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  • Delayed administration of ixazomib modifies the immune response and prevents chronic graft-versus-host disease. Bone marrow transplantation Ramos, T. L., Garcia-Guerrero, E., Caballero-Velazquez, T., Rodriguez-Gil, A., Caracuel-Garcia, R., Nufer, M., Robles-Frias, M. J., Barbado, M. V., Perez-Simon, J. A. 2021

    Abstract

    In this study, we aimed to modify the immune response in the long term after allogeneic bone marrow transplantation (allo-BMT) by using the proteasome inhibitor ixazomib (IXZ) at the late stages of the post-transplant period. This approach facilitated the immune reconstitution after transplantation. IXZ significantly prolonged survival and decreased the risk of chronic graft-versus-host disease (cGvHD) in two different murine models without hampering the graft-versus-leukemia (GvL) effect, as confirmed by bioluminescence assays. Remarkably, the use of IXZ was related to an increase of regulatory T cells both in peripheral blood and in the GvHD target organs and a decrease of effector donor T cells. Regarding B cells, IXZ treated mice had faster recovery of B cells in PB and of pre-pro-B cells in the bone marrow. Mice receiving ixazomib had a lower number of neutrophils in the GvHD target organs as compared to the vehicle group. In summary, delayed administration of IXZ ameliorated cGvHD while preserving GvL and promoted a pro-tolerogenic immune response after allo-BMT.

    View details for DOI 10.1038/s41409-021-01452-1

    View details for PubMedID 34556806

  • Invariant Natural Killer T Cell Subsets Have Diverse Graft-Versus-Host-Disease-Preventing and Anti-Tumor Effects. Blood Maas-Bauer, K., Lohmeyer, J. K., Hirai, T., Lopes Ramos, T., Fazal, F. M., Litzenburger, U. M., Yost, K. E., Ribado, J. V., Kambham, N., Wenokur, A., Lin, P., Alvarez, M., Mavers, M., Baker, J., Bhatt, A. S., Chang, H., Simonetta, F., Negrin, R. S. 2021

    Abstract

    Invariant Natural Killer T (iNKT) cells are a T cell subset with potent immunomodulatory properties. Experimental evidence in mice and observational studies in humans indicate that iNKT cells have antitumor potential as well as the ability to suppress acute and chronic Graft-versus-Host-Disease (GvHD). Murine iNKT cells differentiate during thymic development into iNKT1, iNKT2 and iNKT17 sublineages, which differ transcriptomically and epigenomically, and have subset-specific developmental requirements. Whether distinct iNKT sublineages also differ in their antitumor effect and their ability to suppress GvHD is currently unknown. In this work, we generated highly purified murine iNKT-sublineages, characterized their transcriptomic and epigenomic landscape, and assessed specific functions. We demonstrate that iNKT2 and iNKT17, but not iNKT1 cells, efficiently suppress T cell activation in vitro and mitigate murine acute GvHD in vivo. Conversely, we show that iNKT1 cells display the highest antitumor activity against murine B-cell lymphoma cells both in vitro and in vivo. Thus, we demonstrate for the first time that iNKT sublineages have distinct and different functions, with iNKT1 cells having the highest antitumor activity and iNKT2 and iNKT17 cells having immune-regulatory properties. These results have important implications for the translation of iNKT cell therapies to the clinic for cancer immunotherapy as well as for GvHD prevention and treatment.

    View details for DOI 10.1182/blood.2021010887

    View details for PubMedID 34036317

  • Selective expansion of regulatory T cells using an orthogonal IL-2/IL-2 receptor system facilitates transplantation tolerance. The Journal of clinical investigation Hirai, T., Ramos, T. L., Lin, P., Simonetta, F., Su, L. L., Picton, L. K., Baker, J., Lin, J., Li, P., Seo, K., Lohmeyer, J. K., Wagers, S. B., Mavers, M., Leonard, W. J., Blazar, B. R., Garcia, K. C., Negrin, R. S. 2021; 131 (8)

    Abstract

    Adoptive transfer of Tregs has been shown to improve alloengraftment in animal models. However, it is technically challenging to expand Tregs ex vivo for the purpose of infusing large numbers of cells in the clinic. We demonstrate an innovative approach to engineering an orthogonal IL-2/IL-2 receptor (IL-2R) pair, the parts of which selectively interact with each other, transmitting native IL-2 signals, but do not interact with the natural IL-2 or IL-2R counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this orthogonal IL-2R into Tregs. Upon adoptive transfer in a murine mixed hematopoietic chimerism model, orthogonal IL-2 injection significantly promoted orthogonal IL-2R+Foxp3GFP+CD4+ cell proliferation without increasing other T cell subsets and facilitated donor hematopoietic cell engraftment followed by acceptance of heart allografts. Our data indicate that selective target cell stimulation enabled by the engineered orthogonal cytokine receptor improves Treg potential for the induction of organ transplantation tolerance.

