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 TGFbeta/Activin/Nodal signaling plays a crucial role in repression of human embryonic stem cell (hESC) differentiation under mechanical strain. Strain-induced transcription of TGFbeta1, Activin A, and Nodal, and upregulated Similar to Mothers Against Decapentaplegic homolog (Smad)2/3 phosphorylation in undifferentiated hESC. TGFbeta/Activin/Nodal receptor inhibitor SB431542 stimulated differentiation of hESCs cultured under biaxial strain. Exogenous addition of TGFbeta1, Activin A, or Nodal alone was insufficient to stimulate hESC self-renewal to replicate behavior of hESCs in presence of strain. However, exogenous TGFbeta1 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 TGFbeta1 promoted hESC differentiation whereas inhibition of Activin A by Follistatin promoted hESC differentiation to a lesser extent. Together, these findings show that TGFbeta 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 TGFbeta superfamily ligands.

Nodal promotes growth and invasion in human gliomas

“…It is well known that Nodal regulates cell differentiation in human embryonic stem cells (Besser, 2004;Saha et al, 2008;Smith et al, 2008). However, the role of Nodal on glioma differentiation remained to be analyzed.…”

“…Nodal is an embryonic morphogen that regulates dorsal mesoderm induction, anterior patterning and left-right asymmetry (Brennan et al, 2002;Nonaka et al, 2002;Smith et al, 2008). Nodal also has an important role in maintaining pluripotency of human embryonic stem cells and cancer cells (Vallier et al, 2004(Vallier et al, , 2005Saha et al, 2008). Importantly, Nodal secretion mediated plasticity and tumorigenicity of metastatic melanoma and inhibition of Nodal promoted tumor differentiation and regression (Topczewska et al, 2006;Hendrix et al, 2007;Postovit et al, 2008a, b).…”

Single Mechanosensitive and Ca2+-Sensitive Channel Currents Recorded from Mouse and Human Embryonic Stem Cells

“…Although some variations, both within and between species, may reflect differences in the stretch protocols used (e.g., the cyclic strain frequencies ranged from 10 -60 cycles/minute over durations from 1 hour to 14 days) it is notable that when mESCs and hESCs were both exposed to 14 days of cyclic stretch, opposing effects on pluripotency and differentiation were observed (Saha et al, 2006; Gwak et underlying the different responses. In the case of the strain-induced inhibition of hESC differentiation, it has been proposed that stretch-induced release of transforming growth factorbeta (TGF-) ligands and activation of TGF-/Activin/Nodal signaling produce the inhibition of differentiation and the maintenance of pluripotency (Saha et al, 2008). In the case of mESCs, stretch-induced generation of reactive oxygen species (ROS) and integrin-mediated PI3K-Akt signaling appears to promote differentiation (Schmelter et al, 2006;Heo et al, 2011).…”

Mechanobiology of Human Pluripotent Stem Cells

“…82 On the contrary, the strain contributed to hESC pluripotency, leading to increases in TGFb1, Activin A, and Nodal mRNA levels as well as phosphorylation of SMAD2/3. 83 Each of these signaling molecules has been known to be intimately involved in hESC self-renewal. However, mechanical strain does not obviate the requirement for conditioned medium.…”

“…(B) Flow cytometrical analysis data showing Oct4 expression in hESCs subjected to mechanical strain, Activin A and TGFb1 treatment, and inhibition of TGFb signaling for 7 days. Adapted with permission from Teramura et al 81 and Saha et al 83 …”