(155c) Unraveling the Molecular Regulation of Mesendodermal Differentiation in Human Embryonic Stem Cells

Signaling mediated by the Transforming Growth Factor ? beta (TGF-beta) and Wnt ligands, through the Smad2/3 and beta catenin proteins respectively, plays an important role in regulating human embryonic stem cell (hESC) fate. In particular, Smad2/3 and beta-catenin activity is essential for maintaining the pluripotent hESC state as well as during differentiation to mesendodermal lineages (precursors of clinically relevant cell types such as heart, blood). However, the exact mechanisms through which hESCs interpret Smad2/3 and beta-catenin signals to remain undifferentiated or initiate differentiation remain largely unknown.

We hypothesize that the while Smad2/3 and beta-catenin signaling is active in the undifferentiated state, the activity of Smad2/3 and beta-catenin is restricted. Mesendodermal differentiation is associated with inactivation of mechanisms that restrict Smad2/3 and beta-catenin and a concomitant increase in their transcriptional activity. We have used a combination of conventional biochemical analyses such as immunoblotting as well as quantitative mass spectrometry to identify and subsequently quantify the molecular mechanisms regulating Smad2/3 and beta-catenin signaling. Here we present our results from a systematic analysis of the membrane, cytoplasmic and nuclear fractions of undifferentiated hESCs and cells undergoing mesendodermal differentiatiation.