    View details for DOI 10.1172/JCI139991

    View details for PubMedID 33855972

  • The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34(+) Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability STEM CELLS Preciado, S., Muntion, S., Corchete, L. A., Ramos, T. L., de la Torre, A. G., Osugui, L., Rico, A., Espinosa-Lara, N., Gastaca, I., Diez-Campelo, M., del Canizo, C., Sanchez-Guijo, F. 2019; 37 (10): 1357–68

    Abstract

    Mesenchymal stromal cells (MSC) may exert their functions by the release of extracellular vesicles (EV). Our aim was to analyze changes induced in CD34+ cells after the incorporation of MSC-EV. MSC-EV were characterized by flow cytometry (FC), Western blot, electron microscopy, and nanoparticle tracking analysis. EV incorporation into CD34+ cells was confirmed by FC and confocal microscopy, and then reverse transcription polymerase chain reaction and arrays were performed in modified CD34+ cells. Apoptosis and cell cycle were also evaluated by FC, phosphorylation of signal activator of transcription 5 (STAT5) by WES Simple, and clonal growth by clonogenic assays. Human engraftment was analyzed 4 weeks after CD34+ cell transplantation in nonobese diabetic/severe combined immunodeficient mice. Our results showed that MSC-EV incorporation induced a downregulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the Janus kinase (JAK)-STAT pathway in CD34+ cells. A significant decrease in apoptosis and an increased CD44 expression were confirmed by FC, and increased levels of phospho-STAT5 were confirmed by WES Simple in CD34+ cells with MSC-EV. In addition, these cells displayed a higher colony-forming unit granulocyte/macrophage clonogenic potential. Finally, 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. Stem Cells 2019;37:1357-1368.

    View details for DOI 10.1002/stem.3032

    View details for Web of Science ID 000491263600011

    View details for PubMedID 31184411

    View details for PubMedCentralID PMC6852558

  • Vitamin D Modifies the Incidence of Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation Depending on the Vitamin D Receptor (VDR) Polymorphisms CLINICAL CANCER RESEARCH Carrillo-Cruz, E., Garcia-Lozano, J. R., Marquez-Malaver, F. J., Sanchez-Guijo, F. M., Montero Cuadrado, I., Ferra i Coll, C., Valcarcel, D., Lopez-Godino, O., Cuesta, M., Parody, R., Lopez-Corral, L., Alcoceba, M., Caballero-Velazquez, T., Rodriguez-Gil, A., Bejarano-Garcia, J. A., Lopes Ramos, T., Perez-Simon, J. A. 2019; 25 (15): 4616–23

    Abstract

    The biologically active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (vit D), has immunoregulatory properties via binding vitamin D receptor (VDR). In a prospective trial, we previously reported a reduction in the incidence of chronic GvHD (cGvHD) among patients who received vit D after allogeneic stem cell transplantation (allo-HSCT; Clinical Trials.gov: NCT02600988). Here we analyze the role of patients and donors' VDR SNPs on the immunomodulatory effect of vit D.Patients undergoing allo-HSCT were included in a prospective phase I/II clinical trial (Alovita) in three consecutive cohorts: control (without vit D), low-dose (1,000 IU/day), and high-dose (5,000 IU/day) groups. Vit D was given from day -5 until +100 after transplant. Genotyping of four SNPs of the VDR gene, FokI, BsmI, ApaI, and TaqI, were performed using TaqMan SNP genotyping assays.We observed a decrease in the incidence of overall cGvHD at 1 year after allo-HSCT depending on the use or not of vit D among patients with FokI CT genotype (22.5% vs 80%, P = 0.0004) and among those patients without BsmI/ApaI/TaqI ATC haplotype (22.2% vs 68.8%, P = 0.0005). In a multivariate analysis, FokI CT genotype significantly influenced the risk of cGvHD in patients treated with vit D as compared with the control group (HR 0.143, Pinteraction < 0.001).Our results show that the immunomodulatory effect of vit D depends on the VDR SNPs, and patients carrying the FokI CT genotype display the highest benefit from receiving vit D after allo-HSCT.

