Hematopoietic derived cells do not contribute to osteogenesis as osteoblasts

Despite years of extensive investigation, the cellular origin of heterotopic ossification (HO) has not been fully elucidated. We have previously shown that circulating bone marrow-derived osteoblast progenitor cells, characterized by the immunophenotype CD45 −/CD44 +/CXCR4 +, contributed to the formation of heterotopic bone induced by bone morphogenetic protein (BMP)-2. In contrast, other reports have demonstrated the contribution of CD45 + hematopoietic derived cells to HO. Therefore, in this study, we developed a novel triple transgenic mouse strain that allows us to visualize CD45 + cells with red fluorescence and mature osteoblasts with green fluorescence. These mice were generated by crossing CD45-Cre mice with Z/RED mice that express DsRed, a variant of red fluorescent protein, after Cre-mediated recombination, and then crossing with Col2.3GFP mice that express green fluorescent protein (GFP) in mature osteoblasts. Utilizing this model, we were able to investigate if hematopoietic derived cells have the potential to give rise to mature osteoblasts. Analyses of this triple transgenic mouse model demonstrated that DsRed and GFP did not co-localize in either normal skeletogenesis, bone regeneration after fracture, or HO. This indicates that in these conditions hematopoietic derived cells do not differentiate into mature osteoblasts. Interestingly, we observed the presence of previously unidentified DsRed positive bone lining cells (red BLCs) which are derived from hematopoietic cells but lack CD45 expression. These red BLCs fail to produce GFP even under in vitro osteogenic conditions. These findings indicate that, even though both osteoblasts and hematopoietic cells are developmentally derived from mesoderm, hematopoietic derived cells do not contribute to osteogenesis in fracture healing or HO.