Substrate stiffness affects early differentiation events in embryonic stem cells

TLDR

Embryonic stem cells are a promising source for tissue replacement and a model for early development, and while chemical cues influence their differentiation, evidence shows they also sense and respond to mechanical properties of their environment. The study tested whether varying substrate stiffness on flexible PDMS influences ESC differentiation by culturing cells on substrates of different rigidity. ESCs showed increased spreading, growth, and upregulation of primitive streak genes such as Brachyury, Mixl1, and Eomes on stiffer substrates, and osteogenic differentiation was also enhanced, indicating that substrate stiffness promotes both early and terminal differentiation.

Abstract

Embryonic stem cells (ESC) are both a potential source of cells for tissue replacement therapies and an accessible tool to model early embryonic development. Chemical factors such as soluble growth factors and insoluble components of the extracellular matrix are known to affect the differentiation of murine ESCs. However, there is also evidence to suggest that undifferentiated cells can both sense the mechanical properties of their environment and differentiate accordingly. By growing ESCs on flexible polydimethylsiloxane substrates with varying stiffness, we tested the hypothesis that substrate stiffness can influence ESC differentiation. While cell attachment was unaffected by the stiffness of the growth substrate, cell spreading and cell growth were all increased as a function of substrate stiffness. Similarly, several genes expressed in the primitive streak during gastrulation and implicated in early mesendoderm differentiation, such as Brachyury, Mixl1 and Eomes, were upregulated in cell cultures on stiffer compared to softer substrates. Finally, we demonstrated that osteogenic differentiation of ESCs was enhanced on stiff substrates compared to soft substrates, illustrating that the mechanical environment can play a role in both early and terminal ESC differentiation. Our results suggest a fundamental role for mechanosensing in mammalian development and illustrate that the mechanical environment should be taken into consideration when engineering implantable scaffolds or when producing therapeutically relevant cell populations in vitro.