    View details for DOI 10.1158/1078-0432.CCR-18-3875

    View details for Web of Science ID 000478021200007

    View details for PubMedID 31043390

  • Neuroprotective Effects of Diets Containing Olive Oil and DHA/EPA in a Mouse Model of Cerebral Ischemia NUTRIENTS Gonzalo-Gobernado, R., Irene Ayuso, M., Sansone, L., Jose Bernal-Jimenez, J., Dario Ramos-Herrero, V., Sanchez-Garcia, E., Ramos, T. L., Abia, R., Muriana, F. G., Bermudez, B., Montaner, J. 2019; 11 (5)

    Abstract

    Stroke is one of the leading causes of death worldwide and while there is increasing evidence that a Mediterranean diet might decrease the risk of a stroke, the effects of dietary fat composition on stroke outcomes have not been fully explored. We hypothesize that the brain damage provoked by a stroke would be different depending on the source of dietary fat. To test this, male C57BL/6J mice were fed for 4 weeks with a standard low-fat diet (LFD), a high-fat diet (HFD) rich in saturated fatty acids (HFD-SFA), an HFD containing monounsaturated fatty acids (MUFAs) from olive oil (HFD-OO), or an HFD containing MUFAs from olive oil plus polyunsaturated fatty acids (PUFAs) docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA) (HFD-OO-ω3). These mice were then subjected to transient middle cerebral artery occlusion (tMCAo). Behavioural tests and histological analyses were performed 24 and/or 48 h after tMCAo in order to elucidate the impact of these diets with different fatty acid profiles on the ischemic lesion and on neurological functions. Mice fed with HFD-OO-ω3 displayed better histological outcomes after cerebral ischemia than mice that received an HFD-SFA or LFD. Furthermore, PUFA- and MUFA-enriched diets improved the motor function and neurological performance of ischemic mice relative to those fed with an LFD or HFD-SFA. These findings support the use of DHA/EPA-omega-3-fatty acid supplementation and olive oil as dietary source of MUFAs in order to reduce the damage and protect the brain when a stroke occurs.

    View details for DOI 10.3390/nu11051109

    View details for Web of Science ID 000471021600169

    View details for PubMedID 31109078

    View details for PubMedCentralID PMC6566717

  • Vorinostat synergizes with antioxidant therapy to target myeloproliferative neoplasms EXPERIMENTAL HEMATOLOGY Cardoso, B. A., Ramos, T. L., Belo, H., Vilas-Boas, F., Real, C., Almeida, A. M. 2019; 72: 60–71

    Abstract

    BCR-ABL-negative myeloproliferative neoplasms (MPNs) are driven by JAK-STAT pathway activation, but epigenetic alterations also play an important pathophysiological role. These can be pharmacologically manipulated with histone deacetylase inhibitors (HDACIs), which have proven to be clinically effective in the treatment of MPNs but exhibit dose-limiting toxicity. The treatment of primary MPN cells with vorinostat modulates the expression of genes associated with apoptosis, cell cycle, inflammation, and signaling. The induction of this transcriptional program results in decreased cellular viability, paralleled by a decrease in levels of reactive oxygen species (ROS). In vitro manipulation of ROS levels revealed that the reduction of ROS levels promoted apoptosis. When vorinostat was combined with antioxidant agents, the apoptosis of MPN cells increased in a synergistic manner. The results described here suggest a novel and promising therapeutic strategy combining HDACIs with ROS-reducing agents to treat MPNs.

