(96e) Intracellular Signaling During Early Differentiation of Human Embryonic Stem Cells

Human Embryonic Stem Cells (hESCs) can revolutionize regenerative medicine and provide a means to produce functional cell types for drug evaluation. However, clinical application of hESCs is still in its infancy, largely due to the lack of efficient processes to differentiate them to desired cell types. A quantitative understanding of hESC fate decisions will greatly help in designing efficient processes for differentiation of hESCs to clinically relevant cell types

In this study, we specifically focus on signaling mediated by Transforming Growth Factor-beta (TGF-beta) and Wnt pathways. The TGF-beta and Wnt ligands play an important role in regulating the self renewal and early differentiation of hESCs. However, the exact mechanisms through which hESCs interpret these extracellular stimuli to remain undifferentiated or initiate differentiation remain largely unknown. For instance, TGF-beta and Wnt signaling is necessary for maintenance of undifferentiated hESCs. Yet, these signals are also important mediators of differentiation to the mesendodermal lineages (which give rise to cell types such as heart, lungs).

Using quantitative mass spectrometry, we analyze the quantitative concentration thresholds of nuclear localized Smads and beta-catenin that regulate the self renewal and early differentiation of hESCs. Beta-catenin and Smads interact with multiple proteins inside the nucleus. Further, Smads and beta-catenin compete for the same set of interacting proteins. Using immunoprecipitation and mass spectrometry, we identify the interaction partners of Smads and beta catenin in undifferentiated hESCs and hESCs undergoing early differentiation.