ABSTRACT
-tiated into the precartilaginons stem cells.
ABSTRACT
PURPOSE: The isolated human bone marrow-derived stromal cells (BMSCs) were expanded and showed multipotentiality for differentiation to both adipocytes and osteoblasts. We selected the multiple clones and characterized their proliferation potentials, surface markers and differentiation potentials. MATERIALS AND METHODS: BMSCs were isolated from bone marrow. After forming colonies, colonies were selected and cultured. We performed MTT assay and FACS. Cultured colonies were differentiated into adipocyte and osteoblast. RESULTS: BMSCs were isolated and plated in tissue-culture dishes. The number of clones was about 30. Clones were divided into three groups. Group I was round and small in size, and showed rapid proliferation. Group II showed similar pattern in cell size and proliferation with group I clones during early passage. But, after 4 passages, the clones became flat and larger cells and showed slower proliferation. Finally, group III clones were flat and large cells and replicated slowly in early passage. Group I was fibroblastic in morphology, but Group II changed to flat and larger cells after passage 4. FACS and proliferation assay were performed at passage 5. Group I and Group II cells differed in their expression of the cell-surface epitopes, CD29 and CD105. Group I cells were differentiated into adipocytes better than Group II cells. CONCLUSION: In this study, these results indicated that cell surface markers were expressed differently for clones having different differentiation properties. These clonal BMSCs may also be used for the identification of lineage-determining factors.
Subject(s)
Humans , Adipocytes , Adipogenesis , Bone Marrow , Cell Proliferation , Cell Size , Clone Cells , Epitopes , Fibroblasts , Osteoblasts , Osteogenesis , Population Characteristics , Stromal CellsABSTRACT
OBJECTIVE: Multipotent bone marrow stromal cells have the ability to differentiate toward a variety of connective tissue lineages including cartilage. The future use of adult mesenchymal stem cells (MSCs) for human therapies depends on the establishment of preclinical studies. Therefore, in this preclinical study we demonstrated the expression of MSC surface markers CD29, CD105, and CD44 on human bone marrow derived stromal cells during chondrogenic differentiation. METHODS: Adult human bone marrow was collected from the iliac crest of 7 donors following informed consent. Mononuclear cells were isolated, incubated in monolayers, and embedded in alginate beads for three-dimensional cultures. Cellualr viability was assessed by MTT assay. Flow cytometry of alginate bead cultures was performed on days 0, 7, 14, 21, and 28 using monoclonal antibody against surface molecules, CD105, CD29, CD44, CD34 and CD45. Total contents of collagen and glycosaminoglycan (GAG) of the alginate beads was measured. SPSS 11.0 was used for data analysis. RESULTS: After 7 days of culture, 89% of the cells expressed the human integrin beta 1 antibody, CD29. The CD29-positive cells remained elevated at 83% on days 28. However, while only 18% expressed the type II TGF-beta receptor endoglin, CD105 on day 7, the CD105-positive cells increased abruptly 65% on day 14 remaining elevated up to day 28. The expression of CD44 was maximal in the first passage cell (63%). High concentration of TGF-beta 3 (10 ng/mL) was more favorable for sustaining cell viability than a low concentration (0.5 ng/mL)(n=4, p= 0.002, day 21). The total contents of collagen and GAG in the MSC-alginate beads increased during the three-dimensional culture (n=4, p=0.02, p=0.006) suggesting its differentiation into a chondrogenic lineage. CONCLUSION: CD29 was expressed earlier than CD105 during chondrogenic differentiation of human bone marrow MSC. CD44 expression was highest in the first passage cells and gradually decreased afterwards.