Our study has shown that MSC differentiation is responsive to two important stimuli in the cellular microenvironment: matrix stiffness and growth factors. Matrix stiffness alone can promote a certain lineage over another, i.e. SMC on stiff substrates, and cartilage and fat on soft substrates. These results are largely correlated with the mechanical properties of the native tissue in which these cells reside. For example, native arteries have MPa stiffness, while fat stiffness is at the level of kPa. Although mature cartilage may not be as soft as 1 kPa, this stiffness could be the reminiscent of the stiffness during cartilage development, thereby signaling development toward a cartilage phenotype by matrix synthesis.
It has been shown that matrix stiffness regulates MSC differentiation toward bone, muscle or neuronal lineages when grown on hard, medium or soft substrates that are akin to the native stiffness of their respective tissues [11
]. Here we report the effects of matrix stiffness on smooth muscle, chondrogenic and adipogenic lineages. The difference of these observations could be attributed to the difference in culture medium, e.g., the amount of FBS and other biochemical supplements. In most of our experiments, low FBS was used to avoid the compounding effects of soluble factors in the culture medium. Nevertheless, the fact that the same matrix stiffness modulates the differentiation of cells into multiple lineages suggests that, at least in some cases (e.g., chondrogenic vs. adipogenic), matrix stiffness alone is not sufficient to determine the cell fate. The definitive commitment and terminal differentiation of the cells into a specific lineage may require the cooperation of other differentiation factors. Indeed, the effects of matrix stiffness on the expression of SMC, chondrogenic and adipogenic makers are further defined by TGF-β and adipogenic medium. TGF-β has previously been used to induce stem cell differentiation into either SMC lineage [2
] or chondrogenic lineage [3
]. Our results indicate that this phenomenon can be explained by the growth factor effects working in concert with the physical cue of matrix stiffness. Although matrix stiffness alone is not most effective to drive a specific and terminal differentiation, it is an important determinant and co-factor for cell differentiation induced by soluble biochemical factors. Stem cells need to integrate microenvironmental cues from both soluble biochemical factors and ECM to regulate differentiation.
The differential effects of matrix stiffness on the expression of SMC and chondrogenic markers have significant implications in tissue engineering and regenerative medicine applications. For example, to construct vascular grafts, scaffolds with MPa stiffness are preferred over hydrogels that are not appropriate for SMC differentiation and are not strong enough for a functional vascular graft. On the other hand, when MSCs are engineered for cartilage repair, they are usually grown in hydrogels or as cell pellets [3
], of which the stiffness is low; under these conditions, three-dimensional culture microenvironment and cell-cell interactions could also play a role. The results from this study provide a rational basis for the design and selection of appropriate scaffolds.
Our results also provide important insights into the underlying mechanisms of mechanotransduction. Matrix stiffness does not affect the Smad2/3 signaling pathway, suggesting that it may regulate mechanotransduction in parallel to TGF-β signaling. Rho GTPase plays an important role in actin assembly and focal adhesion formation. Although Rho has been suggested as a mechanotransducer of matrix stiffness, the difference in Rho activity on stiff and soft substrates is not significant under our experimental conditions with low serum. In addition, Rho activation induced SMC markers on a stiff substrate and increased chondrogenic and adipogenic markers on a soft substrate, suggesting that Rho-mediated signaling is important for the expression of all these linage markers but cannot explain the differential expression of these markers regulated by matrix stiffness. As Rho regulates multiple signaling pathways, the basal level of Rho activity may be essential for maintaining the expression of many genes.
If Rho cannot account for the differential effects of matrix stiffness, what causes the decrease of stress fibers on soft substrate and the differential gene expression in MSCs? Our data indicate that the weak cell adhesion on soft substrates is a limiting factor. Soft substrate can only withstand low levels of force exerted by cells. When cells generate traction forces higher than what the substrate can withstand, the matrix may yield and cells could adapt by decreasing the traction force, integrin binding and their own stiffness [16
], which modulate cell spreading and thus differentiation [18
]. The limit of cell adhesion strength on a soft substrate can provide feedback to cells to decrease stress fiber formation, which also explains why Rho activation fails to induce stress fibers on soft substrates. Indeed, cell adhesion is much weaker on soft substrates, and the decrease of cell adhesion strength by blocking integrin binding mimics the effect of soft substrates on the differential expression of SMC, chondrogenic and adipogenic markers in MSCs. In summary, our results support the following hypothesis: a soft matrix only allows weak cell adhesion, which decreases the formation of stress fibers and differentially regulates the expression of SMC, chondrogenic and adipogenic markers. These experiments not only underscore the importance of mechanical cues in collaborating with chemical cues to determine cell fate, but also provide a mechanistic explanation for how mechanical cues can regulate the lineage specification of MSCs and other type of stem cells.
In conclusion, stiff matrix promotes MSC differentiation into SMC lineage, while soft matrix (~1 kPa) promotes MSC differentiation into chondrogenic and adipogenic lineage. Although matrix stiffness is an important determinant of stem cell differentiation, its effect may not be specific for only one lineage, and biochemical factors such as TGF-β are required, together with matrix stiffness, to define a unique differentiation pathway. Furthermore, the weak cell adhesion strength on soft matrix may account for the inhibition of Rho-induced stress fibers and α-actin assembly and the differential effects of matrix stiffness on MSC differentiation.