TGF-β-superfamily signaling regulates embryonic stem cell heterogeneity: self-renewal as a dynamic and regulated equilibrium.

Embryonic stem cells dynamically fluctuate between phenotypic states, as defined by expression levels of genes such as Nanog, while remaining pluripotent. The dynamic phenotype of stem cells is in part determined by gene expression control and dictated by various signaling pathways and transcriptional regulators. We sought to define the activities of two TGF-β-related signaling pathways, bone morphogenetic protein (BMP) and Nodal signaling, in modulating mouse embryonic stem (ES) cell heterogeneity in undifferentiated culture conditions. Both BMP and Nodal signaling pathways were seen to be active in distinct Nanog subpopulations, with subtle quantitative differences in activity. Pharmacological and genetic modulation of BMP or Nodal signaling strongly influenced the heterogeneous state of undifferentiated ES cells, as assessed by dynamic expression of Nanog reporters. Inhibition of Nodal signaling enhanced BMP activity, which through the downstream target Id factors, enhanced the capacity of ES cells to remain in the Nanog-high epigenetic state. The combined inhibition of Nodal and BMP signaling resulted in the accumulation of Nanog-negative cells, even in the presence of LIF, uncovering a shared role for BMP and Nodal signaling in maintaining Nanog expression and repression of differentiation. These results demonstrate a complex requirement for both arms of TGF-β-related signaling to influence the dynamic cellular phenotype of undifferentiated ES cells in serum-based media, and that differing subpopulations of ES cells in heterogeneous culture have distinct responses to these signaling pathways. Several pathways, including BMP, Nodal, and FGF signaling, have important regulatory function in defining the steady-state distribution of heterogeneity of stem cells.