Supplementary Figure 5: The effect of media components on the choice between self-renewal or differentiation of naïve ESCs.

(A) Comparison between the base media N2B27 and RPMI + B27. While there are many differences, some of the largest differences were in the components highlighted in red including Insulin, Glucose, Galactose, L-Alanine, Sodium Pyruvate and Vitamin A. We then addressed these components during ESC self-renewal, and PrE differentiation. (B) Table of ingredients of B27. There is 2× the amount of B27 in RPMI + B27 than in N2B27, therefore one of these components could be enhancing PrE differentiation. Highlighted in red are two components that based on other studies could be responsible, Vitamin A and Galactose. (C) RPMI + B27 contains ready-mixed Glutamax, while N2B27 contains a homemade supplement of Glutamine, therefore we asked whether this could cause the difference in PrE differentiation of the two media. NanogGFP (transcriptional reporter) ESCs were differentiated to PrE for 6 days in either glutamine or Glutamax in RPMI + B27. We did not observe a significant difference. Dot plots are representative of 3 differentiations. (D) To address whether the supplements added to RPMI or N2B27 could affect pluripotency versus PrE differentiation, we asked whether making RPMI + B27 more comparable to N2B27 would reduce PrE differentiation. Thus, we added half the amount of B27, or N2 to RPMI PrE medium and differentiated the cells for 6 days. Both B27 and N2 have a small effect of reducing PrE differentiation, suggesting that there are multiple factors causing a difference in differentiation efficiency between the two media. Dot plots are representative of 3 differentiations. (E) NanogGFP ESCs were differentiated to PrE for 6 days in increasing doses of Sodium Pyruvate (NaPyr), or in a double dose of L-Alanine. Neither Sodium pyruvate nor L-alanine had a large effect on PrE differentiation (extremely high doses of NaPyr reduced PrE differentiation by 1/5, however this may be due to the secondary effects of such a high dose). Dot plots are representative of 3 differentiations. (F) NanogGFP ESCs were differentiated to PrE for 6 days in Galactose, which had little effect on differentiation efficiency. Dot plots are representative of 3 differentiations. (G) Retinoic acid can improve PrE differentiation. Retinoic acid is known to play a role in PrE specification. NanogGFP ESCs were differentiated to PrE for 6 days in DMSO control, or in varying concentrations of the RA inhibitor AGN 193109 (AGN). Cells were analysed for PrE differentiation by flow cytometry, with staining for PDGFRA. Graph shows quantification of percentages of gated cells from FACS data based on NanogGFP expression (ESC) or PDGFRA staining (PrE); n = 3, error bars show ± s.d., P values were calculated by t-test for each in comparison to 0 μM (DMSO) control for either PrE or ESC population. PrE calculations; P values 1 μM P = 0.0017; 10 μM P = 0.0032. ESC calculations; 1 μM P = 0.0060, 10 μM P = 0.0126. Dot plots are representative of 3 differentiations. (H) Antagonism of RA signalling can inhibit differentiation. AGN (1 μM) was added to self-renewing cells and differentiating cells. In both 2i/LIF and NACL there was less spontaneous differentiation to PrE in the presence of AGN. In PrE differentiation, AGN drastically reduced the percentage of differentiated PrE cells. Dot plots are representative of 4 differentiations.