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.

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Fig. 1: Schematic overview of the protocol for derivation of mature kidney glomerular podocytes from human iPS cells.
Fig. 2: Morphological changes of human iPS cells at each stage of differentiation.
Fig. 3: Immunofluorescence staining and scanning electron micrograph of cells during the differentiation process.
Fig. 4: Whole-transcriptome analysis using Affymetrix Human Gene 2.0 ST gene array.
Fig. 5: Design of microfluidic Organ Chip device to recapitulate the structure and function of the kidney glomerular capillary wall.
Fig. 6: Fluorescence microscopy images of the human kidney Glomerulus Chip established from iPS cell–derived podocytes and primary human glomerular endothelial cells.

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Acknowledgements

This work was supported by the Defense Advanced Research Projects Agency under Cooperative Agreement No. W911NF-12-2-0036 and the Wyss Institute for Biologically Inspired Engineering at Harvard University. S.M. was supported by a Dean’s Postdoctoral Fellowship from Harvard Medical School, a UNCF-Merck Postdoctoral Fellowship, a Postdoctoral Enrichment Program Award from the Burroughs Wellcome Fund, and an NIH/NIDDK Nephrology Training Grant (4T32DK007199-39). We thank the Wyss Institute Microfabrication Team for engineering the microfluidic devices, S. Jeanty for providing photographs of the setup for microfluidic Organ Chip culture, P.K. Tetteh for helpful suggestions, and O. Levy and R. Prantil-Baun for comments on the manuscript.

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Contributions

S.M., G.M.C., and D.E.I. conceived the strategy for this study; S.M. designed and performed the experiments; S.M. and D.E.I. wrote the manuscript; N.D. and D.M.C. independently analyzed the microarray data; S.M. interpreted the results; and N.D. generated heatmaps and corresponding statistical datasets. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Donald E. Ingber.

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Competing interests

D.E.I. and S.M. declare that they are authors on a patent pending for methods for the generation of kidney glomerular podocytes from pluripotent stem cells (US patent application 14/950859). D.E.I. declares that he is a founder, holds equity and chairs the scientific advisory board at Emulate Inc. The remaining authors declare no competing interests.

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Related links

1. Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip: https://doi.org/10.1038/s41551-017-0069

2. Monitoring and robust induction of nephron intermediate mesoderm from human pluripotent stem cells: https://doi.org/10.1038/ncomms2378

3. Microfabrication of human organs-on-chips: https://doi.org/10.1038/nprot.2013.137

Integrated supplementary information

Supplementary Figure 1 Expression of integrin subtypes in human iPS cells and an established human glomerular podocyte cell line.

Expression of (a) alpha and (b) beta integrin subtypes in human iPS cells and the immortalized human podocyte cell line (PCL) determined by their ability to bind to surfaces presenting antibodies specific for the indicated integrin receptors. Error bars denote standard deviation of the mean (n=3), and open circles represent individual data points. Immunofluorescence staining of β1 integrin in (c) human iPS cells and (d) PCL. Scale bars, 100 µm

Supplementary Figure 2 Heatmap of top 300 variant genes.

Heatmap of top 300 variant genes between triplicate samples of undifferentiated human iPS cells, human iPS-derived podocytes (hiPS-podocytes) and the immortalized human podocyte cell line (PCL). Each replicate represents an independent experiment. Red color indicate higher expression Z-score. Hierarchical clustering was performed using complete agglomeration method and a Euclidean distance metric

Supplementary information

Combined Supplementary Information

Supplementary Figures 1 and 2 and Supplementary Methods

Supplementary Dataset 1

Lineage characterization of genes up- or downregulated in human iPS-cell–derived podocytes, relative to expression in undifferentiated human iPS cells (PGP1 line)

Supplementary Dataset 2

Lineage characterization of genes up or downregulated in human iPS-cell–derived podocytes, relative to expression in the human immortalized podocyte cell line (PCL)

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Musah, S., Dimitrakakis, N., Camacho, D.M. et al. Directed differentiation of human induced pluripotent stem cells into mature kidney podocytes and establishment of a Glomerulus Chip. Nat Protoc 13, 1662–1685 (2018). https://doi.org/10.1038/s41596-018-0007-8

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