Extended Data Fig. 2. BMP signaling and hPGCLC differentiation.

a, Expression of key lineage markers and BMP ligands in single cells of cultured human embryos (~E11)¹⁶ visualized by Uniform manifold approximation and projection (UMAP) and Louvain clustering. Color coding is as indicated. STB: syncytiotrophoblast; CTB: cytotrophoblast; Epi: epiblast; Hyp: hypoblast. b, Expression of BMP2 in various cell types in a gastrulating human embryo at ~E16¹⁷. c, d, Unsupervised hierarchical clustering (UHC) (c) and cell-type annotation (c, d) based on key marker expression of endoderm cells in a gastrulating human embryo at ~E16 in (b)¹⁷ and expression of BMP ligands in each cell type (d). Numbers of the cells in each cluster are: n = 50 for DE; n = 51 for Hyp; n = 34 for YS. In (b, d), the upper hinges, lower hinges, and middle lines indicate 75 percentiles, 25 percentiles, and median values, respectively. The whiskers are drawn in length equal to the inter-quartile range (IQR) multiplied by 1.5. Data beyond the upper/lower whiskers are shown as dots. e, Expression of BMP ligands in cells composing human embryonic gut at week 6.1¹⁸. Note that BMP ligands are expressed at a high level in colonic (i.e., hindgut) epithelium and mucosal mesoderm. FLC: fibroblasts; SMC: smooth muscle cells. f, g, Representative FACS plots for BTAG expression (f, top) and for FSC/SSC fluorescence of the BT⁻AG⁻ cells (f, bottom), and hPGCLC fold-change (g, left) and the enrichment scores (g, right) of the hPGCLC culture with various concentrations of BMP2 with IWR1 (1.5 μM) in advanced RPMI at c22. The data represent (f)/show (g) 2 (BMP2 5 ng/mL) and 3 (BMP2 0, 10−200 ng/mL) biological replicates. The passages were performed with dilution. Note that there were nearly no de-differentiated cells in the P1 gate under all conditions. h, Immunofluorescence (IF) analysis of the expression of GFP (TFAP2C-EGFP: AG), tdTomato (BLIMP1-tdTomato: BT), and DAZL in hPGCLC-derived cells cultured without (top) or with (bottom) BMP2 (25 ng/ml) at c55. ~19% (5/26) of BT⁺AG⁺ cells were DAZL⁺ in the culture with BMP2, whereas no DAZL⁺ cells were found in the culture without BMP2 (1 biological replicate). Bar, 50 μm.

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