Fig 2: Comparison of the ability of different fibroblast subpopulations to support epidermal growth on DED. (a) Isolation of fibroblast subpopulations by flow cytometry. (b–f) Hematoxylin and eosin staining (scale bars = 100 µm) and (g–k) immunofluorescence staining (scale bars = 200 µm) of DED organotypic cultures (b, g) without fibroblasts or (c, h) seeded with unfractionated (lin–CD90+) fibroblasts, (d, i) CD90+CD39+ (enriched in papillary) fibroblasts, (e, j) CD90+CD39– (depleted of papillary) fibroblasts, and (f, k) CD90+CD36+ (reticular/pre-adipocyte) fibroblasts. Keratin 14 (green) marks keratinocytes, and vimentin (white) marks mesenchymal cells (fibroblasts). Experiments were repeated for a minimum of two biological replicates (donor cells derived from female [age 19 years] breast skin and female [age 37 years] breast skin; DED derived from male [age 47 years] abdominal skin, and representative images are displayed. In the case of the CD90+CD39+ cells, two of three experiments involved additional selection for CD26– cells to further enrich for the papillary cell population (CD90+CD39+CD26–). (l) Quantification of epidermal thickness, (m) density of fibroblasts within 300 µm of the epidermis, and (n) relative abundance of fibroblasts at different depths from the epidermis. adipo, adipocyte; DED, decellularized human dermis; FB, fibroblasts; FSC, forward scatter detector; K14, keratin 14; lin–, lineage negative; ns, not significant; SSC, side scatter detector.

Fig 3: Relationship between LGR5 and reported CSCs markers and stem cell genes. a Analyses of CD133, CD44, CD90, CD24, and EpCAM expression in LGR5+ and LGR5- U251 glioma cells and in LGR5+ and LGR5- 8591 human primary glioma cells by FCM. b U251-GSCs and 8591-GSCs from U251 and 8591 human primary glioma cells through serum-free enrichment (top, scale bar = 100 µm) and immunofluorescence double staining for LGR5 and SOX2 (bottom, scale bar = 50 µm). c The expression levels of LGR5, CSCs markers (CD133, CD44, CD24 and EpCAM) and stem cell genes (SOX2, OCT4 and Nanog) in shLGR5 and shCtrl GSCs determined by Western blot (top). The protein intensity of these markers were determined by densitometric analysis and normalized to the relevant ß-actin value. Error bars represent the mean ± SD of three independent experiments (bottom). *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig 4: Cellular heterogeneity within Wilms tumour organoid cultures.a t-SNE representation of single cells from four Wilms tumour organoid lines (51T, 80T, 88T and 101T). Cells are coloured by organoid of origin (top panel) or clustering (bottom panel). Indicated are the cell types the populations are representing based on marker gene expression (see panel b). e: epithelial, s: stromal, b: blastemal-like. b t-SNE maps showing the colour-coded logged expression levels of several markers for each population demonstrating that different cell types are present in 51T and 88T, whereas 80T and 101T organoids primarily consist of different epithelial subpopulations, which is in line with their histological appearance. c High-resolution 3D imaging of 51T Wilms tumour organoids immunolabeled for E-cadherin (E-cad; red), SIX2 (green) and CD90 (white). Bottom panels depict enlargement from top panel in 3D (left panel) and a representative optical section (others panels). Scale bars, 100 µm (top) and 50 µm (bottom). Images are representative of n = 2 independent experiments. d Immunofluorescence imaging on 51T Wilms tumour tissue sections immunolabeled for E-cadherin (E-cad; red), SIX2 (green) and CD90 (white). Scale bars 100 µm. Images are representative of n = 2 independent experiments.

Fig 5: The localization of vimentin and DAPI was visualized under fluorescence microscopy after immunofluorescence staining. (A) DAPI staining of nuclei (blue). (B) Anti-vimentin antibody (green). (C) the merge of (A and B). Superficial markers of RA-FLSs cells were detected by flow cytometry, with SW982 cells being negative control. (D) RA-FLS without inclusion of CD68 antibody as isotype control. (E) RA-FLS without inclusion of vimentin antibody as isotype control. (F) RA-FLS without inclusion of CD90 antibody as isotype control. (G) RA-FLS showing positive for CD68 (99.53±0.03%, n = 3). (H) RA-FLS showing positive for vimentin (98.67±0.12%, n = 3). (I) RA-FLS showing positive for CD90 (95.57±0.18%, n = 3). (J) SW982 showing negative for CD68. (K) SW982 showing negative for vimentin. (L) most SW982 showing positive for CD90.