| During the course of development, pluripotent stem cells produce multipotent stern cells , the latter bringing out tissue specific stem cells , following with commited progenitor cells and terminal differentiated cells. In recent years, each scape stem cell research has been made great achievements. In 1998, James A.Thomson successfully established human Embryonic Stem CelKESOlines, which set foundation for cloning of human organs and tissues. Now, there has been reports of murine and human embryonic stem cell differentiated into hematopoietic stem/progenitor cells, cardiomyoblast, neuron, epidermal stem cells and pancreatic B cells, etc .Bone marrow containing hematopoietic stem cells has already been known , these's another group stem cells exists in bone marrow named mesenchymal stem cells (MSCs). MSCs retain the potentiality of differentiating into osteoblast, chondroblast, adipoblast, tendonblast, myoblast, cardiomyoblast, neuron, etc , which displays a bright prosperity of its use in tissue enigeering, wound healing , cell transplatation and gene therapy. MSCs can be obtained and expanded easily compared to ESC without any problems of morality , law and ethic , immunology. MSCs have become an area of active investigation of stem cell.Bone marrow mesenchymal stem cells are a group of homogenous cells with unique characteristic. They express several markers. The majority of4cells is in the Go/Gi phase. Some associated genes are intiated by the agents when they exposed into adipogenesis and osteogeneisis inducing mediums. They express AKP, osteocalcin, osteopontin, PPAR Y 2 and aP2, etc.Mesenchymal stem cells originate from various organs and tissues including liver, peripheral blood, dermis, pancreatis, etc. People should pay attention to them.Aims to isolate MSCs from bone marrow aspiration , then culture , and identify the obtained cells by detecting cell surface antigens, cell cycle, and further induce them into adipoblasts and osteoblasts.Materials and Methods1. Isolation and Cultivation of MSCs:35 cases of bone marrow samples were obtained from healthy adult human donors at the Second Affiliated Hospital of Zhejiang University. Each donor was obtained information consent. Mononuclear cells were separated by centrifugation in Percoll solution (density 1.073g/ml, Pharmacia) followed by the method of M.F. Pittenger' s (Science 1999,284:143-147). Then they were seeded in DMEM-LG supplemented with 10% fetal calf-serum (Gibco BRL) and 1% antibiotic-antimyotic solution at a concentration of IxlO6 cells/cm2 . Cultures were maintained at 37癈 in a humidified atmosphere camber containing 5% CC<2. The medium was changed 24 hours and 72 hours, nonadherent cells were removed with change in medium, and twice weekly thereafter. When the cultures reached nearly 90% of confluence, cells were trypsinized with 0.05% trypsin (Sigma), washed, resuspened at a ratio of 1:3. The serum lots selected for MSCs outgrowth from marrow aspiration were chosen for their optimum ability to promote the growth of an adherent, well-spread colony-form cell.2. Immunophenotyping of MSCs:Detections of cell surface antigens were performed on Passages 3 cultures of MSCs using flow cytometry. Cells were detached by 0.05% trypsin, washed, recovered by centrifugation at a density of 1><105 cells/ml. Aliquots (100 v 1 ) of suspension were incubated with respective 20 n 1 of fluorescence conjugated mouse anti human monoclonal antibodies as CD14-PE/CD45-FITC, CD34-FITC/CD45-Percp, CD44-FITC, VLA-1-FITC, HLA-DR-PE ,CD105-FITC, CD29-PE (Beckon Dickinson)and isotype negative controlled mouse anti mouse antibodies IgGl-FITC, IgG2a-PE (Beckon Dickinson). All incubations with antibodies performed for 20 min at room temperature, washed with PBS. Each sample was analyzed by collecting at least 10,000 events on a Beckton Dickinson Vantage instrument using CellQaest Software.3. Cell cycle Status of MSCs:On passage 3 cultures, 2><105 cells were harvested from flasks by treatment with 0.05% trypsin in PBS, permeabil... |
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