Long-term expandable mouse and human-induced nephron progenitor cells enable kidney organoid maturation and modeling of plasticity and disease

Abstract

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.

Keywords: CAKUT; CRISPR screen; NPC; PKD; Wilms tumor; cellular plasticity; congenital anomalies of the kidney and urinary tract; hPSC; human pluripotent stem cell; kidney organoid; nephron progenitor cell; podocyte; polycystic kidney disease.

Figure 1.. p38 inhibition allows the derivation of NPC lines from any mouse strain.

(A) Schematic of mNPC line derivation and applications. (B) Quantification of SIX2⁺/PAX2⁺ cell percentages in cultured in various conditions. Scale bars, 50 μm. (c) Immunostaining of E13.5 mouse kidney section for markers as indicated. Scale bar, 50 μm. (D) Morphology of mNPCs (day 28). Scale bar, 50 μm. (E) Growth curve of mNPCs starting from 5,000 cells. (F and G) Immunofluorescence images (F) and quantification (G) of mNPCs (day 28). Scale bars, 100 μm. (H) Bright-field image showing co-culture of spinal cord (SP) with aggregated mNPCs. Scale bar, 200 μm. (I) Whole-mount immunofluorescence analysis of mNPC-derived nephron structures in (H). Scale bar, 100 μm. (J) Time-course images showing mNPC clonal expansion. Scale bars, 50 μm. (K) Bright-field (BF) and immunofluorescence images of a single cell mNPC clone. Scale bars, 50 μm. (L) Growth curve of a single mNPC. (M) Cloning efficiency of mNPC lines. (N) Whole-mount immunofluorescence analyses of a clonal NPC line-derived organoid. Scale bars, 100 μm. (O) PCA of bulk RNA-seq datasets. (P) Heatmap showing selected marker gene expression. Data are presented as mean ± SD. Each column represents counts from three biological replicates (n=3). See also Figures S1, S2 and Methods S1, and Table S1.

Figure 2.. Plasticity of developing mouse nephron cells with mNPSR-v2 medium.

(A - D) Immunofluorescence images and quantification of the expression of SIX2 and SALL1, from cultured Six2-GFP⁻ cells (A and B) and from cultured postnatal kidney cells (C and D). Note that only fluorescence signals in the nucleus were true SIX2 signals. Membrane-bound signals were non-specific. Scale bars, 100 μm. (E) Schematic showing genetic labeling and FACS isolation of the induced Six2-tdT⁺ cells. (F) qRT-PCR analysis of samples as indicated. (G) Schematic showing genetic labeling, FACS isolation, and culturing of Wnt4-tdT⁺ cells. (H) Flow cytometry gating plot showing the purification of Wnt4-tdT⁺ cells. (I and J) Immunofluorescence images (I) and quantification (J) of the expression of SIX2 and SALL1 in samples as indicated. Scale bars, 100 μm. Data are presented as mean ± SD. Each column represents counts from three biological replicates (n=3). The significance was determined by two-tailed unpaired Student’s t tests; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001. See also Figure S3 and Methods S1.

Figure 3.. Genome-wide CRISPR screen in NPC lines.

(A) Schematic illustrates the workflow of genome-wide CRISPR screen. (B) Box plot showing the distribution of beta scores. Boxes, 25th to 75th percentiles; whiskers, 1st to 99th percentiles. (C) MAGeCKFlute scatterplots of beta scores showing common genes identified from replicates. (D) Top 11 enriched IPA Canonical Pathways from CRISPR screen replicate #1. (E–I) MAGeCKFlute scatterplots of beta scores from two CRISPR screen replicates. (J and K) Normalized read counts of individual sgRNAs at the start and the end of the screen. (L and M) Immunofluorescence images (L) and quantification (M) of NPC marker gene expression upon inhibitor treatment. Scale bars, 50 μm. Data are presented as mean ± SD. Each column represents counts from three biological replicates (n=3). The significance was determined by two-tailed unpaired Student’s t tests; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001. See also Figure S4 and Table S2.

Figure 4.. YAP activation derives long-term expandable human NPC lines.

(A–C) Immunofluorescence images of human fetal kidney sections (11.4wk). Scale bars, 100 μm. (D and E) Immunofluorescence images (E) and quantification (F) of YAP expression in cultured iNPCs. Scale bars, 50 μm. (F) Schematic showing the derivation of iNPC lines and applications. (G) Bright-field image of iNPCs (day 87). Scale bar, 50 μm. (H) Growth curve of iNPCs starting from 5,000 cells. (I and J) Immunofluorescence images (I) and quantification (J) of iNPCs (day 21). Scale bars, 50 μm. (K) Bright-field images showing clonal expansion of iNPCs. Scale bars, 50 μm. (L) Immunofluorescence images of a single cell iNPC clone. Scale bars, 50 μm. (M and N) 3D (M) and 2D (N) PCA plots of bulk RNA-seq data. (O) Heatmap showing gene expression of selected marker genes. “D0-iNPC-SIX2” and “D0-iNPC-SIX2/PAX2” are FACS-purified SIX2⁺ and SIX2⁺/PAX2⁺ iNPCs without further culture; “Pri-SIX2-Neg” are primary SIX2-negative non-NPCs from human fetal kidneys. (P and Q) Bright field (BF) and fluorescence images (P) and quantification (Q) of mCherry expression in iNPCs upon lentiviral overexpression of mCherry (lentiviral OE), or knock-in of mCherry-expressing cassette into AAVS1 allele (CRISPR KI). Scale bars, 50 μm. (R-U) Whole-mount immunofluorescence analyses of nephron organoids generated from iNPCs (day 42). Scale bars, 50 μm. Data are presented as mean ± SD. Each column represents counts from three biological replicates (n=3). The significance was determined by two-tailed unpaired Student’s t tests; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001. See also Figures S5, S6, and Methods S1 and Table S1.

