Human embryonic stem cells (HESCs) are pluripotent cells that are derived from the inner cell mass of blastocyst stage embryos. HESCs can be propagated extensively in culture while retaining the ability to differentiate into different somatic cell types. The pluripotent HESC state is maintained by the interactions between (i) extracellular factors added to cell culture and (ii) endogenous factors secreted by HESCs and differentiated cells in cell culture. Specifically, signaling mediated by fibroblast growth factors (FGFs), Wnts and members of the transforming growth factor - beta (TGF-beta) superfamily have emerged as important regulators of HESC self renewal.

We have previously demonstrated that the effect of endogenous factors and the resultant spatial gradients in signaling can be controlled by manipulating the spatial organization of HESCs in culture. This is achieved through micropatterning of HESC colonies with defined diameters and separation. Control of HESC colony size minimizes the variability arising from endogenous signaling in conventional HESC cultures. Using experimental data generated in our system and simple mechanistic modeling, here we show that the level of Smad2 activity is maintained in a specified range in self-renewing HESCs. Smad2 signaling activity is regulated through intracellular and extracellular control mechanisms. Specifically, we focus on extracellular interactions between Nodal, Lefty, Cripto, Activin and the Activin Receptors and intracellular crosstalk between the PI3 kinase, Wnt and Smad2 signaling pathways.