    View details for DOI 10.1016/j.exphem.2019.02.002

    View details for Web of Science ID 000464090000007

    View details for PubMedID 30769020

  • Selection of Tumor-Specific Cytotoxic T Lymphocytes in Acute Myeloid Leukemia Patients Through the Identification of T-Cells Capable to Establish Stable Interactions With the Leukemic Cells: "Doublet Technology" FRONTIERS IN IMMUNOLOGY Garcia-Guerrero, E., Sanchez-Abarca, L. I., Domingo, E., Ramos, T. L., Bejarano-Garcia, J. A., Gonzalez-Campos, J. A., Caballero-Velazquez, T., Perez-Simon, J. A. 2018; 9: 1971

    Abstract

    The relevance of the immune system in cancer has long been studied. Autologous adoptive T cell therapies, based on the use of tumor infiltrating lymphocytes (TILs), have made great progress in recent years for the treatment of solid tumors, especially melanoma. However, further work is needed to isolate tumor-reactive T cells among patients diagnosed with hematologic malignancies. The dynamics of the interaction between T cells and antigen presenting cells (APC) dictate the quality of the immune responses. While stable joints between target cells and T lymphocytes lead to the induction of T cell activation and immune response, brief contacts contribute to the induction of immune-tolerance. Taking advantage of the strong interaction between target cell and activated T-cells, we show the feasibility to identify and isolate tumor-specific cytotoxic T lymphocytes (CTLs) from acute myeloid leukemia (AML) patients by flow cytometry. Using this technology, CTLs bound through T cell receptor (TCR) to tumor cells can be identified in peripheral blood and bone marrow and subsequently selected and isolated by FACS-based cell sorting. These CTLs display higher percentage of effector cells and marked cytotoxic activity against AML blasts. In conclusion, we have developed a new procedure to identify and select specific cytotoxic T cells in patients diagnosed with acute myeloid leukemia.

    View details for DOI 10.3389/fimmu.2018.01971

    View details for Web of Science ID 000443613700001

    View details for PubMedID 30233577

    View details for PubMedCentralID PMC6129592

  • Biocompatibility of two model elastin-like recombinamer-based hydrogels formed through physical or chemical cross-linking for various applications in tissue engineering and regenerative medicine JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE Ibanez-Fonseca, A., Ramos, T. L., Gonzalez de Torre, I., Ignacio Sanchez-Abarca, L., Muntion, S., Javier Arias, F., Consuelo del Canizo, M., Alonso, M., Sanchez-Guijo, F., Carlos Rodriguez-Cabello, J. 2018; 12 (3): E1450–E1460

    Abstract

    Biocompatibility studies, especially innate immunity induction, in vitro and in vivo cytotoxicity, and fibrosis, are often lacking for many novel biomaterials including recombinant protein-based ones, such as elastin-like recombinamers (ELRs), and has not been extensively explored in the scientific literature, in contrast to traditional biomaterials. Herein, we present the results from a set of experiments designed to elucidate the preliminary biocompatibility of 2 types of ELRs that are able to form extracellular matrix-like hydrogels through either physical or chemical cross-linking both of which are intended for different applications in tissue engineering and regenerative medicine. Initially, we present in vitro cytocompatibility results obtained upon culturing human umbilical vein endothelial cells on ELR substrates, showing optimal proliferation up to 9 days. Regarding in vivo cytocompatibility, luciferase-expressing hMSCs were viable for at least 4 weeks in terms of bioluminescence emission when embedded in ELR hydrogels and injected subcutaneously into immunosuppressed mice. Furthermore, both types of ELR-based hydrogels were injected subcutaneously in immunocompetent mice and serum TNFα, IL-1β, IL-4, IL-6, and IL-10 concentrations were measured by enzyme-linked immunosorbent assay, confirming the lack of inflammatory response, as also observed upon macroscopic and histological evaluation. All these findings suggest that both types of ELRs possess broad biocompatibility, thus making them very promising for tissue engineering and regenerative medicine-related applications.

    View details for DOI 10.1002/term.2562

    View details for Web of Science ID 000427137100014

    View details for PubMedID 28865091

  • Mesenchymal Stromal Cell Irradiation Interferes with the AdipogeniciOsteogenic Differentiation Balance and Improves Their Hematopoietic-Supporting Ability BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Preciado, S., Muntion, S., Rico, A., Perez-Romasanta, L. A., Ramos, T. L., Ortega, R., Borrajo, J., Corchete, L. A., Rodriguez, C., Diez-Campelo, M., Sanchez-Abarca, L. I., Del Canizo, M., Sanchez-Guijo, F. 2018; 24 (3): 443–51

    Abstract

    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.

