Fig. 4: Selected growth factors secreted from M-E cells differentiate hESC-derived endocrine progenitors into CHGA- and INS-positive cells.

a Venn diagram presenting growth factors selected for further testing and their upregulation in M-E17 and M-E20 compared to M-E9, HDFs and HUVECs. b Experimental design, where hESCs were differentiated into endocrine progenitors (EPs) and then incubated with basal media only or with individual M-E derived growth factors at two concentrations (Supplementary Table 3) for 3 days, followed by immunofluorescence analysis. See also Supplementary Fig. 4. c Images of CHGA (green) immunofluorescent (IF) stainings of EPs cultured in basal media only (UT) or in the presence of growth factors. DAPI marks nuclei. Images of high concentration treatment (Supplementary Table 3) are shown. Scale bar = 100 µm. N = 6 independent experiments. d IF quantification of CHGA+ cells after treatment of EPs with growth factors, shown as % out of total (DAPI+) cells. Boxes extend from 25th to 75th percentile, middle lines denote median, while whiskers show minimum and maximum values. N = 6 independent experiments. p values (Dunnett’s multiple comparisons test) are shown for conditions significantly different from the untreated (UT) control. e Images of INS F stainings of EPs cultured in media only (UT) or in the presence of example growth factors. DAPI marks nuclei. Images of high concentration treatment (Supplementary Table 3) are shown. Scale bar = 100 µm. N = 6 independent experiments. f Quantification of INS+ cells after treatment of EPs with growth factors, shown as % out of total (DAPI+) cells based on IF staining. Boxes extend from 25th to 75th percentile, middle lines denote median, while whiskers show minimum and maximum values. N = 6 independent experiments p values (Dunnett’s multiple comparisons test) are shown for conditions significantly different from the untreated (UT) control. g Images of SST IF stainings of EPs cultured in media only (UT) or in the presence of Endocan. Scale bar = 100 µm. N = 3 independent experiments. h Flow cytometry plot with quantification of SST+ cells in EPs cultured in media only (UT) or in the presence of Endocan. N = 3 independent experiments.