Mechanisms Regulating Early Mesendodermal Differentiation of Human Embryonic Stem Cells: A Dissertation

Abstract

Key regulatory events take place at very early stages of human embryonic stem cell (hESC) differentiation to accommodate their ability to differentiate into different lineages; this work examines two separate regulatory events. To investigate precise mechanisms that link alterations in the cell cycle and early differentiation, we examined the initial stages of mesendodermal lineage commitment and observed a cell cycle pause that occurred concurrently with an increase in genes that regulate the G2/M transition, including WEE1. Inhibition of WEE1 prevented the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. These findings reveal a novel G2 cell cycle pause required for endodermal differentiation. A role for phenotypic transcription factors in very early differentiation is unknown. From a screen of candidate factors during early mesendodermal differentiation, we found that RUNX1 is selectively and transiently up-regulated. Transcriptome and functional analyses upon RUNX1 depletion established a role for RUNX1 in promoting cell motility. In parallel, we discovered a loss of repression for several epithelial genes, indicating that RUNX1 knockdown impaired an epithelial to mesenchymal transition during differentiation. Cell biological and biochemical approaches revealed that RUNX1 depletion compromised TGFβ2 signaling. Both the decrease in motility and deregulated epithelial marker expression upon RUNX1 depletion were rescued by reintroduction of TGFβ2, but not TGFβ1. These findings identify novel roles for RUNX1-TGFβ2 signaling in mesendodermal lineage commitment.