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

Abstract

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.

Figure 1. Reporter models of Nanog heterogeneity in ES cells

ES cell cultures from TNG (A) and BNG ES cells (B) were sorted according to GFP expression levels. C) Gene expression of sorted BNG ES cells showed differential expression of pluripotency and differentiation markers. D) GFP expression and cell morphology were analyzed in FACS-purified BNG cells.

Figure 2. Activity of TGF-beta signaling in Nanog subpopulations

A) Expression of TGFbeta regulated genes was lower in GFP-high than GFP-low cells. B) GFP-low and GFP-high cells had similar expression changes to Nodal and BMP signaling inhibition. C) TNG cells were treated in an identical manner and protein was analyzed for levels of phosph-Smad2 and phospho-Smad1/5 activity.

Figure 3. Nanog subpopulation dynamics in response to TGF-beta modulation

A,B) GFP-high sorted cells were cultured and then flow analyzed for GFP expression. SB treatment significantly decreased the number of GFP-low cells. In contrast, both LDN treatment and SB+LDN cotreatment increased the number of GFP-low cells. C) Graph depicts triplicate samples.

Figure 4. Id1 overexpression

A) Id1 overexpression significantly enhanced the percentage of GFP-high cells and significantly attenuated the effects of SB+LDN treatment. B) Graphs summarize data from triplicate experiments. C) Id1 overexpression decreased the effects of LDN treatment on Nanog-GFP expression. D) In KOSR, Id1 induced a similar reduction of GFP-low cells compared to recombinant BMP stimulation.

Figure 5. Inhibition of FGF/MEK signaling

A) PD treatment enhanced GFP-high and -very high cells. B) SB and LDN treatments did not affect levels of pERK while PD treatment vastly reduced pERK. Inhibition of FGF/MEK signaling reduced the phosphorylation of Smad2 and Smad1/5 (C) and decreased the expression of many TGF-beta-related genes (D).

Figure 6. TGF-beta inhibition decreases Nanog

In GFP-high (A) and GFP-low (B) sorted ES cells, combined inhibition of Nodal and BMP signaling increased the percentage of GFP-negative cells. C,D) Under SB+LDN+Smad7 growth conditions, GFP-negative cells with differentiated morphologies were apparent (see bracket). RNA analysis showed Nanog expression after 24 hours (E) and differentiation markers after 6 days (F).

Figure 7. Model for Nodal and BMP function in regulating dynamic heterogeneity of mouse ES cells in serum-based culture

See text for details.