The identification of metabolites that influence specific cell behaviour can be challenging because it is difficult to follow metabolite changes without adding biochemical bias from the media used or from the substrata on which the cells are cultured. Our clients wanted a synthetic hydrogel matrix with simple chemistry and modifiable mechanical properties, to influence stem cell differentiation using solely mechanical cues, in order to facilitate their metabolomics analysis of these differentiation pathways.
As part of this study, perivascular stem cells were cultured on hydrogels of different stiffness; soft (1 kPa), stiff (13 kPa), and rigid (32 kPa), prepared from Biogelx Powder. The cells were assessed for primary differentiation lineages via qPCR after 1 week of culture. Cells were assessed for the expression of β3-tubulin (neural cells), SOX-9 (chondrocytes), and RUNX2 (osteoblasts) on each hydrogel type. Primary expression of β3-tubulin was observed on the soft surface, SOX-9 was observed on the stiff surface, and RUNX2 was observed on the rigid surface after 1 week of culture. Cells were only cultured in basal media with no differentiation-enhancing factors, and so differentiation is purely substrate driven. These experiments clearly demonstrated stem cells undergo purely stiffness-directed fate selection on Biogelx hydrogels, whereby soft gels induce neural marker expression and rigid gels induce osteogenesis.
The study went on to analyse concentration variances of >600 metabolites during differentiation on the stiff and rigid gels (focusing on chondrogenesis and osteogenesis as regenerative targets, respectively), to identify specific lipid metabolites that play key roles driving differentiation. It is envisaged such a strategy could be used discovering cell-directing bioactive metabolites of therapeutic relevance.
“The ability to generate hydrogels over a range of stiffnesses from a single material was a huge advantage for our metabolomics experiments as it allowed us to control stem cell growth and differentiation without the use of different media formulations, which allowed for a direct comparison of metabolite profiles between different target lineages. The simple chemistry of Biogelx hydrogels meant we could understand the influence of the cells’ physical environment alone, and the fact that they are synthetic meant we could rely on consistent results.”