamers increase angiogenesis in vivo after hindlimb ischemia

Producción CientíficaHindlimb ischemia is an unmet medical need, especially for those patients unable to undergo vascular surgery. Cellular therapy, mainly through mesenchymal stromal cell (MSC) administration, may be a potentially attractive approach in this setting. In the current work, we aimed to assess the potential of the combination of MSCs with a proangiogenic elastin-like recombinamer (ELR)–based hydrogel in a hindlimb ischemia murine model. Human bone marrow MSCs were isolated from four healthy donors, while ELR biomaterials were genetically engineered. Hindlimb ischemia was induced through ligation of the right femoral artery, and mice were intramuscularly injected with ELR biomaterial, 0.5 × 106 MSCs or the combination, and also compared to untreated animals. Tissue perfusion was monitored using laser Doppler perfusion imaging. Histological analysis of hindlimbs was performed after hematoxylin and eosin staining. Immunofluorescence with anti–human mitochondria antibody was used for human MSC detection, and the biomaterial was detected by elastin staining. To analyze the capillary density, immunostaining with an anti–CD31 antibody was performed. Our results show that the injection of MSCs significantly improves tissue reperfusion from day 7 (p = 0.0044) to day 21 (p = 0.0216), similar to the infusion of MSC + ELR (p = 0.0038, p = 0.0014), without significant differences between both groups. After histological evaluation, ELR hydrogels induced minimal inflammation in the injection sites, showing biocompatibility. MSCs persisted with the biomaterial after 21 days, both in vitro and in vivo. Finally, we observed a higher blood vessel density when mice were treated with MSCs compared to control (p<0.0001), but this effect was maximized and significantly different to the remaining experimental conditions when mice were treated with the combination of MSCs and the ELR biomaterial (p < 0.0001). In summary, the combination of an ELR-based hydrogel with MSCs may improve the angiogenic effects of both strategies on revascularization of ischemic tissues.Spanish Government (RTI2018-096320-B-C22, FPU16-04015, PID2019-110709RB-I00, PID2020-118669RA-I00)Interreg V España Portugal POCTEP (0624_2IQBIONEURO_6_E), Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y LeónConsejería de Educación de Castilla y León (CAS079P17)Instituto de Salud Carlos III (ISCIII) (PI19/01630)Programs of ISCIII- European Regional Development Fund (RD16/0011/0015, RD21/ 0017/0006

“Micropíldoras” educativas para la formación práctica en las asignaturas de la Unidad Docente de Terapia Celular del Departamento de Medicina

Memoria ID-047 Ayudas de la Universidad de Salamanca para la innovación docente, curso 2020-2021

Mesenchymal stromal cells (MSC) from JAK2+ myeloproliferative neoplasms differ from normal MSC and contribute to the maintenance of neoplastic hematopoiesis

[EN]There is evidence of continuous bidirectional cross-talk between malignant cells and bone marrow-derived mesenchymal stromal cells (BM-MSC), which favors the emergence and progression of myeloproliferative neoplastic (MPN) diseases. In the current work we have compared the function and gene expression profile of BM-MSC from healthy donors (HDMSC) and patients with MPN (JAK2V617F), showing no differences in the morphology, proliferation and differentiation capacity between both groups. However, BM-MSC from MPN expressed higher mean fluorescence intensity (MIF) of CD73, CD44 and CD90, whereas CD105 was lower when compared to controls. Gene expression profile of BM-MSC showed a total of 169 genes that were differentially expressed in BM-MSC from MPN patients compared to HD-MSC. In addition, we studied the ability of BM-MSC to support the growth and survival of hematopoietic stem/progenitor cells (HSPC), showing a significant increase in the number of CFU-GM colonies when MPN-HSPC were co-cultured with MPN-MSC. Furthermore, MPN-MSC showed alteration in the expression of genes associated to the maintenance of hematopoiesis, with an overexpression of SPP1 and NF-kB, and a downregulation of ANGPT1 and THPO. Our results suggest that BM-MSC from JAK2+ patients differ from their normal counterparts and favor the maintenance of malignant clonal hematopoietic cell

Foliar Aspersion of Salicylic Acid Improves Phenolic and Flavonoid Compounds, and Also the Fruit Yield in Cucumber (<i>Cucumis sativus</i> L.)

