Mechanochemical Effects on Extracellular Signal-Regulated Kinase Dynamics in Stem Cell Differentiation.

Understanding how key signaling molecules are coregulated by biochemical agents and physical stimuli during stem cell differentiation is critical but often lacking. Due to the important role of extracellular signal-regulated kinase (ERK), this study has examined its temporal dynamics to determine the coregulation of mechanochemical cues on ERK phosphorylation for smooth muscle cell (SMC) differentiation. To assess ERK1/2 activity, a fluorescence resonance energy transfer-based biosensor was transfected into mesenchymal stem cells. The influences of nanopatterned substrates, growth factors, and drugs on ERK activities were related to their effects on SMC differentiation. Results revealed that nanopatterned substrates significantly increased ERK activity in cells, overriding ERK response from administered biochemical factors. The nanopatterned substrates reduced expression of SMC markers after a 48-h biochemical treatment, except for the combination with ERK inhibitor PD98059 treatment, which enhanced expression of mature SMC marker MYH11. Immunofluorescent staining for focal adhesion proteins, vinculin and zyxin, indicated no significant differences in vinculin cluster distribution or dimension, while the location of zyxin changed from adhesion sites of cell periphery on nonpatterned substrate to actin filaments on nanopatterned substrate. The zyxin-reinforced stress fibers likely enhanced the cytoskeletal tension to increase ERK dynamics. Collectively, results suggest that physical stimuli play a dominating role in initial ERK signaling and early-stage differentiation through focal adhesion changes, and the capability of monitoring signaling events in real time could be exploited to guide the engineering of cell microenvironment.

Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.

Cell-matrix and cell-biomolecule interactions play critical roles in a diversity of biological events including cell adhesion, growth, differentiation, and apoptosis. Evidence suggests that a concise crosstalk of these environmental factors may be required to direct stem cell differentiation toward matured cell type and function. However, the culmination of these complex interactions to direct stem cells into highly specific phenotypes in vitro is still widely unknown, particularly in the context of implantable biomaterials. In this study, we utilized tunable hydrogels based on a simple high pressure CO2 method and silk fibroin (SF) the structural protein of Bombyx mori silk fibers. Modification of SF protein starting water solution concentration results in hydrogels of variable stiffness while retaining key structural parameters such as matrix pore size and ß-sheet crystallinity. To further resolve the complex crosstalk of chemical signals with matrix properties, we chose to investigate the role of 3D hydrogel stiffness and transforming growth factor (TGF-ß1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Our data revealed the potential to upregulate matured vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Overall, our observations suggest that chemical and physical stimuli within the cellular microenvironment are tightly coupled systems involved in the fate decisions of hMSCs. The production of tunable scaffold materials that are biocompatible and further specialized to mimic tissue-specific niche environments will be of considerable value to future tissue engineering platforms. STATEMENT OF SIGNIFICANCE: This article investigates the role of silk fibroin hydrogel stiffness and transforming growth factor (TGF-ß1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Specifically, we demonstrate the upregulation of mature vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Moreover, we demonstrate the potential to direct specialized hMSC differentiation by modulating stiffness and growth factor using silk fibroin, a well-tolerated and -defined biomaterial with an impressive portfolio of tissue engineering applications. Altogether, our study reinforce the fact that complex differentiation protocols may be simplified by engineering the cellular microenvironment on multiple scales, i.e. matrix stiffness with growth factor.