SummaryHeterogeneity within pluripotent stem cell (PSC) populations is indicative of dynamic changes that occur when cells drift between different states. Although the role of metastability in PSCs is unclear, it appears to reflect heterogeneity in cell signaling. Using the Fucci cell-cycle indicator system, we show that elevated expression of developmental regulators in G1 is a major determinant of heterogeneity in human embryonic stem cells. Although signaling pathways remain active throughout the cell cycle, their contribution to heterogeneous gene expression is restricted to G1. Surprisingly, we identify dramatic changes in the levels of global 5-hydroxymethylcytosine, an unanticipated source of epigenetic heterogeneity that is tightly linked to cell-cycle progression and the expression of developmental regulators. When we evaluated gene expression in differentiating cells, we found that cell-cycle regulation of developmental regulators was maintained during lineage specification. Cell-cycle regulation of developmentally regulated transcription factors is therefore an inherent feature of the mechanisms underpinning differentiation.

Suppressing the activity of Gsk3␤ is critical for maintenance of murine pluripotent stem cells. In murine embryonic stem cells (mESCs), Gsk3␤ is inhibited by multiple mechanisms, including its inhibitory phosphorylation on serine 9 by protein kinase B (Akt), a major effector of the canonical phosphatidylinositol 3-kinase (PI3K) pathway. A second PI3K/Akt-regulated mechanism promotes the nuclear export of Gsk3␤, thereby restricting its access to nuclear substrates such as c-myc and ␤-catenin. Although Gsk3␤ shuttles between the nucleus and cytoplasm under self-renewing conditions, its localization is primarily cytoplasmic because its rate of nuclear export exceeds its rate of nuclear import. In this report, we show that Gsk3␤ is exported from the nucleus in a complex with Frat. Loss of PI3K/Akt activity results in dissociation of this complex and retention of Gsk3␤ in the nucleus. Frat continues to shuttle between the nucleus and cytoplasm under these conditions and remains predominantly in the cytoplasm. These results indicate that Frat carries Gsk3␤ out of the nucleus under self-renewing conditions and that PI3K regulates this by promoting its association with Frat. These findings provide new links between PI3K/Akt signaling and regulation of Gsk3␤ activity by Frat, an oncogene previously shown to cooperate with Myc in tumorigenesis.

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The fluorescence ubiquitination cell cycle indicator (FUCCI) system provides a powerful method to evaluate cell cycle mechanisms associated with stem cell self-renewal and cell fate specification. By integrating the FUCCI system into human pluripotent stem cells (hPSCs) it is possible to isolate homogeneous fractions of viable cells representative of all cell cycle phases. This method avoids problems associated with traditional tools used for cell cycle analysis such as synchronizing drugs, elutriation and temperature sensitive mutants. Importantly, FUCCI reporters allow cell cycle events in dynamic systems, such as differentiation, to be evaluated. Initial reports on the FUCCI system focused on its strengths in reporting spatio-temporal aspects of cell cycle events in living cells and developmental models. In this report, we describe approaches that broaden the application of FUCCI reporters in PSCs through incorporation of FACS. This approach allows molecular analysis of the cell cycle in stem cell systems that were not previously possible.