Comparative osteogenesis of equine mesenchymal stem cells isolated from bone marrow, adipose tissue, and synovium

The experiments in this study were carried out to define the osteogenic potential of equine MSCs derived from synovium, bone marrow, and adipose tissue maintained under the same culture conditions. Equine MSCs were isolated from synovium [SYN], bone marrow [BM] and adipose tissue [AD], expanded in monolayer culture for two passages, and then transferred to basal or standard osteogenic medium. At day 7 and 14, the phenotypic status of the cells was primarily evaluated by staining for mineralized matrix (Alizarin Red and Von Kossa) and the enzymatic activity of alkaline phosphatase (ALP). Other secondary indicators of osteogenic differentiation included in this study were mRNA levels of ALP and osteoblast-specific genes Runx2, Osterix, Osteonectin and Osteomodulin. Staining of the mineralized matrix of BM-MSCs cultured under osteogenic conditions, demonstrated a higher uptake of both Alizarin Red (AR) and Von Kossa (VK) stains, restricted to dense cellular aggregation (nodules). Stain up-take in SYN and AD cultures was also limited to infrequent sites of cellular aggregation but was less intense than in BM cultures. In osteogenic cultures, intense ALP activity was present within and immediately around the cell aggregates in BM cultures, whereas staining in the SYN and AD monolayers was far less intense and more diffusely distributed. BM-MSCs cultured under osteogenic conditions increased ALP mRNA expression and ALP activity. Osteogenic AD cultures also significantly increased ALP activity by day 14. In SYN cultures, basal ALP activity was comparatively low and, even under osteogenic conditions, there were negligible changes in ALP activity. These findings suggest that BM-MSCs are more able to undergo osteogenic differentiation than SYN- or AD-MSCs. Bone marrow-derived cells should be preferred for any application focused on bone regeneration