Figure 5.. Single cell multiome analyses of iNPC-derived nephron organoids.

(A) Schematic of the experimental design of multimodal analyses, and the major conclusions. (B) PCA of bulk RNA-seq datasets as indicated. (C) Heatmap showing expression of selected markers from bulk RNA-seq datasets. (D) UMAP projection of iNPC-derived nephron organoid snRNA-seq dataset. (E) UMAP projection of signature gene expression in iNPC-derived nephron organoid. (F) Dot plot of cluster-enriched gene expression in iNPC-derived nephron organoid. (G) UMAP projection of integrated single-cell datasets of iNPC-derived organoids and two other published hPSC-derived kidney organoids. (H) Proportions of cell types identified in (G) in different kidney organoids. Morizane protocol: TT.r1 and TT.r2; Takasato protocol: AN1.1, BJFF, and H9. (I and J) NPHS1 expression in the integrated dataset (G) is shown through a feature plot (I), and a violin plot (J). (K-M) Dot plots of marker gene expression in the proximal tubule population (K), distal tubule population (L), and podocyte population (M) from the integrated dataset (G). (N) Genome browser views of snATAC-seq open chromatin regions of selected genes in iNPC-derived podocyte (COL4A3, COL4A4, and NPHS1) and distal tubule (SLC12A1), as compared to adult kidney’s podocyte and distal tubule. See also Figure S7, Tables S1 and S3.

Figure 6.. hNPSR-v2 reveals human podocyte plasticity.

(A) Schematic of the reprogramming process. (B) Flow cytometry gating plots showing isolation of SIX2-GFP⁻ cells from day 7 nephron organoids (left) and purification of SIX2-GFP⁺ rNPCs upon culture (right). (C) Bright field image of rNPCs (day 13) derived from (B). Scale bar, 50 μm. (D) PCA of bulk RNA-seq data. (E) Flow cytometry gating plots showing isolation of SIX2-GFP⁻/PODXL⁺ podocytes from day 7 nephron organoids (left) and purification of rNPCs upon culture (right). (F) Bright field images of SIX2-GFP⁻/PODXL⁺ podocytes from (E) and the derivative rNPCs. Scale bars, 50 μm. (G-J) Immunofluorescence images and quantification of SIX2-GFP⁻/PODXL⁺ podocytes (G and I) and the derivative rNPCs (H and J). Scale bars, 50 μm. (K) Bright-field (BF) and immunofluorescence images of MAFB-GFP nephron organoid (day 8). Scale bars, left, 200 μm; right, 100 μm. (L) Flow cytometry gating plot showing the enrichment of rNPCs with ITGA8. (M) Bright-field image of rNPC line derived from MAFB-GFP⁺ podocytes. Scale bar, 50 μm. (N and O) Whole-mount immunofluorescence images of rNPC line (from MAFB-GFP⁺ podocytes)-derived organoids. Scale bars, left, 200 μm; right, 50 μm. (P) Flow cytometry gating plots showing isolation of PODXL⁺ primary podocytes from 17.4 week human fetal kidney (left) and enrichment of rNPCs upon culture with ITGA8 (right). (Q) Immunofluorescence images of primary podocyte-derived rNPCs (day 31). Scale bars, 50 μm. (R) Whole-mount immunofluorescence images of rNPC (from primary podocyte)-derived nephron organoid. Scale bars, left, 200 μm; right, 50 μm. (S) Heatmap showing marker gene expression during podocyte-to-NPC reprogramming. (T) Schematic of the model. See also Figures S8, S9, and Table S1.

Figure 7.. PKD modeling and small molecule screening from genome-edited NPCs.

(A and B) Schematic of the experimental protocols. (C) Bright-field and GFP images of mini mNPC aggregates in Aggrewell. Scale bars, 500 μm. (D) Quantification of pHH3⁺ nuclei in LTL⁺/CDH⁺ cells in mini nephron organoids. (E) Heatmap of selected gene expression as determined by qRT-PCR. (F) Metabolic analyses of mini nephron organoids using Seahorse assays at baseline (blank boxes) and at stressed levels (filled boxes). (G) Heatmaps showing the quantification of screen results in cyst formation efficiency (left) and cyst diameter (right). Identified HDAC inhibitors (green), and BRD4 inhibitors (blue). (H) Venn diagram showing the common small molecules identified in (G). (I) Western blot analysis of PC2 expression in candidate PKD2^−/− single cell hPSC clones. (J) Schematic of genotyping results in PKD2^−/− clones. (K) Bright field images showing cyst formation. Scale bars, 500 μm and 200 μm (enlarged pictures, right panels). (L) Bright-field images of organoids upon various small molecule treatment. Scale bars, 200 μm. (M) Quantification of the percentages of cystic organoids shown in (L). (N) Immunofluorescence images of samples in (L) for TUNEL assay. Scale bars, 50μm. Data are presented as mean ± SD. Each column represents counts from three biological replicates (n=3). The significance was determined by two-tailed unpaired Student’s t tests; ns, not significant; *, p<0.05; **, p<0.01. See also Figures S10–S15.