Endogenous biological factors modulated by substrate stiffness regulate endothelial differentiation of mesenchymal stem cells

During the process of tissue regeneration facilitated by stem cells, physical properties of a scaffold affect behavior and activities of the cell. To enhance differentiation of human mesenchymal stem cells (MSCs) into endothelial-like cells (ELCs), we used electrospun fibrous substrates with different stiffness to enhance the differentiation. A simple method of annealing with different lengths of treatment time was employed to modulate stiffness of electrospun fibrous substrates without changing their chemistry. We seeded MSCs on substrates with different stiffness to study how stiffness of a culture substrate affects differentiation of MSCs into ELCs. Results of RT-PCR and western blotting revealed that stiffer substrates with the average surface modulus of 7.82 MPa induced differentiated MSCs to express more VEGF, CD31, and vWF mRNA transcripts and proteins than softer ones with that of 3.8 or 1.44 MPa. We also found that the production of macrophage migration inhibitory factor (MIF) in ELCs was increased with substrate stiffness. After silencing MIF mRNA, MSCs during differentiation showed lower expression levels of VEGF, CD31, and vWF than control cells whereas VEGF-silenced and control cells expressed comparable levels of MIF, indicating that MIF is an upstream molecule regulating VEGF in the mechanism. Our findings provide new insight into how stiffness of a culture substrate regulates differentiation of MSCs into ELCs.