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Generation of proximal tubule-enhanced kidney organoids from human pluripotent stem cells

Nature Protocols volume 18, pages 3229–3252 (2023)Cite this article

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Abstract

Kidney organoids derived from human pluripotent stem cells (hPSCs) are now being used as models of renal disease and nephrotoxicity screening. However, the proximal tubules (PTs), which are responsible for most kidney reabsorption functions, remain immature in kidney organoids with limited expression of critical transporters essential for nephron functionality. Here, we describe a protocol for improved specification of nephron progenitors from hPSCs that results in kidney organoids with elongated proximalized nephrons displaying improved PT maturity compared with those generated using standard kidney organoid protocols. We also describe a methodology for assessing the functionality of the PTs within the organoids and visualizing maturation markers via immunofluorescence. Using these assays, PT-enhanced organoids display increased expression of a range of critical transporters, translating to improved functionality measured by substrate uptake and transport. This protocol consists of an extended (13 d) monolayer differentiation phase, during which time hPSCs are exposed to nephron progenitor maintenance media (CDBLY2), better emulating human metanephric progenitor specification in vivo. Following nephron progenitor specification, the cells are aggregated and cultured as a three-dimensional micromass on an air–liquid interface to facilitate further differentiation and segmentation into proximalized nephrons. Experience in culturing hPSCs is required to conduct this protocol and expertise in kidney organoid generation is advantageous.

Key points

  • This protocol generates kidney organoids with elongated proximalized nephrons displaying improved proximal tubule maturity compared with those generated using standard kidney organoid protocols.

  • Methods for assessing the functionality of the proximal tubules within the organoids and visualizing maturation markers via immunofluorescence are also described.

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Fig. 1: Schematic diagram of the PT-enhanced organoid protocol.
Fig. 2: Brightfield images depicting expected morphologies of hPSC monolayers and resulting PT-enhanced organoids.
Fig. 3: Live confocal images of PT functionality assays for protein uptake and organic cation transport.
Fig. 4: Protein expression and localization in PT-enhanced organoid nephron.

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Data availability

The original research relating to this protocol can be accessed in a previous publication6, the manuscript website (https://kidneyregeneration.github.io/Vanslambrouck2022/), and via the Github repository (https://github.com/KidneyRegeneration/Vanslambrouck2022).

References

  1. Freedman, B. S. et al. Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids. Nat. Commun. 6, 8715 (2015).

    Article  CAS  PubMed  Google Scholar 

  2. Morizane, R. et al. Nephron organoids derived from human pluripotent stem cells model kidney development and injury. Nat. Biotechnol. 33, 1193–1200 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Taguchi, A. & Nishinakamura, R. Higher-order kidney organogenesis from pluripotent stem cells. Cell Stem Cell 21, 730–746 e736 (2017).

    Article  CAS  PubMed  Google Scholar 

  4. Takasato, M. et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526, 564–568 (2015).

    Article  CAS  PubMed  Google Scholar 

  5. Toyohara, T. et al. Cell therapy using human induced pluripotent stem cell-derived renal progenitors ameliorates acute kidney injury in mice. Stem Cells Transl. Med. 4, 980–992 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Vanslambrouck, J. M. et al. Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids. Nat. Commun. 13, 5943 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Howden, S. E., Vanslambrouck, J. M., Wilson, S. B., Tan, K. S. & Little, M. H. Reporter-based fate mapping in human kidney organoids confirms nephron lineage relationships and reveals synchronous nephron formation. EMBO Rep. https://doi.org/10.15252/embr.201847483 (2019).

  8. Takasato, M. et al. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat. Cell Biol. 16, 118–126 (2014).

    Article  CAS  PubMed  Google Scholar 

  9. Takasato, M., Er, P. X., Chiu, H. S. & Little, M. H. Generation of kidney organoids from human pluripotent stem cells. Nat. Protoc. 11, 1681–1692 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Howden, S. E. & Little, M. H. Generating kidney organoids from human pluripotent stem cells using defined conditions. Methods Mol. Biol. 2155, 183–192 (2020).

    Article  CAS  PubMed  Google Scholar 

  11. Tanigawa, S., Taguchi, A., Sharma, N., Perantoni, A. O. & Nishinakamura, R. Selective in vitro propagation of nephron progenitors derived from embryos and pluripotent stem cells. Cell Rep. 15, 801–813 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. de Bakker, B. S., van den Hoff, M. J. B., Vize, P. D. & Oostra, R. J. The pronephros; a fresh perspective. Integr. Comp. Biol. 59, 29–47 (2019).