    View details for DOI 10.1016/j.bbmt.2017.11.007

    View details for Web of Science ID 000427663000005

    View details for PubMedID 29155314

  • HDAC8 overexpression in mesenchymal stromal cells from JAK2(+) myeloproliferative neoplasms: a new therapeutic target? ONCOTARGET Ramos, T. L., Ignacio Sanchez-Abarca, L., Redondo, A., Hernandez-Hernandez, A., Almeida, A. M., Puig, N., Rodriguez, C., Ortega, R., Preciado, S., Rico, A., Muntion, S., Gonzalez Porras, J., Del Canizo, C., Sanchez-Guijo, F. 2017; 8 (17): 28187–202

    Abstract

    Histone deacetylases (HDACs) are involved in epigenetic modulation and their aberrant expression has been demonstrated in myeloproliferative neoplasms (MPN). HDAC8 inhibition has been shown to inhibit JAK2/STAT5 signaling in hematopoietic cells from MPN. Nevertheless, the role of HDAC8 expression in bone marrow-mesenchymal stromal cells (BM-MSC) has not been assessed. In the current work we describe that HDAC8 is significantly over-expressed in MSC from in JAK-2 positive MPN compared to those from healthy-donors (HD-MSC). Using a selective HDAC8 inhibitor (PCI34051), we verified that the subsequent decrease in the protein and mRNA expression of HDAC8 is linked with an increased apoptosis of malignant MSC whereas it has no effects on normal MSC. In addition, HDAC8 inhibition in MPN-MSC also decreased their capacity to maintain neoplastic hematopoiesis, by increasing the apoptosis, cell-cycle arrest and colony formation of JAK2+-hematopoietic cells. Mechanistic studies using different MPN cell lines revealed that PCI34051 induced their apoptosis, which is enhanced when were co-cultured with JAK2V617F-MSC, decreased their colony formation and the phosphorylation of STAT3 and STAT5. In summary, we show for the first time that the inhibition of HDAC8 in MSC from JAK2+ MPN patients selectively decreases their hematopoietic-supporting ability, suggesting that HDAC8 may be a potential therapeutic target in this setting by acting not only on hematopoietic cells but also on the malignant microenvironment.

    View details for DOI 10.18632/oncotarget.15969

    View details for Web of Science ID 000400050000046

    View details for PubMedID 28390197

    View details for PubMedCentralID PMC5438642

  • Assessment of dry eye in a GVHD murine model: Approximation through tear osmolarity measurement EXPERIMENTAL EYE RESEARCH Martinez-Carrasco, R., Ignacio Sanchez-Abarca, L., Nieto-Gomez, C., Martin Garcia, E., Ramos, T. L., Velasco, A., Sanchez-Guijo, F., Aijon, J., Hernandez-Galilea, E. 2017; 154: 64–69

    Abstract

    Dry eye disease is one of the most frequent pathological events that take place in the course of the graft versus host disease (GVHD), and is the main cause of deterioration in quality of life for patients. Thus, demonstration of dry eye signs in murine models of oGVHD is crucial for the validation of these models for the study of the disease. Given the increasing evidence that tear osmolarity is an important player of dry eye disease, our purpose in this study was to validate the use of a reliable method to assess tear osmolarity in mice: the electrical impedance method. Then, we wanted to test its utility with an oGVHD model. Tear volume assessment was also performed, using the phenol red thread test. We found differences in tear osmolarity in mice that received a transplant with cells from bone marrow and spleen (the GVHD group) when compared with mice that only received bone marrow cells (the BM group) at day 7 (362 ± 8 mOsm/l and 345 ± 9 mOsm/l respectively; P < 0.01) and day 21 (348 ± 19 mOsm/l vs. 326 ± 15 mOsm/l; P < 0.05). We found also differences in tear volume at day 14 (2.30 ± 0.61 mm in oGVHD group and 2.89 ± 0.62 mm in BM group; P = 0.06) and at day 21 (2.10 ± 0.30 mm in oGVHD group and 2.89 ± 0.32 mm in BM group; P < 0.01). Besides this, we observed reduction in epithelial thickness between the GVHD and BM groups (37.0 ± 6.2 μm and 43.6 ± 3.3 μm respectively; P < 0.05). These data show the usefulness of the electrical impedance method to measure tear osmolarity in mice. We can also conclude that this oGVHD model mimics the tear film alterations found in human dry eye disease, what contributes to give relevance to this model for the study of GVHD.

