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Fig. 1. Generation of stable human kidney cell lines with proven origin. A: Schematic of the human kidney and the cortex illustrates the source of isolated cells. B: Immunofluorescence staining of PDGFRβ+, CD10+ and CD31+ specific antibodies for cells in cortex of human kidney. C: Enumeration of the workflow from isolating the cells to the generation of genetically tagged cells. D: Representative pictures of genetically labeled immortalized PDGFRβ+, proximal tubular epithelial and endothelial cell lines. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. E: Principal component analysis (PCA) plot of the generated three cell lines. The three cell lines show higher variability from each other, while technical replicates are similar to each other. F: Shared DE genes between isolated cell lines and human bulk RNA seq [13]. G: CD10+ (red) cells stained for cell-specific proximal tubule epithelial markers cubilin (green, left) and CD13 (green, right) including DAPI. H: Heatmap of bulk RNA-sequencing data demonstrating the genes expressed in CD10+. I: P-gp Calcein-AM transporter assay of CD10+ and negative control PDGFRβ+ cells, with or without transporter inhibitor <t>PSC833.</t> ***p < 0.001 (one-way ANOVA analysis followed by Tukey post-test), n = 3 with each 12 replicates. J: Presence of intracellular vWF (red) in CD31+ (green) cells including DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 1. Generation of stable human kidney cell lines with proven origin. A: Schematic of the human kidney and the cortex illustrates the source of isolated cells. B: Immunofluorescence staining of PDGFRβ+, CD10+ and CD31+ specific antibodies for cells in cortex of human kidney. C: Enumeration of the workflow from isolating the cells to the generation of genetically tagged cells. D: Representative pictures of genetically labeled immortalized PDGFRβ+, proximal tubular epithelial and endothelial cell lines. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. E: Principal component analysis (PCA) plot of the generated three cell lines. The three cell lines show higher variability from each other, while technical replicates are similar to each other. F: Shared DE genes between isolated cell lines and human bulk RNA seq [13]. G: CD10+ (red) cells stained for cell-specific proximal tubule epithelial markers cubilin (green, left) and CD13 (green, right) including DAPI. H: Heatmap of bulk RNA-sequencing data demonstrating the genes expressed in CD10+. I: P-gp Calcein-AM transporter assay of CD10+ and negative control PDGFRβ+ cells, with or without transporter inhibitor <t>PSC833.</t> ***p < 0.001 (one-way ANOVA analysis followed by Tukey post-test), n = 3 with each 12 replicates. J: Presence of intracellular vWF (red) in CD31+ (green) cells including DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 1. Generation of stable human kidney cell lines with proven origin. A: Schematic of the human kidney and the cortex illustrates the source of isolated cells. B: Immunofluorescence staining of PDGFRβ+, CD10+ and CD31+ specific antibodies for cells in cortex of human kidney. C: Enumeration of the workflow from isolating the cells to the generation of genetically tagged cells. D: Representative pictures of genetically labeled immortalized PDGFRβ+, proximal tubular epithelial and endothelial cell lines. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. E: Principal component analysis (PCA) plot of the generated three cell lines. The three cell lines show higher variability from each other, while technical replicates are similar to each other. F: Shared DE genes between isolated cell lines and human bulk RNA seq [13]. G: CD10+ (red) cells stained for cell-specific proximal tubule epithelial markers cubilin (green, left) and CD13 (green, right) including DAPI. H: Heatmap of bulk RNA-sequencing data demonstrating the genes expressed in CD10+. I: P-gp Calcein-AM transporter assay of CD10+ and negative control PDGFRβ+ cells, with or without transporter inhibitor <t>PSC833.</t> ***p < 0.001 (one-way ANOVA analysis followed by Tukey post-test), n = 3 with each 12 replicates. J: Presence of intracellular vWF (red) in CD31+ (green) cells including DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 1. Generation of stable human kidney cell lines with proven origin. A: Schematic of the human kidney and the cortex illustrates the source of isolated cells. B: Immunofluorescence staining of PDGFRβ+, CD10+ and CD31+ specific antibodies for cells in cortex of human kidney. C: Enumeration of the workflow from isolating the cells to the generation of genetically tagged cells. D: Representative pictures of genetically labeled immortalized PDGFRβ+, proximal tubular epithelial and endothelial cell lines. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. E: Principal component analysis (PCA) plot of the generated three cell lines. The three cell lines show higher variability from each other, while technical replicates are similar to each other. F: Shared DE genes between isolated cell lines and human bulk RNA seq [13]. G: CD10+ (red) cells stained for cell-specific proximal tubule epithelial markers cubilin (green, left) and CD13 (green, right) including DAPI. H: Heatmap of bulk RNA-sequencing data demonstrating the genes expressed in CD10+. I: P-gp Calcein-AM transporter assay of CD10+ and negative control PDGFRβ+ cells, with or without transporter inhibitor <t>PSC833.</t> ***p < 0.001 (one-way ANOVA analysis followed by Tukey post-test), n = 3 with each 12 replicates. J: Presence of intracellular vWF (red) in CD31+ (green) cells including DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 1. Generation of stable human kidney cell lines with proven origin. A: Schematic of the human kidney and the cortex illustrates the source of isolated cells. B: Immunofluorescence staining of PDGFRβ+, CD10+ and CD31+ specific antibodies for cells in cortex of human kidney. C: Enumeration of the workflow from isolating the cells to the generation of genetically tagged cells. D: Representative pictures of genetically labeled immortalized PDGFRβ+, proximal tubular epithelial and endothelial cell lines. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. E: Principal component analysis (PCA) plot of the generated three cell lines. The three cell lines show higher variability from each other, while technical replicates are similar to each other. F: Shared DE genes between isolated cell lines and human bulk RNA seq [13]. G: CD10+ (red) cells stained for cell-specific proximal tubule epithelial markers cubilin (green, left) and CD13 (green, right) including DAPI. H: Heatmap of bulk RNA-sequencing data demonstrating the genes expressed in CD10+. I: P-gp Calcein-AM transporter assay of CD10+ and negative control PDGFRβ+ cells, with or without transporter inhibitor PSC833. ***p < 0.001 (one-way ANOVA analysis followed by Tukey post-test), n = 3 with each 12 replicates. J: Presence of intracellular vWF (red) in CD31+ (green) cells including DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Journal: Biomaterials

