| It is known that there are two kind of stem cell in bone marrow, one is hematopoietic stem cell (HSC) and the other is Mesenchymal stem cell(MSC). MSC has been proved to have multilineage differentiation potential and can differentiate into hematopoietic stromal cells, adipocytes,fibroblasts,and osteogenic precursor cells,which are comprised of hematopoietic microenvironment. They provide cell-to-cell interactions, expression and presentation of hematopoietic growth factors,and the secretion of extracellular matrix proteins. Bone marrow microenvironment provide a favorable platform for the localization, self-renewal,and differentiation of hematopoietic stem cell. But now the relation between MSC and HSC remain to be unknown. In this study, we try to explore if MSC can regulate hematopoiesis in vitro and what effects it has.In part I research, a bone marrow aspirate was collected and processed using density gradient centrifugation, from which light-density cells were taken and plated in a DMEM media containing 10%FBS. After allowing 1 day for adherence to culture flask, nonadherent cells were removed. After a 14-day primary expansion period, MSC nearly reached confluency, and these cells had a fibroblast-like morphology. Confluent cultures could be processed further by trypsinization and expansion through sequential passages to confluency. MSC at passage 3 were analysed by flow cytometry, MSC were uniformly positive for CD29(99.7%),CD44(99.1%) and CD166(83.2%), negative for CD34,CD45 and HLA-DR. the result verified that there were not hematopoietic cells in these cells isolated and cultured in vitro.There were not any exclusive Immunophenotypic markers which had been found in MSC, sothat it is impossible to identify it directly. The only way to do it is to assess its multipotent differentiation ability. The differentiation ability of MSCs was assessed in our research. Osteogenic differentiation was assessed by incubating the cells with DMEM containing 10% FBS supplemented with 10-8 M dexamethasone, 0.2 mM ascorbic acid, and 10 mM glycerol phosphate, adipogenic differentiation was assessed by incubation with DMEM containing 10% FBS supplemented with 10-7 M dexamethasone, 10-9 M insulin. RT-PCR assay showed that osteoblast and adipocyte expressed osteopontin and LpL respectively after induced culture., cytochemical staining results displayed that osteogenic differentiation was positive for Von Kossa staining and adipogenic differentiation was positive for Oil-red-O. staining. These data also implied that the cells isolated and cultured in vitro were MSC.To investigate hematopoietic regulation ability of MSC in vitro, MSC was used as feeder cells, but they had never been irradiated, because after irradiation, these cells might possibly undergo morphologic , phenotypic , and regulatory changes that make them unpredictable surrogates for their normal cell counterparts. Mononuclear cells (MNC) isolated from umbilical cord blood cells were seeded in 6-well plates in which MSC had been seeded and had reached confluence. Control experiments were also performed by :1) culturing cord blood MNC in the absence of a feeder cell layer, 2) culturing cord blood MNC supplemented with the supernatant of MSCs in the absence of a feeder cell layer .The cocultures were incubated at 37 in 5% CO2 in air in DMEM-LG medium supplemented with 10% FBS.Under this culture condition, MSC can survive and FBS was so poor that the effect of unknown factor in FBS might be minimized. There were few adherent hematopoietic cells could be observed during the first week,most cells were nonadherent and even the number of hematopoietic cells were reduced. After 10 days of coculture, many cells started to attach tightly to MSC and proliferated rapidly over the feeder cells.Some colonies could be seen in the early stage,but later it was difficult to recognize them because of the rapid proliferation of hematopoietic cells which made the colonies overlapped.After 21 days of coculture, the medium was removed and cells were washed twice with PB... |
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