(122g) Role of Substrate Stiffness On ESC Differentiation Into Endoderm Lineage

Early embryonic stem cell differentiation is marked by the formation of 3 germ layers with distinct molecular markers from which all tissues types will arise. Most of these in-vitro inductions are achieved through modulations of the cellular chemical microenvironment . Recently, researchers are studying the effect of mechanical cues such as matrix elasticity on stem cell differentiation. In this study, we are reporting how the cellular mechanical microenvironment affects the differentiation and phenotypic commitment of embryonic stem cells. The mechanical environment was modulated by culturing the cells on a fibrin hydrogel matrix, and subsequently modifying the gelation characteristics to vary the mechanical properties of the fibrin substrate. The selection criteria for the materials were to use a substrate that would promote cell attachment, whose chemistry can be easily manipulated to achieve different mechanical properties, while also maintaining chemical equivalence. The mechanical properties of the fibrin substrate is varied by systematically altering the fibrinogen concentration and furthermore the fibrinogen-thrombin cross-linking ratio.

Detailed mechanical characterization of the different substrates illustrate that substrate stiffness can be modified by either varying the concentration of fibrinogen or the cross-linking ratio. Further SEM analysis of the synthesized gels illustrate the variation of microscopic gel microstructure, such as the fiber thickness, porosity and overall area of fibrin strand available for cell anchorage as a result of altered fibrinogen concentration and cross-linking ratio. It is important to note here that although alteration of both fibrinogen concentration and cross-linking ratio will result in changes in substrate mechanical properties, altered fibrinogen concentration will also lead to modified substrate chemistry. Hence the effect of changing fibrinogen concentration cannot be attributed solely to substrate mechanical effects. Modification of cross-linking ratio, however, will maintain chemical equivalence in the gel and any changes observed in the gel can be attributed solely to substrate mechanical properties.Analysis of the embryonic stem cells cultured on different substrate conditions clearly illustrate strong influence which substrate mechanical properties assert on cellular proliferation and differentiation potential.

Analysis of the embryonic stem cells cultured on different substrate conditions clearly illustrate strong influence which substrate mechanical properties assert on cellular proliferation and differentiation potential. Overall, the ESCs cultured on lower cross-linking substrates were found to be more conducive to proliferation as well as differentiation. For both the 2-dimensional and 3-dimensional culture configurations analyzed in this work, the low-cross-linking substrates elicited a somewhat elevated expression level of most of the gene expression markers tested. However, the level of up-regulation strongly depended of the specific germ layer. It was consistently observed that differential expression of endodermal markers are the strongest in low-cross-linked gels as compared to high cross-linked gels, an effect which was further accentuated in 3-dimensional culture as compared to 2-dimensional culture.

Based on the analysis of both the substrate mechanical properties and the cellular differentiation pattern, it is reasonable to suggest the substrate stiffness to be the principle player in the modulation of cellular differentiation patterning. The presented results are indicative of the fact that lower substrate stiffness values are favoring the preferential differentiation of the embryonic stem cells towards endodermal lineage. The presented experiments also establish that stronger differentiation can be achieved in 3-Dimensional cultures as compared to 2-Dimensional culture configuration