Isolation and characterization of osteogenic lineage cell populations in the mouse bone marrow
| Autor: | Ching-Fang Chang, 張靜芳 |
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| Rok vydání: | 2015 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 103 The studies presented in this thesis was to define a better way to characterize the heterogeneous bone marrow osteogenic lineage cells by using a specific surface marker combination. To this end, we have successfully identified the Sca-1hi multipotent bone marrow stromal cells (MSCs), the Sca-1lo immature osteoprogenitors, and two subsets of late osteogenic cells, Sca-1-CD24med and Sca-1-CD24-/lo, in the mouse endosteal bone preparation, by using a combination of Sca-1 and CD24 antibodies. To our knowledge, it is the first successful finding to define the ‘elusive’ early committed immature osteoprogenitors by flow cytometry and functional assays for osteogenic cell lineage characterizations. In addition, CD24 antigen is a useful cell surface marker for the enrichment and identification of the late mature osteogenic cell subpopulations within the mouse endosteal bone marrow, and exclusion of the contaminating hematopoietic/erythroid cells within the triple negative stromal cell preparation. Both freshly FACS-isolated Sca-1-CD24med and Sca-1-CD24-/lo cells are highly enriched for ALP+ osteoblastic cells and after short-term culture-expansion in conventional MSC culture medium. We also revealed that the presence of differentiation-inducing agents in the primary colony forming unit–fibroblast (CFU-F) assay significantly suppressed colony formation of the Sca-1hi MSCs. Lastly, we have successfully modified the primary CFU-F assay to a two-step CFU-differentiation assay such that clonal suppression mediated by differentiation-inducing agents is minimal. The assay also reveals that both Sca-1lo and Sca-1- cell fractions are committed osteogenic cells and the existence of committed CFU-Oil Red O and bipotential CFU-ALP/Perilipin within the Sca-1hi MSC fraction. In conclusion, we propose a hierarchical relationship of osteogenic cell progression within the mouse bone marrow based on the expression level of Sca-1 and CD24 antigens. We believe that these findings will in the future, accelerate our understanding in bone cell development, bone metabolism, and hematopoiesis regulation. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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