The Effect Of IGF-1R Inducible Knockout To Nestin~+Bone Marrow Mesenchymal Stem Cell Recruitment And Differentiation

Background:Bone remodeling is a process consisting of bone resorption and bone formation. Insulin-life growth factor1(IGF-1) is important for bone formation. While serum IGF-1tends to target cortical bone, matrix IGF-1is a critical coupling factor for maintenance of trabecular bone. The cell signaling mechanisms for proliferation, differentiation and apoptosis of the mesenchymal stem cells (MSCs) in vivo have yet to be elucidated. This experiment clarified the role of IGF-1signaling on bone marrow MSCs using an inducible gene knockout mouse model.Methods:We first established a reporter mouse, Nestin-CreER::ROSA26-EYFPflox/+to confirm previous findings that Nestin expression represents a subset of bone marrow MSCs. We then used the same Nestin-CreER mouse crossed with an Igflrflox/flox mouse, to delete the IGF-1type1receptor (Igflr) in the bone marrow mesenchymal stem cells (MSCs) from3-7weeks of age after the injection of Tamoxifen (0.1mg/kg-Body weight.q3d). We measured the weight, nasoanal length and the femoral length of the mice, and analyzed the secondary spongiosa of the femur using microcomputed tomography at the end of week7. Femur were processed for H&E staining and immunhistochemical staining for Osterix and Osteocalcin to localize and quantify osteoprogenitor cells as well as mature osteoblasts, respectively.Results:YFP staining of the femur in the reporter mice showed that the majority of Nestin-daughter cells were located either in the bone marrow or on the bone surface by immunoflorecent staining, confirming that Nestin-daughter cells differentiate into the osteoblast lineage. In the Nestin-CreER::Igflrflox/flox mice, the size of the knockout mouse was not affected as wild type littermates had similar body weights and nasoanal and femoral lengths. However, bone volume and trabecular bone thickness were decreased in the secondary spongiosa of female knockout mice relative to wild type littermates (P<0.05). Immunohistochemical analysis revealed that a similar number of Osterix+osteoprogenitors were on the bone perimeter (P=0.726), while Osteocalcin+mature osteoblasts on the bone perimeter were significantly decreased in the secondary spongiosa in knockout mice versus wild type littermates (P<0.001).Conclusion:Nestin+bone marrow MSCs predominantly differentiate into osteoblast lineage in vivo postnatally. Deletion of Igflr in MSCs results in impaired bone formation during bone remodeling secondary to impaired osteoblast differentiation, while MSC recruitment is not affected.