Extended Data Fig. 8: Validation of BMP modulation, comparative functional modulation in mouse, and divergent roles for BMP in naïve pluripotency.

a, Violin plots of BMP2/4/6 expression. N = 9862 single cells. b-c, Immunofluorescence images and quantification of primed hESCs cultured in control conditions (n = 2825), 25 ng/ml BMP2 (n = 2184), 25 ng/ml BMP4 (n = 1681), 50 ng/ml BMP6 (n = 889), or 50 ng/ml BMP6 + 200 nM LDN193189 (n = 1406). d-e, Bayesian coefficients ± credible interval for counts presented in Fig. 5b-d (d) and Fig. 5f-h (e). f, Immunofluorescence images of mouse embryos recovered at E3.5 and cultured in vitro for 48 h in control (N = 19), 50 ng/ml BMP6 (N = 11), or 200 nM LDN193189 (N = 13) conditions. g-h, Quantification of the number epiblast (g) and primitive endoderm/visceral endoderm (h) from f. i, Immunofluorescence images of mouse embryos recovered at E5.0 and cultured in vitro for 36 h in control (N = 8), BMP6 (N = 10) or LDN193189 (N = 8) conditions. j, Quantification of the proportion of embryos that successfully made an egg cylinder and have a Cer1-positive DVE/AVE. k-l, Quantification of the epiblast length (k) and area (l). m, Immunofluorescence images and quantification of hESCs treated for 48 hours with control conditions (PXGL n = 2336, primed n = 7796) or 200 nM LDN193189 (PXGL n = 2398, primed n = 6500, N = 2 experiments). n, Pearson regression correlation coefficients for ID1 and ID3 expression with naïve pluripotency markers. o, Immunofluorescence and quantification of AP2γ in hESCs (LDN: n = 1695 untreated cells; n = 1426 LDN treated cells; N = 2 experiments). p, Immunofluorescence and quantification of Otx2 in mESCs cultured in either 2iLif naïve conditions (n = 1570 cells), N2B27 (n = 3602 cells), or N2B27 + LDN (n = 1064 cells). N = 3 experiments. q, Immunofluorescence images and quantification of pSMAD1.5.9 in YSLCs (control: n = 1150; LDN: n = 827; N = 2 experiments). For box plots, the box encompasses the 25^th-75^th quartiles with the median marked by the central line. The minimum and maximum are denoted by the whiskers. For violin plots, the mean is marked by the red line. Statistical tests: (c, k-l, p) One-way ANOVA with Tukey-Kramer post-hoc; (e-f) two-sided Mann-Whitney test, (m, o, q) two-sided unpaired T-test; (o) Coefficients were tested (cor.test; two-tailed) and corrected for multiple hypothesis testing with the Benjamini-Hochberg method; ****p < 0.0001. ***p < 0.001. **p < 0.01. *p < 0.05. Unmarked pairwise comparisons are not significant (ns). Exact p-values presented in Supplemental Table 8. Scale bars: (b, m, o-q) 50 µm, (f, i) 100 µm.

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