The aim of this research is to evaluate the effect of foliar application of salicylic acid (SA) on the yield and phytochemical content in hydroponically grown cucumber (Cucumis sativus L.). (1) Background: The importance of Mexico&#8217;s cucumber production is based on its cultivation in recent decades as one of the major winter crops; in addition, the production of vegetables under hydroponic systems has increased significantly during the last few years, with cucumber being one of the vegetables with a high economic potential. (2) Methods: A completely randomized experimental design with 15 repetitions was used. SA at five doses (0.075, 0.1, 0.15, 0.25, and 0.5 mM) and one control (distilled water) was sprinkled weekly on cucumber plants. The evaluated variables were yield (total fruit weight per plant), fruit parameters (length, size and firmness), and nutraceutical quality of cucumber. (3) Results: Low concentrations of SA improve the yield and high concentrations decrease it, but the nutraceutical quality of fruits is improved, as compared to the control treatment. (4) Conclusions: In order to obtain a higher content of bioactive compounds without affecting the yield and commercial quality of cucumber fruits, it is advisable to use the average concentration (0.15 mM) of SA

Mesenchymal Stromal Cell Irradiation Interferes with the Adipogenic/Osteogenic Differentiation Balance and Improves Their Hematopoietic-Supporting Ability

[EN]Bone marrow mesenchymal stromal cells (MSCs) are precursors of adipocytes and osteoblasts and key regulators of hematopoiesis. Irradiation is widely used in conditioning regimens. Although MSCs are radioresistant, the effects of low-dose irradiation on their behavior have not been extensively explored. Our aim was to evaluate the effect of 2.5 Gy on MSCs. Cells from 25 healthy donors were either irradiated or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Apoptosis was evaluated by annexin V/7-amino-actinomycin staining. Gene expression profiling and reverse transcriptase (RT)-PCR of relevant genes was also performed. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Our results showed that immunophenotypic characterization and viability of irradiated cells was comparable with that of control cells. Gene expression profiling showed 50 genes differentially expressed. By RT-PCR, SDF-1 and ANGPT were overexpressed, whereas COL1A1 was downregulated in irradiated cells (P = .015, P = .007, and P = .031, respectively). Interestingly, differentiation of irradiated cells was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of genes involved in osteogenesis as SPP1 (P = .039) and lower of genes involved in adipogenesis, CEBPA and PPARG (P = .003 and P = .019), together with an increase in the mineralization capacity (Alizarin Red) was observed in irradiated cells. After differentiation, adipocyte counts were decreased in irradiated cells at days 7, 14, and 21 (P = .018 P = .046, and P = .018, respectively). Also, colony-forming unit granulocyte macrophage number in long-term bone marrow cultures was signifi- cantly higher in irradiated cells after 4 and 5 weeks (P = .046 and P = .007). In summary, the irradiation of MSCs with 2.5 Gy improves their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis

Mesenchymal Stromal Cell Irradiation Interferes with the Adipogenic/Osteogenic Differentiation Balance and Improves Their Hematopoietic-Supporting Ability.

Bone marrow mesenchymal stromal cells (MSCs) are precursors of adipocytes and osteoblasts and key regulators of hematopoiesis. Irradiation is widely used in conditioning regimens. Although MSCs are radio-resistant, the effects of low-dose irradiation on their behavior have not been extensively explored. Our aim was to evaluate the effect of 2.5 Gy on MSCs. Cells from 25 healthy donors were either irradiated or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Apoptosis was evaluated by annexin V/7-amino-actinomycin staining. Gene expression profiling and reverse transcriptase (RT)-PCR of relevant genes was also performed. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Our results showed that immunophenotypic characterization and viability of irradiated cells was comparable with that of control cells. Gene expression profiling showed 50 genes differentially expressed. By RT-PCR, SDF-1 and ANGPT were overexpressed, whereas COL1A1 was downregulated in irradiated cells (P = .015, P = .007, and P = .031, respectively). Interestingly, differentiation of irradiated cells was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of genes involved in osteogenesis as SPP1 (P = .039) and lower of genes involved in adipogenesis, CEBPA and PPARG (P = .003 and P = .019), together with an increase in the mineralization capacity (Alizarin Red) was observed in irradiated cells. After differentiation, adipocyte counts were decreased in irradiated cells at days 7, 14, and 21 (P = .018 P = .046, and P = .018, respectively). Also, colony-forming unit granulocyte macrophage number in long-term bone marrow cultures was significantly higher in irradiated cells after 4 and 5 weeks (P = .046 and P = .007). In summary, the irradiation of MSCs with 2.5 Gy improves their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis