Evaluation of nestin or osterix promoter-driven cre/loxp system in studying the biological functions of murine osteoblastic cells. Am J Transl Res 2016;8(3):1447-59
Date
05/18/2016Pubmed ID
27186271Pubmed Central ID
PMC4859630Abstract
OBJECTIVE: To compare Osterix and Nestin-Cre/Loxp system in studying the biological functions of murine osteoblastic cells including primary osteoblasts (OBs) and osteolineage mesenchymal progenitor cells (MPCs).
METHODS: We isolated primary osteoblasts (OBs) from neonatal Nestin-cre-R26-loxP-YFP (Nes-OBs) and Osterix-cre-R26-loxP-YFP (Osx-OBs) mice and bone marrow mesenchymal stromal cells (BMMSCs) from the adults (termed as Nes-BMMSCs and Osx-BMMSCs). Then we detected the percentage of YFP(+) subpopulation in Nes/Osx-OBs and the percentage of CD45(-)YFP(+) progenitor population in Nes/Osx-BMMSCs and sorted them out (termed as Nes/Osx-YFP(+) OBs and Nes/Osx-CD45(-)YFP(+) MPCs) by using the sorting machine. We also analyzed the expression of surface antigens on Nes/Osx-YFP(+) OBs and Nes/Osx-CD45(-)YFP(+) MPCs by Flow cytometry. PDGF-BB induced proliferation of Nes/Osx-YFP(+) OBs and Nes/Osx-CD45(-)YFP(+) MPCs was measured by H3-Thymidine incorporation assay. We then did OB maturation and mineralization assays of Nes/Osx-YFP(+) OBs and CFU and multi-lineage differentiation assays of Nes/Osx-CD45(-)YFP(+) MPCs.
RESULTS: YFP(+)% in Nes-OBs and Osx-OBs and CD45(-)YFP(+)% in Nes-BMMSCs and Osx-BMMSCs was respectively 5.56%±3.56% (n=5), 10.12%±2.7% (n=4), 1.29%±0.98% (n=13) and 16.38%±6.98% (n=17). Both Nes-YFP(+) OBs and Osx-YFP(+) OBs were positive for CD51. Nes/Osx-CD45(-)YFP(+) MPCs were positive for CD51, CD105 and Sca1, and negative for CD31 and CD45. PDGFR expression in Osx-YFP(+) OBs was a bit higher than that in Nes-YFP(+) OBs, and slightly higher in Osx-CD45(-)YFP(+) MPCs than in Nes-CD45(-)YFP(+) MPCs. Proliferation ability of Nes/Osx-YFP(+) OBs increased dramatically after stimulated with PDGF-BB for 48 h, while it was not statistically significant that PDGF-BB induced the increase of proliferation ability in either Nes-CD45(-)YFP(+) MPCs or Osx-CD45(-)YFP(+) MPCs. We observed that no significant difference of OB maturation and mineralization ability existed between Nes-YFP(+) OBs and Osx-YFP(+) OBs, and there was little difference of self-renewal and multi-lineage differentiation potential between Nes-CD45(-)YFP(+) MPCs and Osx-CD45(-)YFP(+) MPCs, either.
CONCLUSION: Both Nestin and Osterix could be selected as useful markers for the osteoblastic cells, while Osterix was a prior choice due to larger number of Osterix-expressing cells than Nestin-expressing cells in distinct subpopulations of bone-forming cells.
