Fig 2: WNT signalling gradient influences nephron alignment and directionality.a Analyses of scRNAseq datasets for PT-enhanced D13 + 14 organoid replicates (top left and bottom panels) and D13 monolayer replicates (top left and right panels) as classified by DevKidCC. Top left dot plot depicts expression of stroma compartment markers28 and WNT antagonists in D13 + 14 and D13 samples. DevKidCC’s DotPlotCompare (bottom panel) shows the comparison of D13 + 14 to other relevant published datasets (cortical stroma [CS] and unassigned populations only). Dot colour and size in dot plots represent unscaled gene expression and percentage of cells expressing each gene, respectively. Violin plot (top right) depicts the similarity scores for all unassigned cells within D13 monolayer replicates as predicted by the Azimuth label transfer method (https://azimuth.hubmapconsortium.org/) with the human developmental reference dataset67, where cells are grouped by population with the highest similarity score. Population abbreviations in all panels: nephron progenitor cell (NPC), endothelium (Endo), stroma progenitor (SP), cortical stroma (CS), medullary stroma (MS), enteric nervous system (ENS). b Confocal immunofluorescence images of replicate standard organoids bioprinted in a patch conformation and in contact with either agarose beads soaked in PBS (control; left panel) or in 10 µM IWR-1 (right panel). Clusters of beads are outlined with magenta dashed lines. Organoids are stained with markers of epithelium (EPCAM; green), proximal tubule (LTL; blue), and podocytes of the glomeruli (NPHS1; grey). Scale bars represent 100 µm (all top panels) and 200 µm (bottom tile panels). c Top panel shows and example image from (b, left panel [PBS control, replicate 3]) illustrating the image annotation approach used to segment and quantify the proportion of nephron structures (NPHS1 + [grey], EPCAM + [green], and LTL + [blue]) within a defined region 200 pixels from any bead (white outline). Solid colours represent masks for beads and nephrons. Scale bar represents 200 µm. Bottom panel of (c) shows quantification of PBS control and IWR-1 treated organoid images from (b) using approach illustrated in top panel images of (c), with n = 3 replicates per condition. Total area and nephron area values are shown in pixels (106 px). Percentage (%) of each structure (NPHS1+, EPCAM+, LTL+) are shown as a proportion of the total nephron area. P values were calculated using a two-sided t-test without adjustment for multiple comparisons and are indicated below each plot. Source data are provided as a Source data file.

Fig 3: Analysis of pancreatic cancer stem cell surface markers CD24, CD44, and EpCAM in PANC-1 and MIA-PaCa-2. The presence of CD24, CD44, and EpCAM were determined by flow cytometry and immunocytochemistry examination. (A) The percentage of triple-positive cells was 7.2% in PANC-1 and 0% in MIA-PaCa-2 through flow cytometry analysis. (B) In PANC-1 analyzed with immunocytochemistry examination, the percentage of CD24+, EpCAM+, and CD24+CD44+EpCAM+ cells was 17.3 ± 3.7%, 46.0 ± 3.5% and 16.8 ± 3.6%, while in MIA-PaCa-2, the percentage was 11.5 ± 0.7%, 38.7 ± 0.5% and 11.4 ± 0.7%, respectively. (C) Images of PANC-1 and MIA-PaCa-2 cells stained with anti-human CD44, anti-human EpCAM, and anti-human CD24 antibody. The right panel was the merged images. Data are presented as the mean ± standard deviation (SD) of three independent experiments. * P < 0.05 in comparison with the corresponding cells using Student’s t-test.

Fig 4: Percentage of hPGCLCs differentiated from hiPSCsThe hiPSC sublines for each participant (n = 3 biological replicates) were each differentiated a minimum of three times.(A) Fluorescence-activated cell sorting (FACS) plots and quantification of hPGCLC percentages at day 4 of aggregate differentiation from twin pair MZT01 and MZT02 (t test, n = 10, p = 0.36).(B) Twin pair MZT04 and MZT06 (t test, n = 10, p = 0.73).(C) MZT05. The hPGCLC population is identified as double-positive for EPCAM and ITGA6 (circle). Data are represented as mean ± SEM with each circle on the graph, indicating the hPGCLC population. Statistical significance was calculated using a t test.

Fig 5: Gating strategy for CTC detection. At first, speedbeads and debris are excluded. Hoechst staining intensity of the gated events is displayed in a 2D plot. Hoechstpositive nucleated cells (blue) are then separated by their fluorescence intensities for EpCAM–EGFR–AF488 and CD45–AF647. CTCs are defined as Hoechstpositive/CD45negative/EpCAM–EGFRpositive. Single CTCs are detected in the CD45negative/EpCAM–EGFRpositive gate (green), whereas cluster of leukocytes and CTCs are detected in the CD45positive/EpCAM–EGFRpositive gate (purple).