Fig 2: EBN induces phosphorylation of GATA3 and its nuclear translocation. (A) After 16-h serum starvation, cultured differentiated keratinocytes were pre-incubated with or without SB203580 (10 µM) for 2 h, followed by different time of applied IL-4 (a positive control, 100 ng/mL) or EBN digest (100 µg/mL). Total GATA3 (T-GATA3) and phosphorylated GATA3 (P-GATA3) (both at ~48 kDa) were evaluated by western blot assays. (B) The quantification of the increased fold of P-GATA3/T-GATA3 expression levels from (A) after different time of applied IL-4 and EBN digest, with or without SB203580 pre-treatment, are shown. Values are expressed in mean ± SEM, n = 3. Statistically significant results are marked with * p < 0.05, ** p < 0.01, and *** p < 0.001 against the control. (C) After 16-h serum starvation, cultured differentiated keratinocytes were pre-incubated with or without SB203580 (10 µM) for 2 h, followed by 240-min treatment of IL-4 (100 ng/mL, a positive control), EBN extract (100 µg/mL) and EBN digest (100 µg/mL). Phosphorylated GATA3 protein localization in keratinocytes was determined by immunofluorescence assays. n = 3. Bar = 20 µm. (D) The quantification of the percentage of nuclear to cytosol level of P-GATA3 intensities under different treatments from (C) are shown. Values are expressed in mean ± SEM, n = 3. Statistically significant results are marked with * p < 0.05, ** p < 0.01, and *** p < 0.001 against the control or as indicated.

Fig 3: EBN induces nucleus localizations of GATA3 and PPAR? proteins. The immunofluorescent staining of (A) GATA3 or (B) PPAR? in cultured differentiated keratinocytes after 24-h treatments of EBN extract (100 µg/mL) and EBN digest (100 µg/mL) are shown. IL-4 at 100 ng/mL and linoleic acid (C18:2) at 30 µM were adopted as positive controls in GATA3 and PPAR? expression. Bar = 20 µm. (C) The quantification of the percentage of nuclear GATA3 to cytoplasmic GATA3, or PPAR?, are shown. Values are expressed in mean ± SEM, n = 3. Statistically significant results are marked with * p < 0.05, ** p < 0.01, and *** p < 0.001 against the control.

Fig 4: Inhibiting TGFß signalling induces loss of pluripotency in naïve hPSCs.(a) RT-qPCR expression analysis of pluripotency-associated genes and TGFß-associated genes in naïve hPSCs (H9 NK2 line) following SB-431542 treatment (t2iLGö + SB). Expression levels are shown as fold changes relative to day 0. (b) Schematic showing the integration of a single-step optimised inducible knock-down targeting construct into the AAVS1 locus of H9 hPSCs, enabling the expression of SMAD2 and SMAD3 short hairpin RNAs (shRNAs) under the control of a tetracycline inducible promoter. ZFN: zinc-finger nucleases; 5’-HAR/3’-HAR: upstream/downstream homology arm; H1-TO: Tetracycline-inducible H1 Pol III promoter carrying one tet operon after the TATA box; CAAG: CMV early enhancer, chicken ß-actin and rabbit ß-globin hybrid promoter; TetR: Tetracycline-sensitive repressor protein; SA: splice acceptor; Puro, Puromycin resistance; pA, polyadenylation signal. Schematic adapted from Bertero et al., 2016. (c) RT-qPCR analysis of gene expression levels in SMAD2/3 inducible knock-down (iKD) H9 naïve hPSCs following 5 days of tetracycline (tet) treatment. Expression levels are shown for each gene as fold change relative to iKD -tet. Cells were cultured in t2iLGö medium. (d) Phase contrast pictures of H9 NK2 naïve hPSCs after 5, 7, and 10 days of SB treatment in t2iLGö medium. Scale bars: 400 µm. (e) RT-qPCR analysis of trophoblast (HAND1, TP63, MMP2, and SDC1) and pluripotency (POU5F1, NANOG) gene expression levels in naïve hPSCs following long-term (14 days) SB treatment in t2iLGö medium. Expression levels are shown as fold changes relative to day 0 samples, n = two biological replicates. (f) Immunofluorescence microscopy showing the downregulation of NANOG (green) in naïve hPSCs following 3 and 5 days of SB treatment. DAPI signal in blue. White arrowheads indicate colonies displaying heterogeneous expression of NANOG. Scale bars: 50 µm. (g) Immunofluorescence microscopy for OCT4 (red), HAND1 (green), GATA3 (cyan), and DAPI (blue) in naïve hPSCs following 3 and 5 days of SB treatment in t2iLGö medium. Scale bars: 50 µm. (h) Immunofluorescence microscopy for GATA3, HLA-G, SDC1 (magenta), CK19 and CK7 (yellow), and DAPI (blue) in naïve-derived trophoblast stem cells (TS), extravillous trophoblast (EVT), and syncytiotrophoblast (STB). Scale bars: 50 µm. (i) RT-qPCR analysis of gene expression levels in naïve-derived trophoblast stem cells (TS), extravillous trophoblast (EVT) and syncytiotrophoblast (STB) compared to undifferentiated naïve hPSCs. Expression levels are shown for each gene relative to the housekeeping gene RPLP0. RT-qPCR data show the mean ± SD of three biological replicates (unless specified otherwise) and were compared to their relative control using an ANOVA with Tukey's or Šídák's multiple comparisons test (*p = 0.05, **p= 0.01, ***p= 0.001, ****p= 0.0001). Figure 3—source data 1.Numerical data that are represented in Figure 3.

