Xinlin Su1, Mei Yu2, Guixing Qiu3, Yongwei Zheng2, Yuhong Chen2, Renren Wen2, Guoping Fu2, Wen Zhu2, Jun Chen3, Nan Wu4, Pei Ma5, Weisheng Chen3, Zhihong Wu6, Demin Wang2. 1. Department of Orthopaedics Surgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical SciencesBeijing 100730, China; Blood Research Institute, Blood Center of WisconsinMilwaukee, WI53226, USA. 2. Blood Research Institute, Blood Center of Wisconsin Milwaukee, WI53226, USA. 3. Department of Orthopaedics Surgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences Beijing 100730, China. 4. Department of Orthopaedics Surgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical SciencesBeijing 100730, China; Beijing Key Laboratory for Genetic Research of Skeletal DeformityBeijing 100730, China. 5. Department of Orthopaedics Surgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical SciencesBeijing 100730, China; State key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical CollegeBeijing 100050, China. 6. Department of Orthopaedics Surgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical SciencesBeijing 100730, China; Beijing Key Laboratory for Genetic Research of Skeletal DeformityBeijing 100730, China; Central Laboratory, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical SciencesBeijing 100730, China.
Abstract
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.
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.
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