Article Title: A bioprinted and scalable model of human tubulo-interstitial kidney fibrosis.

doi: 10.1016/j.biomaterials.2024.123009

Figure Lengend Snippet: Fig. 1. Generation of stable human kidney cell lines with proven origin. A: Schematic of the human kidney and the cortex illustrates the source of isolated cells. B: Immunofluorescence staining of PDGFRβ+, CD10+ and CD31+ specific antibodies for cells in cortex of human kidney. C: Enumeration of the workflow from isolating the cells to the generation of genetically tagged cells. D: Representative pictures of genetically labeled immortalized PDGFRβ+, proximal tubular epithelial and endothelial cell lines. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. E: Principal component analysis (PCA) plot of the generated three cell lines. The three cell lines show higher variability from each other, while technical replicates are similar to each other. F: Shared DE genes between isolated cell lines and human bulk RNA seq [13]. G: CD10+ (red) cells stained for cell-specific proximal tubule epithelial markers cubilin (green, left) and CD13 (green, right) including DAPI. H: Heatmap of bulk RNA-sequencing data demonstrating the genes expressed in CD10+. I: P-gp Calcein-AM transporter assay of CD10+ and negative control PDGFRβ+ cells, with or without transporter inhibitor PSC833. ***p < 0.001 (one-way ANOVA analysis followed by Tukey post-test), n = 3 with each 12 replicates. J: Presence of intracellular vWF (red) in CD31+ (green) cells including DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Cells were treated for 1 h with 5 μM P-gp transporter inhibitor PSC833 (Tocris Biosciences, Bristol, UK) in KH buffer at 37 ◦C, 5 % (v/v) CO2, and afterwards exposed for 1 h to 1 μM Calcein-AM (Life Technologies Europe BV) with or without 5 μM PSC833 in KH buffer.

Techniques: Isolation, Immunofluorescence, Staining, Labeling, Generated, RNA Sequencing, Negative Control