JOURNAL OF BONE AND MINERAL RESEARCH Volume 9, Number zyxwvutsrqp 11, 1994 Mary Ann Liebert, Inc., Publishers zyxwvutsrqp Responsiveness of Clonal Limb Bud Cell Lines to Bone Morphogenetic Protein 2 Reveals a Sequential Relationship Between Cartilage and Bone Cell Phenotypes VICKI ROSEN, JOHN NOVE, JEFFREY J. SONG, R. SCOTT THIES, KAREN COX, and JOHN M. WOZNEY ABSTRACT There is growing evidence to suggest that BMPs are among the signals necessary to create the embryonic skeleton, but how these regulatory molecules enter the pathways of embryonic bone formation remains to be defined. The earliest steps of endochondral bone formation, consisting of mesenchymal condensation and chondrogenesis, have been shown to result directly from BMP-2 action. To determine whether the transition from chondrogenesis to osteogenesis occurring later in endochondral bone formation is also the result of BMP activity, we tested the effects of BMP-2 on immortalized endochondral skeletal progenitor cells derived from mouse limb bud. The cell lines established by this process were found to fall into three general categories: undifferentiated skeletal progenitor cells, which in the presence of BMP-2 first express cartilage matrix proteins and then switch to production of bone matrix proteins; prechondroblast-like cells that constitutively express a subset of markers associated with chondrogenesis and, in the presence of BMP-2, shut off synthesis of these molecules and are induced to produce bone matrix molecules; and osteoblast-like cells that are not significantly affected by BMP-2 treatment. These data suggest that BMP-2 initiates the differentiation of limb bud cells into cells of both the cartilage and bone lineages in a sequential manner, making BMP-2 a potent regulator of skeletal cell differentiation. INTRODUCTION tw WKI'EHKATE SKELETON is composed of a myriad of T individual bony structures, each with a unique shape. size. and location. Although the outward appearance of these bones difiers greatly. the cell types resident within each bone are the same. At the cellular level, formation of each individual bone follows one of two morphogenic paths: direct conversion of mesenchyme to bone tissue (intramembranous bone formation) or conversion of mesenchyme to cartilage, removal of the cartilage, and replacement by bone (endochondral bone forma- tion). Why two paths for skeletal formation exist and the relationship between progenitor cells and regulatory molecules in these pathways have yet to be discovered. There is growing evidence to suggest that bone morphoge- netic proteins (BMPs) are among the signals necessary to create the embryonic skeleton. but how these regulatory molecules enter the pathways of embryonic bone formation remains to be defined. Individual BMPs. including BMP-2. have been local- ized by in situ hybridization in mouse embryos at sites undergo- ing skeletal formation."-" In addition. mutant mice deficient in at least one of the BMPs, BMP-5. demonstrate impaired carti- lage and bone formation during embryogenesis, as well as impaired fracture healing as adult^.'^ zyxw -" In vitro. cells isolated from the developing skeleton are capable of responding to the BMPs, and BMPs can induce these embryonic cells to differen- tiate into osteoblast-like and chondroblast-like cell types."-"' These results correlate well with in vivo data on BMP activity: it is now well established that the soluble factors present in bone matrix that are responsible for bone-inductive activity are members of the BMP family."""' These BMPs, when im- planted in bone defects or in nonbony sites in adult animals, Genetics Institute. Inc.. Cambridge, Massachusetts. 1759