    View details for DOI 10.1016/j.exer.2016.11.004

    View details for Web of Science ID 000397548100008

    View details for PubMedID 27818317

  • Mesenchymal stromal cells (MSC) from JAK2+ myeloproliferative neoplasms differ from normal MSC and contribute to the maintenance of neoplastic hematopoiesis. PloS one Ramos, T. L., Sánchez-Abarca, L. I., Rosón-Burgo, B. n., Redondo, A. n., Rico, A. n., Preciado, S. n., Ortega, R. n., Rodríguez, C. n., Muntión, S. n., Hernández-Hernández, Á. n., De Las Rivas, J. n., González, M. n., González Porras, J. R., Del Cañizo, C. n., Sánchez-Guijo, F. n. 2017; 12 (8): e0182470

    Abstract

    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 (HD-MSC) 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 cells.

    View details for DOI 10.1371/journal.pone.0182470

    View details for PubMedID 28796790

    View details for PubMedCentralID PMC5552029

  • Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients PLOS ONE Muntion, S., Ramos, T. L., Diez-Campelo, M., Roson, B., Ignacio Sanchez-Abarca, L., Misiewicz-Krzeminska, I., Preciado, S., Sarasquete, M., de las Rivas, J., Gonzalez, M., Sanchez-Guijo, F., del Canizo, M. 2016; 11 (2): e0146722

    Abstract

    Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.

    View details for DOI 10.1371/journal.pone.0146722

    View details for Web of Science ID 000369548500001

    View details for PubMedID 26836120

    View details for PubMedCentralID PMC4737489

  • MSC surface markers (CD44, CD73, and CD90) can identify human MSC-derived extracellular vesicles by conventional flow cytometry CELL COMMUNICATION AND SIGNALING Ramos, T. L., Ignacio Sanchez-Abarca, L., Muntion, S., Preciado, S., Puig, N., Lopez-Ruano, G., Hernandez-Hernandez, A., Redondo, A., Ortega, R., Rodriguez, C., Sanchez-Guijo, F., del Canizo, C. 2016; 14: 2

    Abstract

    Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. In the last few years it has been shown that the beneficial effects of hMSC may be due to paracrine effects and, at least in part, mediated by extracellular vesicles (EV). EV have emerged as important mediators of cell-to-cell communication. Flow cytometry (FCM) is a routine technology used in most clinical laboratories and could be used as a methodology for hMSC-EV characterization. Although several reports have characterized EV by FCM, a specific panel and protocol for hMSC-derived EV is lacking. The main objective of our study was the characterization of hMSC-EV using a standard flow cytometer.Human MSC from bone marrow of healthy donors, mesenchymal cell lines (HS-5 and hTERT) and a leukemic cell line (K562 cells) were used to obtain EV for FCM characterization. EV released from the different cell lines were isolated by ultracentrifugation and were characterized, using a multi-parametric analysis, in a conventional flow cytometer. EV characterization by transmission electron microscopy (TEM), western blot (WB) and Nano-particle tracking analysis (NTA) was also performed.EV membranes are constituted by the combination of specific cell surface molecules depending on their cell of origin, together with specific proteins like tetraspanins (e.g. CD63). We have characterized by FCM the EV released from BM-hMSC, that were defined as particles less than 0.9 μm, positive for the hMSC markers (CD90, CD44 and CD73) and negative for CD34 and CD45 (hematopoietic markers). In addition, hMSC-derived EV were also positive for CD63 and CD81, the two characteristic markers of EV. To validate our characterization strategy, EV from mesenchymal cell lines (hTERT/HS-5) were also studied, using the leukemia cell line (K562) as a negative control. EV released from mesenchymal cell lines displayed the same immunophenotypic profile as the EV from primary BM-hMSC, while the EV derived from K562 cells did not show hMSC markers. We further validated the panel using EV from hMSC transduced with GFP. Finally, EV derived from the different sources (hMSC, hTERT/HS-5 and K562) were also characterized by WB, TEM and NTA, demonstrating the expression by WB of the exosomal markers CD63 and CD81, as well as CD73 in those from MSC origin. EV morphology and size/concentration was confirmed by TEM and NTA, respectively.We described a strategy that allows the identification and characterization by flow cytometry of hMSC-derived EV that can be routinely used in most laboratories with a standard flow cytometry facility.