Fig 5: Extended monolayer culture in CDBLY2 increases SIX2+ progenitor contribution to nephrons and proximalisation.a Confocal immunofluorescence of D7 + 14 (standard protocol) and D13 + 14 (extended protocol) organoids derived from the SIX2Cre/Cre:GAPDHdual lineage tracing iPSC line. Images depict merged and separated channels showing lineage-traced SIX2+ cells (mCherry; red), distal tubules (ECAD; green), podocytes (NPHS1; grey) and proximal tubules (LTL; blue). Scale bars represent 100 µm. b Flow cytometry of SIX2Cre/Cre:GAPDHdual lineage tracing organoids derived from extended (13 day + CDBLY2) and standard (7 day + E6 media) differentiations depicting SIX2-derived mCherry + (red) cell contribution to the EPCAM + (nephron) population. Flow plots shown in top panels are representative of the replicates across multiple experiments. Percentage mCherry contributions from flow cytometry are depicted in bar graph (bottom panel), where error bars represent SEM from n = 4 biological replicates across 3 independent experiments. Statistical significance was determined using an unpaired t test. Asterisks represent two-tailed P values adjusted for multiple comparisons using the Holm–Sidak method, alpha = 0.05 (*P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001 [Experiment 1: P = 0.000924; Experiment 2: P = 0.004899; Experiment 3: P = 0.004845]). Source data are provided as a Source data file. c Confocal immunofluorescence of D13 + 14 organoids demonstrating (left panel) aligned nephron morphology with nephron segmentation makers (nephron epithelium [EPCAM; green], distal tubule/Loop of Henle [SLC12A1; red], proximal tubules [LTL; blue], and podocytes [NPHS1; grey]) and (right panel) the presence of few GATA3 + connecting segment/ureteric epithelium structures (red), co-stained for nephron epithelium (EPCAM; green), podocytes (NPHS1; grey), and proximal tubules (LTL; blue). Inset in right panel shows GATA3 and LTL alone. Scale bars in (i) and (ii) represent 200 µm and 100 µm, respectively. d Confocal immunofluorescence of a D7 + 14 (standard) organoid depicting homogenous distribution of podocytes (NPHS1; grey), proximal tubules (LTL; blue), and nephron epithelium (EPCAM; green), as well as the presence of extended segments of centralised GATA3 + (red [nuclear]) connecting segment/ureteric epithelium (also highlighted in insets). Scale bar represents 200 µm.