Nestin expression in mesenchymal stromal cells: regulation by hypoxia and osteogenesis.

BACKGROUND: The intermediate filament protein nestin is used as a marker for neural stem cells, and its expression is inversely correlated with cellular differentiation. More recently, nestin expression has also been described in other cell types including multipotential mesenchymal stromal cells (MSCs). In this study, we examined the expression of nestin in equine, canine and human bone marrow-derived MSCs undergoing osteogenic differentiation, to determine whether nestin levels were attenuated as the cells acquired a more mature phenotype. In addition, the expression of nestin may be under the influence of cellular hypoxia, as nestin expression is known to increase in areas of ischemic tissue damage. Therefore, we also examined the effects of hypoxia on expression of nestin in human MSCs and examined a role for hypoxia inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in the response. Additionally, we quantified the temporal expression of nestin in the fracture callus during bone regeneration, a site that has been characterized as hypoxic. RESULTS: There were no significant changes in nestin expression in MSCs during osteogenic differentiation. There was a significant increase in expression of nestin mRNA and protein in human MSCs in response to hypoxia (1% O2) or the chemical hypoxia mimetic desferroxamine. This may be due to upregulation of VEGF under hypoxia, as treatment of cells with the VEGF receptor antagonist CPO-P11 attenuated hypoxia-induced nestin expression. A significant increase in nestin mRNA expression was observed in the fracture callus of mice three and seven days post fracture. CONCLUSIONS: Nestin was not a selective marker for MSCs, as its expression was maintained during osteogenic differentiation, in all species examined. Furthermore our data suggest that nestin expression can be induced by hypoxia, and that this increase in nestin is partially regulated by HIF-1α and VEGF. Interestingly, nestin levels were significantly upregulated at the fracture site. Further studies are required to understand the role of nestin in bone cell biology and ultimately bone regeneration.

Mobilization of endogenous stem cell populations enhances fracture healing in a murine femoral fracture model.

BACKGROUND AIMS: Delivery of bone marrow-derived stem and progenitor cells to the site of injury is an effective strategy to enhance bone healing. An alternate approach is to mobilize endogenous, heterogeneous stem cells that will home to the site of injury. AMD3100 is an antagonist of the chemokine receptor 4 (CXCR4) that rapidly mobilizes stem cell populations into peripheral blood. Our hypothesis was that increasing circulating numbers of stem and progenitor cells using AMD3100 will improve bone fracture healing. METHODS: A transverse femoral fracture was induced in C57BL/6 mice, after which they were subcutaneously injected for 3 d with AMD3100 or saline control. Mesenchymal stromal cells, hematopoietic stem and progenitor cells and endothelial progenitor cells in the peripheral blood and bone marrow were evaluated by means of flow cytometry, automated hematology analysis and cell culture 24 h after injection and/or fracture. Healing was assessed up to 84 d after fracture by histomorphometry and micro-computed tomography. RESULTS: AMD3100 injection resulted in higher numbers of circulating mesenchymal stromal cells, hematopoietic stem cells and endothelial progenitor cells. Micro-computed tomography data demonstrated that the fracture callus was significantly larger compared with the saline controls at day 21 and significantly smaller (remodeled) at day 84. AMD3100-treated mice have a significantly higher bone mineral density than do saline-treated counterparts at day 84. CONCLUSIONS: Our data demonstrate that early cell mobilization had significant positive effects on healing throughout the regenerative process. Rapid mobilization of endogenous stem cells could provide an effective alternative strategy to cell transplantation for enhancing tissue regeneration.