Osterix Over-Expression Is Insufficient to Stimulate Osteogenesis in Equine Adipose-Derived MSCS

Introduction: Mesenchymal stem cell (MSC) osteogenesis is driven by two transcription factors, RUNX2 and Osterix (OSX). MSCs, by definition, are capable of multilineage differentiation, but the osteogenic superiority of bone marrow-derived (BM) MSCs, in comparison to progenitors from other sources, such as adipose tissue (ADI), is well established. This study compared baseline and inducible RUNX2 and OSX expression in equine ADI- and BM-MSCs and assessed the phenotypic impact of OSX overexpression in ADI-MSC osteogenesis.

Materials and Methods: BM- and ADI-MSCs from four adult horses were expanded in monolayer. Basal RUNX2 and OSX expression was assessed in “passage 3” cells (paired t-tests). ADI cells were transferred to osteogenic medium ± an OSX-expressing adenovirus (20 and 100 MOIs). Osteogenesis was assessed by Alizarin Red staining, alkaline phosphatase (ALP) mRNA and activity, and qPCR assessment of an osteogenic gene panel (ANOVAs).

Results: Basal RUNX2 expression was similar in ADI- and BM-MSCs. In contrast, OSX levels were significantly higher in BM-MSCs. Osteogenic induction of RUNX2 was also similar, but only BM-MSCs significantly upregulated OSX. OSX overexpression in ADI cells increased ALP mRNA levels but not enzymatic activity. Bone sialoprotein, osteocalcin, osteomodulin, osteonectin, and osteopontin expression was not consistently altered by OSX overexpression. There was minimal Alizarin Red stain up-take in ‘osteogenic’ ADI-MSC cultures.

Discussion/Conclusion: OSX overexpression is not sufficient to significantly improve ADI-MSC osteogenesis. The differential osteogenic capacities of ADI- and BM-MSCs may be due in part to differences in their RUNX2-OSX response axes, but other factors are clearly also important.

Acknowledgment: This study was performed with IACUC approval.

No conflict of interest has been declared by the author(s).