Directed differentiation of human induced pluripotent stem cells into mature kidney podocytes and establishment of a Glomerulus Chip

Abstract

Protocols have been established to direct the differentiation of human induced pluripotent stem (iPS) cells into nephron progenitor cells and organoids containing many types of kidney cells, but it has been difficult to direct the differentiation of iPS cells to form specific types of mature human kidney cells with high yield. Here, we describe a detailed protocol for the directed differentiation of human iPS cells into mature, post-mitotic kidney glomerular podocytes with high (>90%) efficiency within 26 d and under chemically defined conditions, without genetic manipulations or subpopulation selection. We also describe how these iPS cell-derived podocytes may be induced to form within a microfluidic organ-on-a-chip (Organ Chip) culture device to build a human kidney Glomerulus Chip that mimics the structure and function of the kidney glomerular capillary wall in vitro within 35 d (starting with undifferentiated iPS cells). The podocyte differentiation protocol requires skills for culturing iPS cells, and the development of a Glomerulus Chip requires some experience with building and operating microfluidic cell culture systems. This method could be useful for applications in nephrotoxicity screening, therapeutic development, and regenerative medicine, as well as mechanistic study of kidney development and disease.

Figure 1|

Schematic overview of the protocol for derivation of mature kidney glomerular podocytes from human iPS cells. The diagram shows sequential differentiation stages in the protocol. The concentrations of growth factors and signaling molecules are shown. Also shown are the markers used to characterize the cells at each stage of the differentiation protocol. Black square indicates a pause point, where the protocol may be paused and the intermediate mesoderm cells could be stored by cryopreservation. GSC, goosecoid; WT1, Wilm’s tumor 1; OSR1, odd-skipped related transcription factor 1; BMP-7, bone morphogenetic protein 7; VEGF, vascular endothelial growth factor; ECM, extracellular matrix. Figure modified with permission from Reference 5.

Figure 2|

Morphological changes of human iPS cells at each stage of differentiation. Representative bright field images show human iPS cells before and after dissociation on day 0 when differentiation is initiated, mesoderm cells after 2 days of differentiation, intermediate mesoderm cells at around 10 days and after20 days of culture with two passages, as well as the terminally differentiated podocytes. An example of an image of podocytes derived from a population of cells containing a poorly dissociated colony (dotted red circle) is also shown. Int. mesoderm, intermediate mesoderm; hiPS-podocyte, human iPS cell-derived podocytes. Scale bar, 100 μm.

Figure 3|

Immunofluorescence staining and scanning electron micrograph of cells during the differentiation process. Human iPS cell-derived mesoderm cells expressing hand1, goosecoid, and brachyury (a), intermediate mesoderm cells expressing WT1 and Pax2 markers (b), and podocytes expressing lineage characterization markers nephrin, WT1 and podocin, as well as the associated protein ApoL1 (c). (d) High magnification image of iPS cell-derived podocytes showing the development of foot processes that are positive for podocin. (e) Scanning electron micrograph of human iPS-derived podocytes showing the development of primary and secondary foot processes. Figures d, e modified with permission from Reference 5. Scale bars, (a-c) 100 μm, (d) 25 μm, (e) 2 μm.

Figure 4|

Whole transcriptome analysis using Affymetrix Human Gene 2.0 ST gene array. (a) Global gene expression profile of triplicate samples of undifferentiated human iPS cells, iPS-derived podocytes, and an immortalized human glomerular podocyte cell line (PCL). (b) Expression levels of genes involved in pluripotency, development of nephrogenic progenitors, or lineage specification and functional maturation of kidney glomerular podocytes. Each replicate represents an independent experiment. See also Supplementary Data 1 and 2. Heatmaps depict genes that are up-regulated (red), down-regulated (blue), and unchanged (white).

Figure 5|

Design of microfluidic Organ Chip device to recapitulate the structure and function of the kidney glomerular capillary wall. (a) Schematic representation of the glomerular capillary wall showing podocyte and endothelial cell layers separated by the GBM to form capillary and urinary compartments. (b) Photograph of the microfluidic device engineered from PDMS. Scale bar, 5 mm. (c) Schematic representation of the microfluidic kidney Glomerulus Chip with microchannels replicating the urinary and capillary compartments of the glomerulus. The GBM is mimicked by using a porous and flexible PDMS membrane functionalized with the protein laminin. Cyclic mechanical strain was applied to cell layers by stretching the flexible PDMS membrane using vacuum. Example photographs of the experimental setup for Glomerulus Chip cultures show (d) a single Organ Chip microfluidic device connected to two reservoirs containing cell culture media for the urinary (right) and capillary (left) channels, (e) multiple Organ Chips placed on a cartridge built-in-house for handling of microfluidic devices, (f) photograph of the programmable vacuum regulator system built-in-house, (g) complete setup of the microfluidic cell culture system with the Glomerulus Chips connected to media reservoirs, inlet and outlet tubing, vacuum lines for stretching, and the peristaltic pump, as well as receptacles for media outflow. Figure modified with permission from Reference 5.

Figure 6|

Fluorescence microscopy images of the human kidney Glomerulus Chip established from iPS cell-derived podocytes and primary glomerular endothelial cells. (a) Side and (b) cross-sectional view of 3D reconstructed confocal images of the human Glomerulus Chip showing the iPS cell-derived podocytes and endothelial cells in their respective layers after differentiation and co-culture on opposing sides of the flexible ECM-coated PDMS membrane. (c) Additional immunofluorescence confocal images showing a top view of both cell layers (left), the endothelial cell layer only (middle), and the human iPS cell-derived podocyte layer (right). Scale bars, 100 μm. Figure modified with permission from Reference 5.