Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip

Abstract

An in vitro model of the human kidney glomerulus - the major site of blood filtration - could facilitate drug discovery and illuminate kidney-disease mechanisms. Microfluidic organ-on-a-chip technology has been used to model the human proximal tubule, yet a kidney-glomerulus-on-a-chip has not been possible because of the lack of functional human podocytes - the cells that regulate selective permeability in the glomerulus. Here, we demonstrate an efficient (> 90%) and chemically defined method for directing the differentiation of human induced pluripotent stem (hiPS) cells into podocytes that express markers of the mature phenotype (nephrin+, WT1+, podocin+, Pax2-) and that exhibit primary and secondary foot processes. We also show that the hiPS-cell-derived podocytes produce glomerular basement-membrane collagen and recapitulate the natural tissue/tissue interface of the glomerulus, as well as the differential clearance of albumin and inulin, when co-cultured with human glomerular endothelial cells in an organ-on-a-chip microfluidic device. The glomerulus-on-a-chip also mimics adriamycin-induced albuminuria and podocyte injury. This in vitro model of human glomerular function with mature human podocytes may facilitate drug development and personalized-medicine applications.

Fig. 1. Efficient differentiation of kidney podocytes from human iPS cells

(A) Schematic overview of the timeline for directed differentiation of hiPS cells into podocytes. BMP7, Bone morphogenetic protein 7; VEGF, Vascular endothelial growth factor; RA, Retinoic acid; L511, Laminin 511; L511-E8, Laminin 511 E8 fragment; L21, Laminin 521. (B) Low (top) and high (bottom) magnification bright field images of cells at each stage of differentiation (bars, 100 and 50 μm in top and bottom, respectively). (C) Bight field images and (D) scatter plot of the diameter (y-axis) of dissociated (non-adhered) hiPS cells, hiPS-derived podocytes, and immortalized human podocytes. Bar, 50 μm; error bars represent the standard deviation of the mean, n = 20 cells; n.s., not significant; ***, p <0.0001. (E) Flow cytometry analysis for the expression of pluripotency and podocyte markers in hiPS cells, hiPS-derived podocytes, and immortalized human podocytes. Representative plots showing expression levels of the Oct4 pluripotency marker, Wilm’s tumor 1 (WT1) kidney cell marker, nephrin podocyte-specific marker, and dual expression of WT1 and nephrin. (F) Quantitative representation of flow cytometry analysis. Y-axis represents the percentage of cells positive for Oct4 (beige), WT1 (purple), nephrin (green) and dual positive for WT1 and nephrin (red). Error bars represent standard deviation of the mean, n = 3 independent experiments.

Fig. 2. Human iPS-derived podocytes express markers characteristic of the mature phenotype

(A) Representative fluorescence microscopic images of intermediate mesoderm, hiPS-derived podocytes, and human immortalized podocytes immunostained for Pax2, WT1 and nephrin. (B) Quantification of hiPS-derived podocytes indicate upregulation of podocyte markers (nephrin, WT1, and podocin), with a corresponding decrease in the pluripotency marker Oct4. The decrease in progenitor cell markers Pax2 and OSR1, and lack of EdU incorporation in hiPS-derived podocytes indicate that the cells are post-mitotic and terminally differentiated, as in mature podocytes. Error bars represent standard deviation of the mean, n = 3; hpf, high power field; OSR1, odd-skipped related transcription factor protein 1; EdU, 5-ethynyl-2′-deoxyuridine. (C) Immunofluorescence microscopic images of hiPS-derived podocytes and immortalized human podocytes immunostained for PKC λ/I, a putative trafficker of nephrin to cell surface. (D) Western blot analysis of total and phosphorylated PKC λ/I proteins levels in hiPS cells, hiPS-derived podocytes, and human immortalized podocytes. (E) Quantification of phosphorylated PKC λ/I levels from Western blots. Error bars represent standard deviation of the mean, n = 3; *, p <0.05; **,p <0.001; ***, p <0.0001; bars, 100 μm.

Fig. 3. Human iPS-derived podocytes exhibit primary and secondary cell processes, and enhanced molecular uptake of exogenous albumin

(A) Top panel, fluorescence microscopic images of hiPS-derived podocytes and human immortalized podocytes immunostained for podocin (green). Bottom panel, scanning electron microscopy images of hiPS-derived podocytes and human immortalized podocytes. (B) hiPS-derived podocytes and human Immortalized podocytes immunostained for FcRn (magenta), a receptor for albumin and IgG transport. (C) Confocal microscopic images of hiPS cells, hiPS-derived podocytes, and human immortalized podocytes (Imm. podocytes) exposed to exogenous albumin (green). White arrowheads indicate cells that exhibit albumin uptake. (D) Quantification of albumin-positive cells from data shown in C. Error bars represent standard deviation of the mean, n = 3; ***, p < 0.0001; hpf, high power field. Scale bars: (A) 50 μm (top panel), 10 μm (bottom panel), (B) 100 μm, (C) 50 μm.

