title: Hematopoietic derived cells do not contribute to osteogenesis as osteoblasts.
abstract: 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.
authors.affiliation: Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Children's Hospital, Columbus, OH 43205, USA. Electronic address: Satoru.Otsuru@nationwidechildrens.org.
authors.forename: Satoru
authors.initials: S
authors.lastname: Otsuru
authors.affiliation: Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Children's Hospital, Columbus, OH 43205, USA; Division of Hematology/Oncology/BMT, Nationwide Children's Hospital, Columbus, OH 43205, USA.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Overholt
authors.affiliation: Division of Oncology/Blood and Marrow Transplantation, The Children's Hospital of Philadelphia and The University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Olson
authors.affiliation: Division of Oncology/Blood and Marrow Transplantation, The Children's Hospital of Philadelphia and The University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Ted J
authors.initials: TJ
authors.lastname: Hofmann
authors.affiliation: Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Children's Hospital, Columbus, OH 43205, USA.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Guess
authors.affiliation: Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Children's Hospital, Columbus, OH 43205, USA.
authors.forename: Victoria M
authors.initials: VM
authors.lastname: Velazquez
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kaito
authors.affiliation: Department of Oncology and Hematology, University-Hospital of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Dominici
authors.affiliation: Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Children's Hospital, Columbus, OH 43205, USA; Division of Hematology/Oncology/BMT, Nationwide Children's Hospital, Columbus, OH 43205, USA. Electronic address: Edwin.Horwitz@nationwidechildrens.org.
authors.forename: Edwin M
authors.initials: EM
authors.lastname: Horwitz
mesh_terms: D000818:Animals; D001842:Bone and Bones / Q000166:cytology; D002454:Cell Differentiation / Q000235:genetics; D002469:Cell Separation; D002478:Cells, Cultured; D003433:Crosses, Genetic; D005260:Female; D005434:Flow Cytometry; D017102:Fracture Healing; D005786:Gene Expression Regulation; D006410:Hematopoiesis / Q000235:genetics; D019426:Integrases / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008297:Male; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009999:Ossification, Heterotopic / Q000235:genetics / Q000473:pathology; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D010012:Osteogenesis / Q000235:genetics
chemical_list: C045073:Cre recombinase; D019426:Integrases; D017493:Leukocyte Common Antigens
keywords: Bone lining cells; CD45; Heterotopic ossification; Osteoblasts