    View details for DOI 10.1186/s12964-015-0124-8

    View details for Web of Science ID 000368053800001

    View details for PubMedID 26754424

    View details for PubMedCentralID PMC4709865

  • PTPN13 and beta-Catenin Regulate the Quiescence of Hematopoietic Stem Cells and Their Interaction with the Bone Marrow Niche STEM CELL REPORTS Lopez-Ruano, G., Prieto-Bermejo, R., Ramos, T. L., San-Segundo, L., Ignacio Sanchez-Abarca, L., Sanchez-Guijo, F., Antonio Perez-Simon, J., Sanchez-Yaguee, J., Llanillo, M., Hernandez-Hernandez, A. 2015; 5 (4): 516–31

    Abstract

    The regulation of hematopoietic stem cells (HSCs) depends on the integration of the multiple signals received from the bone marrow niche. We show the relevance of the protein tyrosine phosphatase PTPN13 and β-catenin as intracellular signaling molecules to control HSCs adhesiveness, cell cycling, and quiescence. Lethally irradiated mice transplanted with Lin(-) bone marrow cells in which PTPN13 or β-catenin had been silenced showed a significant increase of long-term (LT) and short-term (ST) HSCs. A decrease in cycling cells was also found, together with an increase in quiescence. The decreased expression of PTPN13 or β-catenin was linked to the upregulation of several genes coding for integrins and several cadherins, explaining the higher cell adhesiveness. Our data are consistent with the notion that the levels of PTPN13 and β-catenin must be strictly regulated by extracellular signaling to regulate HSC attachment to the niche and the balance between proliferation and quiescence.

    View details for DOI 10.1016/j.stemcr.2015.08.003

    View details for Web of Science ID 000364990900007

    View details for PubMedID 26344907

    View details for PubMedCentralID PMC4624939

  • Effects of MSC Coadministration and Route of Delivery on Cord Blood Hematopoietic Stem Cell Engraftment CELL TRANSPLANTATION Carrancio, S., Romo, C., Ramos, T., Lopez-Holgado, N., Muntion, S., Prins, H. J., Martens, A. C., Brinon, J. G., San Miguel, J. F., del Canizo, M. C., Sanchez-Guijo, F. 2013; 22 (7): 1171–83

    Abstract

    Hematopoietic stem cell transplantation (HSCT) using umbilical cord blood (UCB) progenitors is increasingly being used. One of the problems that may arise after UCB transplantation is an impaired engraftment. Either intrabone (IB) injection of hematopoietic progenitors or mesenchymal stem cell (MSC) coadministration has been proposed among the strategies to improve engraftment. In the current study, we have assessed the effects of both approaches. Thus, NOD/SCID recipients were transplanted with human UCB CD34+ cells administered either intravenously (IV) or IB, receiving or not bone marrow (BM)-derived MSCs also IV or IB (in the right femur). Human HSC engraftment was measured 3 and 6 weeks after transplantation. Injected MSCs were tracked weekly by bioluminescence. Also, lodgment within the BM niche was assessed at the latter time point by immuno-fluorescence. Our study shows regarding HSC engraftment that the number of BM human CD45+ cells detected 3 weeks after transplantation was significantly higher in mice cotransplanted with human MSCs. Moreover, these mice had a higher myeloid (CD13+) engraftment and a faster B-cell (CD19+) chimerism. At the late time point evaluated (6 weeks), human engraftment was higher in the group in which both strategies were employed (IB injection of HSC and MSC coadministration). When assessing human MSC administration route, we were able to track MSCs only in the injected femurs, whereas they lost their signal in the contralateral bones. These human MSCs were mainly located around blood vessels in the subendosteal region. In summary, our study shows that MSC coadministration can enhance HSC engraftment in our xenogenic transplantation model, as well as IB administration of the CD34+ cells does. The combination of both strategies seems to be synergistic. Interestingly, MSCs were detected only where they were IB injected contributing to the vascular niche.

    View details for DOI 10.3727/096368912X657431

    View details for Web of Science ID 000322501300007

    View details for PubMedID 23031585