(685a) Role of Substrate Stiffness Modulation in Controlling Morphology and Differentiation of Embryonic Stem Cells

The effects of cellular microenvironment in modulating stem cell morphology and phenotype commitment have been an intense and intriguing area of research. While the effect of chemical constituents of the cellular microenvironment has been relatively well studied, the effect of mechanical microenvironment on stem cell fate has only recently been explored. In mesenchymal stem cells (MSCs) modification of matrix elasticity was recently reported to induce tissue specific lineage commitment: stiffer materials induce bone while softer materials induce neurogenic commitment. In our studies we have investigated the effect of substrate stiffness on the morphology and phenotype commitment of pluripotent embryonic stem cell, which is likely to unravel new pathways for stem cell based regenerative medicine. Two chemically different biologic based natural polymer systems have been explored as prototype substrates for modulating the mechanical characteristics.

Differentiation of the mouse embryonic stem cells (ESC) is accordingly induced by culturing them on (a) alginate and (b) fibrin substrate. The effect of mechanical microenvironment is explored by maintaining the chemical composition invariant in each system, while modifying the substrate stiffness by altering the gel cross-linking concentration. The embryonic stem cells attained significantly different morphology when cultured on these substrates of varying stiffness. Such effects of substrate stiffness on cellular morphology is well studied in fibroblasts and endothelial cells, which demonstrate a flattened and spread-out shape in stiff matrices, while tending to shrink in substrates exhibiting lower stiffness. Quite interestingly our experiments with embryonic stem cells elicited an opposite behavior with the cells attaining a spread-out morphology in substrates of lower stiffness and shrinking in higher stiffness. This behavior was however, independent of the chemical composition and structure of the substrate, and was consistently observed in both alginate and fibrin substrates.

The differentiation patterns of the ES cells are also altered by the substrate mechanical properties. The pluripotency markers, Oct4, Nanog and Sox2, are significantly suppressed when cells are cultured on stiffer matrices. Detailed analysis of the germ layer transcription factors determined the phenotype commitment of the differentiating population. Cells cultured on higher stiffness substrates consistently up-regulated the mesoderm markers: Brachyury, Fgf8, Goosecoid; those on lower stiffness had a higher expression level of endodermal markers: AFP, Hnf4, Hnf3β, Cldn6 and ectodermal markers: Pax6, Nestin, Fgf5, Nodal.

Our studies reveal interesting dynamic response of ESCs to changes in the mechanical microenvironment of the substrates. We believe these findings will be significant in understanding its effects on cellular morphology and phenotype commitment.