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

Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53706, and WiCell Research Institute, Madison, Wisconsin 53707

Correspondence: Address reprint requests to Sean Palecek, PhD, Dept. of Chemical and Biological Engineering, 1415 Engineering Dr., University of Wisconsin-Madison, Madison, WI 53706. Tel.: 608-262-8931; Fax: 608-262-5434; E-mail: palecek{at}engr.wisc.edu.

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.