Background: We show that a side population (SP) of primitive bone marrow stem cells can differentiate to osteoblasts, which then are available to support the formation of new bone. We postulate that such cells underlie the production of osteoblasts in vivo and can be manipulated ex vivo to commit exclusively to osteoblast differentiation.

Objective/Hypothesis: The level of osteogenic stem cells decreases during aging, which may contribute to osteoporosis. The ability to increase the number of functional osteoblasts in bone by transplantation of SP cells might offset this deficiency and help attenuate the disease phenotype. We hypothesize that SP cells, which can be isolated from the patient's bone marrow, can be forced to differentiate exclusively to osteoblasts by vector-enforced expression of master transcription factors of osteoblast differentiation.

Specific Aims: (1) To determine the extent to which SP cells can engraft and differentiate into mesenchymal stem cells and osteoblasts in murine models of acute bone injury and chronic bone disease. We propose to transplant genetically marked SP cells from GFP or Rosa mice into these model systems and assess their contribution to new bone formation, using immunohistochemical-staining methods and Y chromosome marking techniques to detect mesenchymal stem cells and osteoblasts derived from SP cells. This assessment of the regenerative capacity of these stem cells will provide much needed insight into the roles of SP cells in bone reconstitution. (2) To test in vitro the hypothesis that SP cell commitment to the osteoblast lineage can be switched on by up-regulating the osteoblast-specific transcription factor Cbfa1. We propose to transduce SP cells with an adenoviral vector expressing a Cbfa1 transgene. These experiments are needed to validate our central strategy (SP cells can be directed to the osteoblast lineage through constitutive expression of Cbfa1) before we embark on more ambitious testing in animal models. (3) To determine whether SP cells directed ex vivo to the osteoblast lineage can over-ride competing differentiation signals, engraft specifically in bone, and produce increased rates of bone formation.

Study Design: Primitive stem cells underlie the production of osteoblasts, but little is known about their responses to recruitment and differentiation signals associated with bone injury and disease or their ex vivo inducibility for therapeutic purposes. We hypothesize that bone marrow SP cells can be induced to differentiate exclusively along the osteoblast pathway, affording an abundant source of osteoprogenitors for bone repair and remodeling.

Relevance: Upon completion of these aims, we will have defined the role of SP cells in mammalian osteogenesis and determined the feasibility of driving them toward osteoblast differentiation for therapeutic purposes.