Lineage-Specific Early Differentiation of Human Embryonic Stem Cells Requires a G2 Cell Cycle Pause

Abstract

Human embryonic stem cells (hESCs) have an abbreviated G1 phase of the cell cycle that allows rapid proliferation and maintenance of pluripotency. Lengthening of G1 corresponds to loss of pluripotency during differentiation. However, precise mechanisms that link alterations in the cell cycle and early differentiation remain to be defined. We investigated initial stages of mesendodermal lineage commitment in hESCs, and observed a cell cycle pause. Transcriptome profiling identified several genes with known roles in regulation of the G2/M transition that were differentially expressed early during lineage commitment. WEE1 kinase, which blocks entry into mitosis by phosphorylating CDK1 at Y15, was the most highly expressed of these genes. Inhibition of CDK1 phosphorylation by a specific inhibitor of WEE1 restored cell cycle progression by preventing 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. Our findings identify a novel G2 cell cycle pause that is required for endodermal differentiation and provide important new mechanistic insights into early events of lineage commitment.

Transient RUNX1 Expression during Early Mesendodermal Differentiation of hESCs Promotes Epithelial to Mesenchymal Transition through TGFB2 Signaling

“…We next examined the levels of total RUNX1 transcripts during endodermal (D'Amour et al., 2005), mesodermal (Lian et al., 2013), and ectodermal (Tonge and Andrews, 2010) differentiation of hESCs to determine whether expression is lineage specific (Figure 1E); lineages were confirmed using markers as previously described (VanOudenhove et al., 2016). RUNX1 was expressed during both endodermal and mesodermal, but not ectodermal differentiation, confirming the mesendodermal specificity.…”

“…We induced mesendodermal differentiation (Figure 1A) as described in Experimental Procedures, and ensured mesendodermal commitment of hESCs by evaluating the expression of known mesendoderm markers (Mahmood and Aldahmash, 2015, Tada et al., 2005, VanOudenhove et al., 2016). As expected T , MIXL1 , and MESP1 were upregulated (Figure 1B).…”

“…The female H9 (WA09) line and the male H1 (WA01) of hESCs were maintained on Matrigel and differentiated as previously reported (VanOudenhove et al., 2016). In short, mesendoderm differentiation was induced with Knockout DMEM, containing 20% heat-inactivated defined fetal bovine serum (FBS), 1 mM L-glutamine with 1% (v/v) 2-mercaptoethanol, and 0.1 mM non-essential amino acids.…”

“…RNA was extracted using TRIzol and prepared for microarray analysis as described previously (VanOudenhove et al., 2016). All target preparation and microarray hybridization/scanning was performed in the VGN Microarray Facility at the University of Vermont.…”

Cell cycle-balanced expression of pluripotency regulators via cyclin-dependent kinase 1

“…Some studies have reported relatively uniform expression of pluripotency TFs during cell cycle both at mRNA and protein level (Shin et al, 2016;Singh et al, 2013), while others have revealed fluctuations (Gonzales et al, 2015;Van Der Laan et al, 2014;Tsubouchi et al, 2013). Given the interconnection between ESC fate choices and cell cycle (Gonzales et al, 2015;Jääger et al, 2019;Van Oudenhove et al, 2016;Pauklin and Vallier, 2013;Petruk et al, 2017), we aimed to map mESC proteome dynamics during cell cycle on a global scale.…”