Signalling pathway crosstalk stimulated by L-proline drives mouse embryonic stem cells to primitive-ectoderm-like cells

ABSTRACT The amino acid L-proline exhibits growth factor-like properties during development – from improving blastocyst development to driving neurogenesis in vitro. Addition of 400 μM L-proline to self-renewal medium drives naïve mouse embryonic stem cells (ESCs) to early primitive ectoderm-like (EPL) cells – a transcriptionally distinct primed or partially primed pluripotent state. EPL cells retain expression of pluripotency genes, upregulate primitive ectoderm markers, undergo a morphological change and have increased cell number. These changes are facilitated by a complex signalling network hinging on the Mapk, Fgfr, Pi3k and mTor pathways. Here, we use a factorial experimental design coupled with statistical modelling to understand which signalling pathways are involved in the transition between ESCs and EPL cells, and how they underpin changes in morphology, cell number, apoptosis, proliferation and gene expression. This approach reveals pathways which work antagonistically or synergistically. Most properties were affected by more than one inhibitor, and each inhibitor blocked specific aspects of the naïve-to-primed transition. These mechanisms underpin progression of stem cells across the in vitro pluripotency continuum and serve as a model for pre-, peri- and post-implantation embryogenesis.

Significance is denoted as *P < 0.05.

Fig. S1 .
Fig. S1.Representative western blots show changes in pathway phosphorylation.A. Naïve ESCs was calculated by measuring the integrated intensity of the band or bands with Image Studio.The integrated intensity of an adjacent paired region was subtracted to account for background signal.Worked examples are shown for proteins with a one or two bands (e.g., p-Rps6) and multiple bands (p-4ebp1).Development: doi:10.1242/dev.201704:Supplementary information Development • Supplementary information

Fig. S2 .
Fig. S2.Morphology data by inhibitor combination.Naïve ESCs were cultured over 6 days in 330 U/mL LIF + 400 μM L-proline with combinations of five inhibitors (U: U0126; S: SU5402; L: LY294002; R: rapamycin; P: PF-4708671).Morphology scoring was performed on day 6.Conditions containing L+R (navy) were considered non-viable at day 2 and no data is available for this combination.Data is shown as mean and SEM with individual data points.Data were analysed using one-way ANOVA with Dunnett's multiple comparisons test to cells grown in 330 U/mL LIF + 400 μM L-proline + DMSO, *P < 0.05.

Fig. S3 .
Fig. S3.Cell number data by inhibitor combination.Naïve ESCs were cultured over 6 days in 330 U/ mL LIF + 400 μM L-proline with combinations of five inhibitors (U: U0126; S: SU5402; L: LY294002; R: rapamycin; P: PF-4708671).Cells were counted at passaged on day 2, 4, and 6.Conditions containing L+R (navy) were considered non-viable at day 2 and no data is available for this combination at days 4 and 6.Data is shown as mean and SEM with individual data points.Data were analysed using one-way ANOVA with Dunnett's multiple comparisons test to cells grown in 330 U/mL LIF+ 400 μM L-proline + DMSO, *P < 0.05.

Fig. S8 .
Fig. S8.Alternative model for proliferation data.Naïve ESCs were cultured over 6 days in 330 U/mL LIF + 400 μM L-proline with combinations of five inhibitors (U: U0126; S: SU5402; L: LY294002; R: rapamycin; P: PF-4708671).At days 2, 4 and 6 proliferation was measured.Data was averaged across biological replicates where n ≥ 3. To correct bimodal input data, data was binned into octiles each representing an equal proportion of the data.A. Data was modelled using either MLR, MLR with two-way interaction terms or a BRANNGP.Fit of each model is shown comparing the actual fit with the prediction from the model.B. Coefficients for each variable ± SEM for standard MLR.C.