Fig. 7: Long-term WNT5A treatment inhibits BMP signaling in hESC-derived EPs.

a BMP pathway-associated gene expression after 5 days WNT5A treatment of EPs compared to untreated (UT) control. Downregulation of BMP ligands, effectors and BMP antagonist upregulation suggests decreased BMP activity. Log₂ Z-score of normalized RNA-sequencing expression values are shown. b qRT-PCR verification of selected gene expression after 5-day WNT5A treatment. Mean ± 95% CI and p values (unpaired two-tailed t-tests) are shown. N = 3 independent experiments. AU arbitrary units. c Dual pathway luciferase system to assess activity of Jun (AP1) and Bmp (Smad) pathways. Details in “Methods”. d Quantification of relative AP1 and Smad activity in WNT5A-treated EPs. Anisomycin (Anis) and BMP4 positive controls for AP1 and Smad, respectively. Mean ± SEM and p values (unpaired two-tailed t-test) are shown. N = 3 independent experiments. e Immunofluorescent analysis of phospho-Smad1/5 cellular localization in INS+ cells. p-Smad1/5 is mostly in cytoplasm (CYT) of INS+ cells, while in nucleus (NC) in INS- cells. N = 4 independent experiments. f Quantification of cytoplasmic and nuclear p-Smad1/5 in INS+ and INS- cell. Mean ± SEM and a p value (unpaired two-tailed t-test) are shown. N = 3 independent experiments, ≥10 images per experiment. g post-FACS imaging of single EPs to detect p-Smad1/5 and INS cellular localization. The majority of cells expressing nuclear p-Smad1/5 (top) do not express INS, N = 4 independent experiments. h Quantification of CHGA+ and INS+ cells after 3-day EP treatment with Gremlin1 (Grem1), WNT5A or in combinations. Mean ± SEM and p values (unpaired two-tailed t-tests) are shown. N = 3 independent experiments. i INS immunostaining of EPs treated for 3 days with WNT5A, Grem1, and Grem1 + WNT5A. Scale bar = 50 µm. N = 6 independent experiments. j GSIS analysis after 5-day EP treatment with WNT5A alone or in combination with Grem1 compared to untreated EPs (UT) or M-E20 coculture. Graph represents ELISA measurements. N = 4 (glucose) or 3 (KCl) independent experiments. Mean ± 95% CI, and significant p values (Tukey’s multiple comparison test) are shown. k Model of WNT5A role in human pancreatic niche during β-cell differentiation. Pancreatic stage-specific M-E cells secrete WNT5A and Grem1, which activate JNK/c-JUN/AP1 and inhibit BMP pathways leading to CHGA, INS upregulation and GCG downregulation in hESC-derived EPs. DAPI marks nuclei.