RESUMEN
Various families of ion channels have been characterized in mesenchymal stem cells (MSCs), including some members of transient receptor potential (TRP) channels family. TRP channels are involved in critical cellular processes as differentiation and cell proliferation. Here, we analyzed the expression of TRPM8 channel in human bone marrow MSCs (hBM-MSCs), and its relation with osteogenic differentiation. Patch-clamp recordings showed that hBM-MSCs expressed outwardly rectifying currents which were increased by exposure to 500 µM menthol and were partially inhibited by 10 µM of BCTC, a TRPM8 channels antagonist. Additionally, we have found the expression of TRPM8 by RT-PCR and western blot. We also explored the TRPM8 localization in hBM-MSCs by immunofluorescence using confocal microscopy. Remarkably, hBM-MSCs treatment with 100 µM of menthol or 10 µM of icilin, TRPM8 agonists, increases osteogenic differentiation. Conversely, 20 µM of BCTC, induced a decrease of osteogenic differentiation. These results suggest that TRPM8 channels are functionally active in hBM-MSCs and have a role in cell differentiation.
RESUMEN
BACKGROUND AND OBJECTIVE: The characteristics of human hematopoietic stem cells are conditioned by the microenvironment of the bone marrow, where they interact with other cell populations, such as mesenchymal stem cells and endothelial cells; however, the study of this microenvironment is complex. The objective of this work was to develop a 3D culture system by magnetic levitation that imitates the microenvironment of human HSC. METHODS AND RESULTS: Human bone marrow-mesenchymal stem cells, umbilical cord blood-hematopoietic stem cells and a non-tumoral endothelial cell line (CC2811, Lonzaâ) were used to develop organotypic multicellular spheres by the magnetic levitation method. We obtained viable structures with an average sphericity index greater than 0.6, an average volume of 0.5 mm3 and a percentage of aggregation greater than 70%. Histological studies of the organotypic multicellular spheres used hematoxylin and eosin stains, and an evaluation of vimentin expression by means of immunohistochemistry demonstrated an organized internal structure without picnotic cells and a high expression of vimentin. The functional capacity of human hematopoietic stem cells after organotypic multicellular spheres culture was evaluated by multipotency tests, and it was demonstrated that 3D structures without exogenous Flt3L are autonomous in the maintenance of multipotency of human hematopoietic stem cells. CONCLUSIONS: We developed organotypic multicellular spheres from normal human cells that mimic the microenvironment of the human hematopoietic stem cells. These structures are the prototype for the development of complex organoids that allow the further study of the biology of normal human stem cells and their potential in regenerative medicine.
RESUMEN
BACKGROUND AND OBJECTIVES: The role of bone marrow-mesenchymal stem cells (BM-MSC) in leukaemic cell control is controversial. The purpose of this work was to evaluate BM-MSC role regarding the viability, proliferation and immunophenotype of normal B-cell precursors from control (Ct) patients and leukaemic cells from B-acute lymphoblastic leukaemia (B-ALL) patients. PATIENTS AND METHODS: BM-MSC were isolated and characterised from voluntary donors. Mononuclear cells isolated from Ct and B-ALL bone marrow samples were cultured in the presence or absence of BM-MSC for 7days. Cell viability was determined with LIVE/DEAD and proliferation index evaluated by CFSE labelling. Cell population immunophenotypes were characterised by estimating CD19, CD10, CD20 and CD45 antigens by flow cytometry. RESULTS: After co-culture, B-ALL cells exhibited higher viability (20-40%) as compared to just cells (3-10%). Ct and B-ALL absolute cell counts were higher in the presence of BM-MSC (Ct: 25/mm(3)cf8/mm(3), B-ALL: 15/mm(3)cf3/mm(3)). Normal B-cell subpopulations in co-culture had increased expression of CD19 and CD10 (Pre-pre B) and CD45 and CD20 antigens (Pre-B). B-ALL cells co-cultured with BM-MSC showed an increase in CD19 and CD20, although the greatest increase was observed in the CD10 antigen. CONCLUSIONS: Lymphoid cell maintenance, at early stages of differentiation, was significantly promoted by BM-MSC in normal and leukaemic cells. Co-cultures also modulated the expression of antigens associated with the B-ALL asynchronous phenotype as CD10 co-expressed with CD19 and CD20. To our knowledge, this is the first time that CD10, CD19 and CD20 leukaemic antigens have been reported as being regulated by BM-MSC.
Asunto(s)
Linfocitos B/patología , Células Madre Mesenquimatosas/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Células de la Médula Ósea/patología , Estudios de Casos y Controles , Proliferación Celular , Forma de la Célula , Supervivencia Celular , Análisis por Conglomerados , Técnicas de Cocultivo , Humanos , Inmunofenotipificación , Recuento de Linfocitos , Células Madre Multipotentes/patología , Fenotipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunologíaRESUMEN
The purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increased CD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.