| Objective: investigati ng the method and conditions of isolation, proliferation of multipotent mesenchymal stem cells(MSCs) from human umbilical cord blood in vitro, and inducing MSCs differentiation to osteoblasts and adipocytes directly then identificating them. Method: human umbilical cord blood was collected in asepsis condition, isolated by density gradient centrifugation, or sedimented red cell with methylcellulose then centrifugated,. or obtained by negative immunodepletion of CD34+. These isolated cells(mononuclear cells) were used to carry on plastic adherent culture. To obtain single cell-derived colonies, these cells were proliferated clonally in medium which consists of L-DMEM or Mesencult? medium(+10% fetal calf serum(FCS) respectively), then tested for their differentiation potentiality to osteoblasts and lipoblasts. Result: The way that mononuclear cells were isolated by sedimented then centrifugated and cultured in Mesencult? medium+10%FCS are most available, These adhesive cells were obviously short rod-shape or shuttle-shape, single core, long enation, strong reflection and adhesion, and formed colonies in 3～4 weeks, The colonies form well in third-passage cells. The mononuclear cells obtained by only centrifugalized in density gradient were difficulty to form colony, and isolated by immunomagnetic beads were difficulty to culture. The surface antigens of these colonies cells present CD29, CD59, CD71 but not the CD34, CD45 and HLA-DR etc. The osteoblasts induced from the colonies which could be stained by alizarin red in cytoplasm and adipocytes could accumulated lipid vacuoles stained by oil red in cytoplasm. Thus MSCs can be identified.Conclusion: The MSCs from human umbilical cord blood can be isolated and proliferated in vitro. the way that red cell were eliminated by methylcellulose sediment and cultured by Mesencult? medium+10% FCS is the valid method of isolation. These proliferated colonies cells present matrix cell immunophenotypes, can differentiate into osteoblasts and adipocytes. Objective: investigating the method and the conditions of isolation, proliferation multipotent mesenchymal stem cells(MSCs) from the subendothelial layer of umbilical cord vein, and inducing differention to osteoblasts and adipocytes directly in vitro. Method: Umbilical cords were collected and processed within 4 hours after normal deliveries. The cord vein was washed out with phosphate buffered solution. Then the vessel was filled with 0.1% collagenase solution, 6～8 hours late, suspended cells from the subendothelial layer were gained and centrifuged, Finally, the cells were cultured in polystyrene dishes. A simple cell-derived colonies were obtained, then tested for their immunophenotype and cell differentiation to osteoblast and lipoblast. Result: From the subendothelial layer of umbilical cord vein we can get adherent cells. Those cells which became obviously short rod-shape or shuttle-shape, could be proliferated and formed single cell-derived colonies cells. They presented matrix cell immunophenotype. These colonies cells could differentiate into osteoblasts which produced mineralized matrices in cytoplasm, stained by alizarin red, and differentiate into the adipocytes which accumulated lipid vacuoles and could be demonstrated by morphology, and stained by oil red. Conclusion: The MSCs can be isolated from the subendothelial layer of umbilical cord vein and proliferated it in vitro. These proliferated colonies cells present matrix cell immunophenotypes, and can differentiate into osteoblasts and adipocytes.
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