A newly identified mechanism involved in regulation of human mesenchymal stem cells by fibrous substrate stiffness

Abstract

Stiffness of biomaterial substrates plays a critical role in regulation of cell behavior. Although the effect of substrate stiffness on cell behavior has been extensively studied, molecular mechanisms of regulation rather than those involving cytoskeletal activities still remain elusive. In this study, we fabricated aligned ultrafine fibers and treated the fiber with different annealing temperatures to produce fibrous substrates with different stiffness. Human mesenchymal stem cells (hMSCs) were then cultured on these fibrous substrates. Our results showed that annealing treatment did not change the diameter of electrospun fibers but increased their polymer crystallinity and mechanical properties. The mRNA expression of RUNX2 was upregulated while the mRNA expression of scleraxis was downregulated in response to an increase in substrate stiffness, suggesting that increased stiffness favorably drives hMSCs into the osteogenic lineage. With subsequent induction of osteogenic differentiation, osteogenesis of hMSCs on stiffer substrates was increased compared to that of the cells on control substrates. Cells on stiffer substrates increasingly activated AKT and YAP and upregulated transcript expression of YAP target genes compared to those on control substrates, and inhibition of AKT led to decreased expression of YAP and RUNX2. Furthermore, macrophage migration inhibitory factor (MIF) was increasingly produced by the cell on stiffer substrates, and knocking down MIF by siRNA resulted in decreased AKT phosphorylation. Taken together, we hereby demonstrate that simply using the annealing approach can manipulate stiffness of an aligned fibrous substrate without altering the material chemistry, and substrate stiffness dictates hMSC differentiation through the MIF-mediated AKT/YAP/RUNX2 pathway.

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

“…Interestingly, fibrous substrates particularly the 2H group supported endothelial differentiation of MSCs more effectively than polystyrene culture plates. We also found that the expression level of MIF , a chemokine regulated by tissue stiffness and culture stiffness, showed the same trend with that of CD31, vWF, or VEGF , suggesting that MIF may be involved in the regulation of differentiation from MSCs to ELCs.…”

A biomaterial approach to cell reprogramming and differentiation

“…Substrate stiffness dictates hMSC differentiation through the MIF-mediated AKT/YAP/RUNX2 pathway. Once a stem cell contacts a stiffer substrate, intracellular migration inhibitory factor (MIF) increases and starts a pathway towards AKT/YAP signaling inside the nucleus which promotes osteogenic development (Figure 7) [96]. Other regulation mechanisms such as the p190RhoGAP pathway have been characterized proving the intricacies and intertwining intracellular signaling that occurs once a cell comes into contact with a substrate with certain rigidity.…”

Outer–inner dual reinforced micro/nano hierarchical scaffolds for promoting osteogenesis

“…The relevance of YAP/vinculin activity on the osteogenic effect of progenitor cells has been reported. 40,41 As osteogenic markers, Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) were potentially affected by multiple pathways, such as integrin and stiffness-related vinculin/YAP pathways in the current scenario. Therefore, in order to access the general osteogenic effect of different scaffolds, western blot was performed to compare the expression level of RUNX2 and OCN in BMSCs seeded on different scaffolds for 7 days in osteogenic medium.…”