Fig. 5: WNT5A is expressed in the human pancreatic niche during development and promotes INS expression in vitro.

a Human Wk16.3 pancreas stained for WNT5A and PECAM1 (left panel) or VIM (right panel). DAPI marks nuclei. Scale bar = 100 µm. N = 4 independent experiments. b Quantification of PECAM1+WNT5A+ or VIM+WNT5A+ cells in Wk16 pancreas in percentage of total PECAM+ or VIM+ cells, respectively. Data are shown as mean ± 95% CI. N = 4 independent experiments. c hESC-derived EPs after ectopic WNT5A expression (pWNT5A), were stained for WNT5A and INS. DAPI marks nuclei. Backbone plasmid served as mock control (pCDNA). Scale bar = 50 µm. N = 4 independent experiments. d Quantification of INS+ cells after ectopic WNT5A expression (pWNT5A) in EPs. Mean ± 95% CI and p values (unpaired two-tailed t-tests vs. pCDNA control) are shown. N = 4 independent experiments.) e WNTA KO in M-E17 cells. WNT5A KO in M-E cells was confirmed by immunofluorescence for WNT5A and control staining for VIM. DAPI marks nuclei). Scale bar = 100 µm. N = 3 independent experiments. See Supplementary Fig. 8C, D for KO design. f M-E20 or WNT5A KO (W5A KO) M-E cells were cocultured with EPs for 4 days, and INS+ cells were quantified from IF and normalized to no coculture control (EP W/O). EP and M-E20 coculture increased INS+ cell number by 4.5-fold compared to EP W/O, while coculture with WNT5A KO M-E caused 2-fold decrease in INS+ cells for both pairs of sgRNAs used to target WNT5A locus (KO1 + 2 or KO3 + 4). The low induction of INS+ cells with WNT5A KO M-E cells was partially rescued by WNT5A recombinant protein (blue). Mean ± 95% CI and p values (Tukey’s multiple comparisons test) are shown. N = 3 independent experiments. g Spheroids consisting of EPs and wild-type or WNT5A KO (W5A KO) M-E cells were stained for INS and VIM, showing induction of INS expression only in presence of control M-E cells, while only few cells express INS in the presence of WNT5A KO cells. Scale bar = 50 µm. N = 6 independent experiments.