IADR Abstract Archives
Peripheral clock gene, Npas2 in MSC maintenance and bone regeneration
Objectives: It has been reported that circadian clock genes optimize cell proliferation and differentiation in stem cell populations during wound healing. We have previously identified that a circadian clock molecule neuronal PAS domain 2 (Npas2) played a critical role in tooth extraction wound healing. The objective of this study was to elucidate the mechanism for mesenchymal stem cell (MSC) to maintain the regenerative activity by Npas2 modulation.
Methods: Mouse calvarial critical bone defect was created in C57B6/J (WT) and Naps2 KO mice, and bone regeneration in the defect was assessed by histology (HE staining) and mCT analyses. MSCs were isolated from bone marrow or adipose tissues of mice with WT and Npas2 KO mutation. The expression of stemness marker genes (Nanog and Klf4) and the cell proliferation ability of MSC were evaluated by real time RT-PCR and WST-1 cell proliferation assay, respectively. In vitro osteogenic, adipogenic and chondrogenic differentiations were compared between MSCs of WT and Npas2 KO mice.
Results: Four weeks after critical bone defect was created, the accelerated new bone formation was observed in Naps2 KO mice. The bone volume in the defect of Naps2 KO mice was significantly larger than that of WT mice. Naps2 KO MSC showed the increased proliferative activity (ANOVA; P<0.05). After culturing in growth medium for 3 weeks, Nanog and Klf4 gene expression of Npas2 KO MSCs were significantly higher than WT MSC (ANOVA; P<0.05). Multi-lineage differentiation capabilities in Npas2 KO MSC were significantly higher than WT MSC.
Conclusions: These results suggest that peripheral clock gene Npas2 KO enhances and maintains the stemness of MSC in vitro and plays a role in regulating bone regeneration in vivo. These findings represent an important step toward to understand the novel mechanism of maintaining stem cells in situ, which provides a rationale for therapeutic application of alveolar bone regenerative medicine.
Methods: Mouse calvarial critical bone defect was created in C57B6/J (WT) and Naps2 KO mice, and bone regeneration in the defect was assessed by histology (HE staining) and mCT analyses. MSCs were isolated from bone marrow or adipose tissues of mice with WT and Npas2 KO mutation. The expression of stemness marker genes (Nanog and Klf4) and the cell proliferation ability of MSC were evaluated by real time RT-PCR and WST-1 cell proliferation assay, respectively. In vitro osteogenic, adipogenic and chondrogenic differentiations were compared between MSCs of WT and Npas2 KO mice.
Results: Four weeks after critical bone defect was created, the accelerated new bone formation was observed in Naps2 KO mice. The bone volume in the defect of Naps2 KO mice was significantly larger than that of WT mice. Naps2 KO MSC showed the increased proliferative activity (ANOVA; P<0.05). After culturing in growth medium for 3 weeks, Nanog and Klf4 gene expression of Npas2 KO MSCs were significantly higher than WT MSC (ANOVA; P<0.05). Multi-lineage differentiation capabilities in Npas2 KO MSC were significantly higher than WT MSC.
Conclusions: These results suggest that peripheral clock gene Npas2 KO enhances and maintains the stemness of MSC in vitro and plays a role in regulating bone regeneration in vivo. These findings represent an important step toward to understand the novel mechanism of maintaining stem cells in situ, which provides a rationale for therapeutic application of alveolar bone regenerative medicine.