Substrate elasticity regulates adipose-derived stromal cell differentiation towards osteogenesis and adipogenesis through β-catenin transduction.

It is generally recognised that mesenchymal stem cells (MSCs) can differentiate into multiple lineages through guidance from the biophysical properties of the substrates. However, the precise biophysical mechanism that enables MSCs to respond to substrate properties remains unclear. In the current study, polydimethylsiloxane (PDMS) substrates with different stiffnesses were fabricated and the way in which the elastic modulus of the substrate regulated differentiation towards osteogenesis and adipogenesis in adipose-derived stromal cells (ASCs) was explored. Initially, a cell morphology change by SEM was observed between the stiff and soft substrates. The cytoskeleton stains including microfilament by F-actin and microtubule by α- and β-tubulin further showed a larger cell spreading area on the stiff substrate. Then the expression of vinculin, in charge for the linkage of adhesion molecules to the actin cytoskeleton, was enhanced on the stiff substrate. This change in focal adhesion plaque further triggered intracellular β-catenin signaling and promoted its nuclear translocation especially on the stiff substrate. The influence of β-catenin signaling on direct differentiation to osteogenic lineages was through direct binding between its downstream protein, Lef-1, and the osteogenic transcriptional factors, Runx2 and Osx, while on differentiation to adipogenic lineages was through modulating the expression of PPARγ. The imbalance of stiffness-induced β-catenin signaling finally induced a stronger osteogenesis and a weaker adipogenesis on the stiff substrate relative to those on the soft substrate. This study indicates the importance of stiffness on ASC differentiation and could help to increase understanding of the mechanism underlying molecular signal transduction from mechanosensing, mechanotransducing to stem cell differentiation. STATEMENT OF SIGNIFICANCE: Mesenchymal stem cells can differentiate into multiple lineages, such as adipogenesis, myogenesis, neurogenesis, angiogenesis and osteogenesis, through influence of biophysical properties of the extracellular matrix. However, the precise bio-mechanism that triggers stem cell differentiation in response to matrix biophysical properties remains unclear. In the current study, we provide a series of experiments involving the characterization of cell morphology, microfilament, microtubule and adhesion capacity of adipose-derived stromal cells (ASCs) in response to substrate stiffness, and further elucidation of cytoplasmic β-catenin-dependent signal transduction, nuclear translocation and resultant promoter activation of transcriptional factors for osteogenesis and adipogenesis. This study provides an explanation on deeper understanding of bio-mechanism underlying substrate stiffness-triggered β-catenin signal transduction from active mechanosensing, mechanotransducing to stem cell differentiation.