Patient-iPSC-Derived Kidney Organoids Show Functional Validation of a Ciliopathic Renal Phenotype and Reveal Underlying Pathogenetic Mechanisms

Abstract

Despite the increasing diagnostic rate of genomic sequencing, the genetic basis of more than 50% of heritable kidney disease remains unresolved. Kidney organoids differentiated from induced pluripotent stem cells (iPSCs) of individuals affected by inherited renal disease represent a potential, but unvalidated, platform for the functional validation of novel gene variants and investigation of underlying pathogenetic mechanisms. In this study, trio whole-exome sequencing of a prospectively identified nephronophthisis (NPHP) proband and her parents identified compound-heterozygous variants in IFT140, a gene previously associated with NPHP-related ciliopathies. IFT140 plays a key role in retrograde intraflagellar transport, but the precise downstream cellular mechanisms responsible for disease presentation remain unknown. A one-step reprogramming and gene-editing protocol was used to derive both uncorrected proband iPSCs and isogenic gene-corrected iPSCs, which were differentiated to kidney organoids. Proband organoid tubules demonstrated shortened, club-shaped primary cilia, whereas gene correction rescued this phenotype. Differential expression analysis of epithelial cells isolated from organoids suggested downregulation of genes associated with apicobasal polarity, cell-cell junctions, and dynein motor assembly in proband epithelial cells. Matrigel cyst cultures confirmed a polarization defect in proband versus gene-corrected renal epithelium. As such, this study represents a "proof of concept" for using proband-derived iPSCs to model renal disease and illustrates dysfunctional cellular pathways beyond the primary cilium in the setting of IFT140 mutations, which are established for other NPHP genotypes.

Keywords: CRISPR/Cas9; IFT140; cilia; functional genomics; gene correction; induced pluripotent stem cells; kidney organoid; nephronophthisis.

Figure 1

Whole-Exome Sequencing (WES) Leads to Revision of Clinical Phenotype (A) Proband retinal photograph demonstrating retinitis pigmentosa. (B) Renal ultrasound demonstrating echogenic kidney with loss of corticomedullary differentiation forms the basis of the eponymous diagnosis of SLS before WES. (C) Trio WES identifies compound-heterozygous IFT140 variants (asterisks indicate sequenced individuals). (D) IFT140 structure with variant loci identified. (E) Conservation of both amino acid loci across vertebrate species. (F) After the identification of IFT140 variants, a skeletal survey found cone-shaped epiphyses in the phalanges of the hand and foot (white boxes), flattened femoral heads (black arrowhead), and widened femoral neck (white arrowheads) consistent with MSS.

Figure 2

Gene Correction, Transcript Analysis, and Differentiation to Kidney Organoid (A) The gene-correction template for the c.634G>A variant contains the corrected 1 bp substitution (blue) and a synonymous downstream 3 bp substitution (green) for ease of identification by Sanger sequencing. Successfully reprogrammed clones were screened for unsuccessful (red nuclei, PR) or successful (green nuclei, GC) gene correction. (B) DNA sequencing demonstrates the c.634G>A variant in the PR clone (red arrowhead) and successful correction of this variant in the GC clone (green arrowhead), as well as the synonymous 3 bp change in this allele (gray shading). (C) Sanger-sequenced RNA (copy DNA) transcripts suggest that the c.634G>A allele is not detectable as a stable transcript, indicated by a single chromatogram peak at both the c.634G>A locus (red arrowhead) and the c.2176C>G variant (blue arrowhead) in the PR clone. The double chromatogram peak at the synonymous 3 bp substitution (gray) and the c. 2176C>G variant locus (not gene-corrected; blue) in the GC clone indicates that gene correction has rescued this transcripts. (D) qPCR identifies 50% relative mRNA expression in PR iPSCs compared with control cells (data represent the mean + 95% CI; p = 0.0001). (E) Capillary western blot reveals 43% relative IFT140 expression in PR differentiated renal epithelial cells compared with GC cells (data represent the mean + 95% CI; p < 0.001). (F) Bright-field images of kidney organoids on day 25 of culture (day 18 of aggregate culture). Scale bars, 1 mm. (G) Immunofluorescence images of whole organoids demonstrate balanced expression of glomerular precursors (NPHS1, white), proximal tubule, (LTL, blue), distal tubule (CDH1, green), and collecting duct (CDH1, green; GATA3, red). Scale bars, 1 mm (low-magnification images) and 50 μm (high-magnification images).

Figure 3

Proband Organoid Tubules Demonstrate Abnormal Cilia, which Are Rescued by Gene Correction (A) Cilia of CDH1⁺ tubular epithelial cells demonstrate classical morphology associated with defective retrograde IFT: short with swollen ciliary tips (left and middle panels; scale bars, 2 μm). Individual cilia were isolated from these images (right panels; scale bars, 500 nm) and shuffled for blinded scoring of morphology. (B) Quantitative output of blinded cilia-morphology analysis demonstrates predominantly clubbed cilia in PR organoids (59%) and predominant wild-type morphology in gene-corrected cilia (61%) (n = 900; p < 0.0001, Chi-square test). (C) PR cilia were shorter than GC cilia at every culture time point, such that an increasing difference was noted with time in culture (n = 600 cilia per condition; p < 0.0001, Welch’s t test; error bars represent the mean + 95% CI). (D) IFT components IFT140, WDR19, and IFT88 were all found to accumulate in the PR ciliary tip. Gene-corrected organoids displayed a wild-type distribution (scale bar, 1 μm).

Figure 4

RNA-Seq of Epithelial Sub-fractions of Organoids Demonstrates Dysfunctional Cellular Processes Resulting from IFT140 Variants (A) Diagrammatic representation of epithelial MACS from dissociated organoids. Dissociated organoids were incubated with magnetic beads conjugated with EPCAM antibody and passed through a magnetic field, trapping epithelial cells that could be gently eluted for downstream application after removal of the magnetic field. (B) Principal-component analysis of primary RNA-seq data demonstrates clustering of PR and GC replicates. (C) Heatmap demonstrates consistency of gene expression across replicates. (D) Top GO terms output from ToppGene gene-list enrichment of set 1 (combined up- and downregulated genes with significant differential expression, adjusted p < 0.01; top graph) and set 2 (genes with log fold change > 0.7 favoring GC and significant differential expression < 0.05; bottom graph). Bars represent the −log₁₀(p value), and the adjacent fraction represents the number of genes within the dataset (numerator) and the number of genes within the GO gene list (denominator). Similar GO terms with a near-identical DGE representation and GO term were filtered out to avoid repetition. Colors are as follows: blue, GO molecular function; purple, GO biological process; and orange, GO cellular component.

Figure 5

Spheroid Culture Demonstrates Functional Assay of Disturbed Apicobasal Polarity in Proband Organoid Epithelium (A and B) Heatmaps demonstrating significant downregulation of components of (A) the apical part of the cell and (B) the apical cell junction in the PR line (red, expression above the mean; blue, expression below the mean). (C) Immunofluorescent images from spheroid culture of EPCAM⁺ MACS-sorted epithelial cells demonstrate examples of poorly polarized structures (PR), well-polarized structures (GC), and reference images from IMCD3 cells. Scale bars, 10 μm. (D) PR EPCAM⁺ cells were less able to establish polarized spheroids than GC controls (PR = 34.0% [25.4–43.7], GC = 53.0% [43.2–62.5]; p = 0.01; error bars represent 95% CI). The asterisk signifies adjusted p < 0.01.