UC San Diego

During development embryonic stem cells use a variety of external cues to direct their differentiation. In particular, definitive endoderm specification coincides with signaling from soluble factors and the extracellular matrix. By contrast, in vitro differentiation methods often rely exclusively on growth factors to direct cell fate and so the role of extracellular matrix signaling is not well understood. Thus this dissertation examines how extracellular matrix enhances soluble factor signaling during definitive endoderm differentiation. Soluble factors used to direct definitive endoderm specification (e.g. Activin A) are shown to induce embryonic stem cells to activate traction forces and assemble a lineage- specific extracellular matrix that is rich in fibronectin and laminin. By contrast, pluripotent embryonic stem cells do not assemble substantial extracellular matrix or activate contractility. Traction forces are shown to be required for definitive endoderm specification and laminin is shown to improve differentiation in a dose-dependent manner. Extracellular laminin and contractility promote Activin A signaling by inhibiting SMAD7 expression and allowing nuclear accumulation of phosphorylated-SMAD2, respectively. This work demonstrates a role for extracellular matrix in regulating the signaling pathways that guide early embryonic stem cell fate