Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development

Abstract

A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin⁺ podocin⁺ ZO-1⁺ ) and microvillus-rich apical membranes (podocalyxin⁺ ), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration. Stem Cells 2017;35:2366-2378.

Keywords: Adhesion receptors; Biophysics; Cell adhesion; Developmental biology; Differentiation; Focal segmental glomerulosclerosis; Foot processes; Gene targeting; Genome editing; Kidney; Nephrin; Nephrogenesis; Pluripotent stem cells; Podocalyxin; Podocin; Slit diaphragm.

Figure 1. Marker localization in hPSC-podocytes and CLS podocytes

(A) Representative wide-field fluorescence image of kidney organoids showing tubule (LTL) and podocyte (PODXL) populations co-localized with the tight junction protein ZO-1. Zoom shows dashed white boxed region where a tubule (tub) forms a junction with a cluster of podocytes (pod). Arrowhead indicates tubule-like cells bordering the podocyte cluster. (B) NPHS1-GFP organoids on days 12 and 15 of differentiation. Red channel is included as a negative control for autofluorescence. (C) Representative confocal optical sections and (D) averaged raw fluorescence intensities in line scans drawn through podocytes, showing co-localization of podocin (NPHS2) with nephrin (NPHS1) or (E–F) with podocalyxin (PODXL) in human glomeruli and kidney organoids. Zoom of boxed regions are shown at right for the merged images. White dashed arrows demonstrate how line scans were drawn. Line scans are 24 μm through individual cells (n = 10). bpp, bits per pixel (raw intensities). (G) Representative confocal images of kidney tissues (left) or human kidney organoids (right) showing co-localization of nephrin with antibodies raised against PAX2 and (H) PAX8. Arrowheads indicate PAX2 or PAX8 co-localization with nephrin at the edge of the podocyte cluster. Scale bars, 100 μm.

Figure 2. Junctions migrate basally during maturation of hPSC-podocytes and CLS podocytes

(A) Representative confocal optical sections (top) and averaged raw fluorescence intensity line scans (bottom) showing ZO-1 and NPHS1 localization in developing human kidneys and (B) human kidney organoids. Zoom of boxed regions without WT1 (human kidneys), or without WT1 and nephrin (human kidney organoids), are shown below images. Apical and basal sides of the epithelium are labeled in merged images. White dashed arrows demonstrate how line scans were drawn. Line scans are 24 μm through individual cells (n = 10). Y axes are the same throughout. Scale bars, 20 μm.

Figure 3. The ultrastructure of hPSC-podocytes resembles CLS podocytes in vivo

(A) Representative TEM images of tubules (Tub) and podocytes (Podo) in human kidney organoids. J, junctions; m, microvilli; L, lumen. Arrows indicate tubule-like cells bordering the podocyte aggregate. (B) Developing glomeruli at different stages in human kidneys. Note similarity between maturing CLS podocytes and hPSC-podocytes at highest magnification. Scale bars, 10 μm.

Figure 4. Podocalyxin regulates microvillus formation and cell spacing in hPSC-podocytes

(A) Representative TEM images of control and PODXL^−/− hPSC-podocytes. Progressive zooms are shown for boxed regions in TEM images. m, microvilli; J, junctions. (B) Counts of microvillus number in PODXL^−/− hPSC-podocytes, compared to non-mutant controls. Each point represents a single podocyte (n = 3 experiments). (C) Quantification of separation distance between cell lateral membranes in these hPSC-podocytes, with associated p-values. (D) Confocal section showing podocalyxin co-localization with filamentous actin at high magnification. Arrowhead highlights podocalyxin concentration between adjacent hPSC-podocytes. Boxed area is shown in zoom and highlights microvilli. Scale bars, 10 μm.

Figure 5. Podocalyxin regulates microvillus formation and cell spacing in mouse podocytes

(A) Schematic of knockout mouse generation and analysis. (B) TEM images of podocytes from Podxl^−/− prenatal mice (E18.5), or age-matched littermate controls. Images from two representative experiments are shown. Zoom is shown to the right for dashed boxed regions. m, microvilli; J, junctions. (C) Quantification of microvillus number and (D) cell separation in Podxl^−/− podocytes and littermate controls, with associated p-values. Each point represents a single podocyte (n = 4 control and 5 Podxl^−/− mice). Scale bars, 5 μm.

Figure 6. Loss of podocalyxin from endothelial cells does not significantly affect the morphology of glomerular endothelial cells or podocytes

(A) TEM micrographs of glomerular endothelial cells (endo) and podocytes (podo) in Cdh5-driven podocalyxin knockout mice, compared to controls. Fenestrae (f); microvillus (mv); foot process (fp); podocyte (Pod); endothelial cell (EC). (B) Quantification of microvillus formation (microvilli per unit length of apico-lateral membrane) iand foot process frequency (continuous membrane events proximal to the glomerular basement membrane separated by a slit diaphragm per length of unit basement membrane) in podocytes from Cdh5-Cre mice. (C) Representative wide-field immunofluorescence images of podocalyxin expression in kidney organoids containing endothelial cells (CD31) and proximal tubules (LTL). Zoom shows close-up of white dashed boxed region. Arrows indicated faint podocalyxin staining in endothelial cells, relative to the underlying layer of podocytes (Pod). Scale bars, 5 μm (A) or 100 μm (C).

Figure 7. Podocalyxin increases cell-cell spacing

(A) Schematic of optical tweezers experiment. Steps are shown top to bottom. Cells are depicted as green spheres. (B) Quantification of gap widths measured with optical tweezers in control or PODXL^−/− cells. Three distinct subclones are shown (average ± s.e.m., n ≥ 7 cells per subclone, p = 4.39 × 10⁻¹⁷). (C) Top gene ontology terms, (D) differentially expressed genes, and (E) upstream regulators in PODXL^−/− hPSCs, relative to isogenic controls. Data were obtained from three separate experiments (different days), each including 2–3 cell lines of each genotype. (F) Schematic of podocalyxin function. In the absence of podocalyxin, podocytes remain closely apposed and form lateral junctions through which urine cannot be filtered (left). During CLS maturation (right), podocalyxin-coated microvilli (green) form on the apical and lateral surfaces of podocytes. Sialic acid residues (negative charges) on podocalyxin act as an anti-adhesive, separating cell membranes and promoting basal migration of junctional complexes (red) to basal slits, through which urine is filtered. (G) Zoom-in of podocyte cell surfaces in close contact, showing the application of Coulomb’s law. (H) Table of parameters for the estimation of surface area and microvillus charge for each membrane, with (I) accompanying charge calculation for two membranes containing ten podocalyxin molecules per microvillus.