Fig. 4. Modeling the human glomerular capillary wall with an organ-on-a-chip microfluidic device

(A) Schematic representation of glomerular capillary wall with podocytes and endothelial cells separated by glomerular basement membrane (GBM). Exemplary directional flow of molecules from the capillary lumen to urinary space is shown by arrowed line. (B) Photograph (left) and schematic (right) of a microfluidic organ-on-a-chip device with microchannels replicating the urinary and capillary compartments of the glomerulus. The glomerular basement membrane is replicated using a porous and flexible PDMS membrane functionalized with the ECM protein laminin. Cyclic mechanical strain was applied to cell layers by stretching the flexible PDMS membrane using vacuum. (C) Bright field microscopic images of hiPS-derived podocytes (left) differentiated in the organ-on-a-chip microdevice, and primary human glomerular endothelial cells (right) cultured on opposite side of the flexible membrane. (D) Fluorescence microscopic images of hiPS-derived podocytes differentiated in organ-on-a-chip microfluidic device with human glomerular endothelial cells (not shown) cultured on opposite side of the PDMS membrane. Cells were differentiated under fluid flow only (no strain), or fluid flow and 10% mechanical strain (10% strain). Cells were immunostained for nephrin (green) and counterstained with DAPI (blue). (E) Graphical representation of the ratio of cytoplasmic to nuclear nephrin in hiPS-derived podocytes that were differentiated on-chip with or without mechanical strain. Error bars represent the standard deviation of the mean, n = 3. (F) 3D reconstructed views of the tissue-tissue interface formed by hiPS-derived podocytes (top layer, green) and human glomerular endothelial cells (bottom layer, magenta) showing that cyclic application of 10% strain enhanced the extension of podocyte cell processes through the pores of the flexible ECM-coated PDMS membrane so that they insert on the abluminal surface of the underlying glomerular endothelium. Error bars represent standard deviation of the mean, n = 4. Scale bars, 100 μm; * p < 0.05.

Fig. 5. Microfluidic organ-on-a-chip device reconstitutes kidney glomerular capillary function in vitro

(A) Secretion of VEGF-A by hiPS-podocytes differentiated in the microfluidic glomerulus-on-a-chip. (B) Quantification of the glomerular filtration (Urinary Clearance) of albumin and inulin molecules that were continuously infused over a period of 6 hours into the capillary channel of the glomerulus chip lined by hiPS-derived podocytes and human glomerular endothelial cells. Results are compared to in vivo values estimated based on the surface area of glomerular capillaries in vivo. (C) Filtration of albumin and inulin in control microfluidic chips lacking human kidney podocytes quantified over 6 hours of continuous infusion using the methods described above. (D) Immunofluorescence microscopic views showing the production and distribution of the basement membrane protein collagen IV (green) in the microfluidic glomerulus chip in the presence or absence of physiological cyclic mechanical strain (lower panels shows a side view of the glomerulus chip; Nuclei were counterstained with DAPI blue). (E) Quantification of collagen IV production by podocyte and endothelium layers in the glomerulus chip with or without mechanical strain as described in D. (Scale bar, 100 μm. For panels A, B, C and E, error bars represent standard deviation of the mean; n = 4; n.s., not significant; * p < 0.05; ** p < 0.001*** p < 0.0001).

Fig. 6. Human glomerulus-on-a-chip mimics adriamycin-induced kidney glomerular injury

(A) Phase contrast images of the hiPS-derived podocyte layer within the glomerulus chip continuously infused for 5 days with different concentrations of adriamycin through the underlying vascular channel lined by glomerular endothelial cells. (B) Fluorescence microscopic images of the podocyte and endothelial cell layers within control and adriamycin-treated glomerulus chips stained with phalloidin (yellow) and counterstained with DAPI (blue). (C) Dose-dependent effects of adriamycin exposure on cell adhesion within the podocyte and endothelial cell populations in the glomerulus chip measured after 5 days of drug treatment using fluorescence microscopic quantification of DAPI-stained cells. (D) Quantification of the dose-dependent effects of adriamycin exposure on glomerular filtration (urinary clearance) of albumin and inulin in the glomerulus chip. (E) Quantification of uptake of exogenous albumin in the urinary channel by the hiPS-derived podocyte layer after adriamycin-induced injury (Scale bar, 100 μm. For panels C, D, and E, error bars represent S.D., n = 3; n.s., not significant; * p < 0.05; ** p < 0.001*** p < 0.0001).