Utility of recombinant Hedgehog proteins and RANKL to generate bone cell models in vitro

Abstract

Abstract Bone remodeling is a lifelong process that begins during the embryonic stage and continues after birth. The two main bone cells involved in bone remodeling are osteoblast (bone formation) and osteoclast (bone resorption). The strict balance between bone formation and bone resorption is maintained by several cytokines and growth factors, including RANKL, the Hedgehog family of proteins (SHH, DHH, and IHH), and M-CSF secreted by bone cells, as well as systemic hormones. Any imbalance in bone remodeling might result in bone related diseases. Osteoblast differentiation was first studied using mouse mesenchymal cell line C3H10T1/2 cells. Recombinant SHH, DHH, and IHH were able to induce osteoblast differentiation of C3H10T1/2 cells. Bone morphogenetic protein 9 (BMP-9) enhanced the differentiation of C3H10T1/2 cells induced by Hedgehogs. Osteoclasts are multinuclear cells that are derived from hematopoietic precursors of the monocyte/macrophage lineage and their differentiation depends on osteoblast-produced RANKL and M-CSF. In order to study osteoclast differentiation, and the role of different cytokines in bone homeostasis; murine bone marrow derived macrophages (BMDM) were obtained and treated with mouse GM-CSF for 7 and 14 days. FACS analysis of cellular markers and functional assays confirmed the identity of macrophages. Subsequently, BMDM were differentiated to osteoclast with mouse M-CSF and RANKL. The multinucleated cells were identified by cell morphology and functional TRAP detection. Similar results were obtained with RAW264.7 cells, a mouse macrophage cell line. Our results confirm that BMDM and osteogenic cells lines are good models to study cytokines involved in cell differentiation and bone remodeling.