Identification and in vitro screening of osteogenic metabolites through supplement-free nanovibration-driven mesenchymal stem cell differentiation: Matrix Biology Europe – July 2018 meeting

Introduction: There is a need for the development of effective tissue engineering approaches to produce bone. In the laboratory, these approaches typically involve osteogenic differentiation of mesenchymal stem cells (MSCs) through medium supplementation. We recently developed a supplement-free osteogenic differentiation protocol through nanovibrational stimulation of MSCs1. Here, we hypothesised that nanovibrational differentiation of MSCs would allow metabolomic analysis of differentiation without confounding exogenous medium supplements. We aimed to investigate MSC nanovibration-driven osteogenesis in 2D and 3D cultures, identify key osteogenic metabolites and metabolomic processes and investigate their osteogenic potential by supplementing these pathways in vitro.

Materials and Methods: Human MSCs were cultured in standard tissue culture well plates (2D) or in type I collagen gels (3D) and cultured over 28 days in three groups—nanovibrational stimulation, osteogenic media (dexamethasone) and MSC expansion media. Differentiation was tracked through changes in gene expression (qPCR) and protein expression (immunofluorescent staining (IFS)). At key points, cell metabolomic analysis was performed (LC-MS; ZIC-pHILIC). We selected the most promising metabolite during osteogenic differentiation. This was synthesised along with several chemical analogs. The osteogenic potential of these metabolites was then investigated through gene and protein expression following supplementation to 2D and 3D MSC cultures.

Results: Nanovibration upregulated key osteogenic genes in both 2D and 3D cultures comparably to osteogenic media, including early upregulation of RUNX2 (2D × 14.5, P < 0.05; 3D × 11.5, P < 0.05) followed by maturation marker osteopontin (2D × 19, P < 0.05; 3D × 7.2, P < 0.05). Corresponding increases in osteogenic proteins were also observed. Metabolomic analysis identified several key networks, with cholesterol sulphate (CS) identified as a promising metabolite target. When CS and several analogs were supplemented at 1 μM to 2D and 3D cultures, they induced osteogenic gene and protein expression, comparably to osteogenic media, while having less off-target effects. In particular, fludrocortisone and fludrocortisone acetate significantly increased osteogenic marker expression, even versus osteogenic media.

Discussion: Nanovibration is an exciting tool for the supplement-free study of MSC osteogenic differentiation, while this work also validates a targeted metabolite supplementation approach for controlling cell fate decisions, which may prove cheaper and more specific than conventional approaches.