TGFβ/Activin/Nodal Pathway in Inhibition of Human Embryonic Stem Cell Differentiation by Mechanical Strain

Abstract

Cyclic biaxial mechanical strain has been reported to inhibit human embryonic stem cell differentiation without selecting against survival of differentiated or undifferentiated cells. We show that TGFβ/Activin/Nodal signaling plays a crucial role in repression of human embryonic stem cell (hESC) differentiation under mechanical strain. Strain-induced transcription of TGFβ1, Activin A, and Nodal, and upregulated Similar to Mothers Against Decapentaplegic homolog (Smad)2/3 phosphorylation in undifferentiated hESC. TGFβ/Activin/Nodal receptor inhibitor SB431542 stimulated differentiation of hESCs cultured under biaxial strain. Exogenous addition of TGFβ1, Activin A, or Nodal alone was insufficient to stimulate hESC self-renewal to replicate behavior of hESCs in presence of strain. However, exogenous TGFβ1 and Activin A in combination partially replicated the self-renewing phenotype induced by strain but when combined with strain did not further stimulate self-renewal. In presence of mechanical strain, addition of a neutralizing antibody to TGFβ1 promoted hESC differentiation whereas inhibition of Activin A by Follistatin promoted hESC differentiation to a lesser extent. Together, these findings show that TGFβ superfamily activation of Smad2/3 is required for repression of spontaneous differentiation under strain and suggest that strain may induce autocrine or paracrine signaling through TGFβ superfamily ligands.