TGFβ/Activin/Nodal pathway in inhibition of human embryonic stem cell differentiation by mechanical strain

Cyclic biaxial mechanical strain has been reported to inhibit human embryonic stem cell differentiation without selecting against survival of differentiated or undifferentiated cells. Weshow that TGF beta/Activin/Nodal signaling plays a crucial role in repression of human embryonic stem cell (hESC) differentiation under mechanical strain. Strain-induced transcription of TGF beta 1, Activin A, and Nodal, and upregulated Similar to Mothers Against Decapentaplegic homolog (Smad)2/3 phosphorylation in undifferentiated hESC. TGF beta/Activin/Nodal receptor inhibitor SB431542 stimulated differentiation of hESCs cultured under biaxial strain. Exogenous addition of TGF beta 1, Activin A, or Nodal alone was insufficient to stimulate hESC self-renewal to replicate behavior of hESCs in presence of strain. However, exogenous TGF beta 1 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 TGF beta 1 promoted hESC differentiation whereas inhibition of Activin A by Follistatin promoted hESC differentiation to a lesser extent. Together, these findings show that TGF beta 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 TGFb superfamily ligands.