Differential expression and phenotypic activities of osterix in equine mesenchymal stem cell populations

Bone is one of the few tissues in vertebrates that possess the innate capacity for authentic regeneration, yet approximately 10% of fractures fail to heal adequately. There is considerable overlap in the cellular and molecular events occurring during embryological bone development and fracture repair. Mesenchymal stem cell (MSC) precursors undergo osteogenesis to give rise to osteoblasts, which are responsible for bone matrix synthesis and mineralization. Osteogenesis is regulated by two master transcription factors, runt-related transcription factor 2 (RUNX2) and Osterix (OSX), with interplay from signaling pathways such as the Bone Morphogenetic Protein (BMP) network. MSCs isolated from bone marrow (BM) and adipose tissue (ADI) are most commonly utilized in human and veterinary regenerative medicine applications. BM-MSCs have shown superior osteogenic capacity to ADI-MSCs in many studies, yet sample collection and culture techniques often vary between species. The overall objective of this dissertation was to determine factors that influence the in vitro osteogenic capacity differences of equine adult ADI- and BM-MSCs in order to standardize techniques and provide insights into avenues for improving osteogenesis in less-osteogenic MSC populations. The first study investigated the hypotheses that in vitro cell viability, proliferation, and osteogenic differentiation of MSCs collected from horses under general anesthesia and after euthanasia would be impaired compared to MSCs collected from horses during sedation. The second set of experiments characterized the RUNX2 and OSX response axes of ADI- and BM-MSCs during osteogenesis to test the hypotheses that 1) the relative osteogenic capacities are consistent with resting and inducible RUNX2 and OSX transcript levels, and 2) increasing OSX expression in ADI-MSCs improves their osteogenic capacity. The third series of experiments evaluated the comparative BMP signaling activities in ADI- and BM-MSCs to determine whether 1) osteogenic BMP ligand expression differs, 2) BMP inhibition in BM-MSCs impairs osteogenesis, and 3) BMP supplementation to ADI-MSCs improves osteogenesis. The results indicate that donor status during MSC collection does not affect in vitro cell viability, proliferation, and osteogenic capacity. BM-MSCs have higher OSX expression and intrinsic BMP signaling in both resting and osteogenic conditions than ADI-MSCs. Endogenous BMP activity is necessary for osteogenesis in BM-MSCs, and exogenous BMP supplementation, but not OSX over-expression, was sufficient to improve the osteogenic capacity of ADI-MSCs. In summary, OSX expression and BMP signaling significantly influence the in vitro osteogenic capacity of MSC populations