    Article  PubMed  Google Scholar 

  13. Georgas, K. M., Chiu, H. S., Lesieur, E., Rumballe, B. A. & Little, M. H. Expression of metanephric nephron-patterning genes in differentiating mesonephric tubules. Dev. Dyn. 240, 1600–1612 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Mugford, J. W., Sipila, P., Kobayashi, A., Behringer, R. R. & McMahon, A. P. Hoxd11 specifies a program of metanephric kidney development within the intermediate mesoderm of the mouse embryo. Dev. Biol. 319, 396–405 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Tiedemann, K., Welling, L. W. & Basto, P. Structural and functional comparison of mesonephric and metanephric proximal tubules. Pediatr. Nephrol. 1, 297–305 (1987).

    Article  CAS  PubMed  Google Scholar 

  16. Dressler, G. R. Advances in early kidney specification, development and patterning. Development 136, 3863–3874 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Brown, A. C. et al. Role for compartmentalization in nephron progenitor differentiation. Proc. Natl Acad. Sci. USA 110, 4640–4645 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Brown, A. C., Muthukrishnan, S. D. & Oxburgh, L. A synthetic niche for nephron progenitor cells. Dev. Cell 34, 229–241 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li, Z. et al. 3D culture supports long-term expansion of mouse and human nephrogenic progenitors. Cell Stem Cell 19, 516–529 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tanigawa, S., Sharma, N., Hall, M. D., Nishinakamura, R. & Perantoni, A. O. Preferential propagation of competent SIX2+ nephronic progenitors by LIF/ROCKi treatment of the metanephric mesenchyme. Stem Cell Rep. 5, 435–447 (2015).

    Article  CAS  Google Scholar 

  21. Chung, E., Deacon, P. & Park, J. S. Notch is required for the formation of all nephron segments and primes nephron progenitors for differentiation. Development 144, 4530–4539 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Duvall, K. et al. Revisiting the role of Notch in nephron segmentation confirms a role for proximal fate selection during mouse and human nephrogenesis. Development https://doi.org/10.1242/dev.200446 (2022).

  23. Surendran, K. et al. The contribution of Notch1 to nephron segmentation in the developing kidney is revealed in a sensitized Notch2 background and can be augmented by reducing Mint dosage. Dev. Biol. 337, 386–395 (2010).

    Article  CAS  PubMed  Google Scholar 

  24. Ryan, M. J. et al. HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney. Kidney Int. 45, 48–57 (1994).

    Article  CAS  PubMed  Google Scholar 

  25. Racusen, L. C. et al. Cell lines with extended in vitro growth potential from human renal proximal tubule: characterization, response to inducers, and comparison with established cell lines. J. Lab. Clin. Med. 129, 318–329 (1997).

    Article  CAS  PubMed  Google Scholar 

  26. Orosz, D. E. et al. Growth, immortalization, and differentiation potential of normal adult human proximal tubule cells. Vitr. Cell Dev. Biol. Anim. 40, 22–34 (2004).

    Article  CAS  Google Scholar 

  27. Wieser, M. et al. hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics. Am. J. Physiol. Ren. Physiol. 295, F1365–F1375 (2008).

    Article  CAS  Google Scholar 

  28. Chandrasekaran, V. et al. Generation and characterization of iPSC-derived renal proximal tubule-like cells with extended stability. Sci. Rep. 11, 11575 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kaminski, M. M. et al. Direct reprogramming of fibroblasts into renal tubular epithelial cells by defined transcription factors. Nat. Cell Biol. 18, 1269–1280 (2016).

    Article  CAS  PubMed  Google Scholar 

  30. Kandasamy, K. et al. Prediction of drug-induced nephrotoxicity and injury mechanisms with human induced pluripotent stem cell-derived cells and machine learning methods. Sci. Rep. 5, 12337 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Narayanan, K. et al. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells. Kidney Int. 83, 593–603 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Heussner, A. H. & Dietrich, D. R. Primary porcine proximal tubular cells as an alternative to human primary renal cells in vitro: an initial characterization. BMC Cell Biol. 14, 55 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Jenkinson, S. E. et al. The limitations of renal epithelial cell line HK-2 as a model of drug transporter expression and function in the proximal tubule. Pflug. Arch. 464, 601–611 (2012).

    Article  CAS  Google Scholar 

  34. Khundmiri, S. J., Chen, L., Lederer, E. D., Yang, C. R. & Knepper, M. A. Transcriptomes of major proximal tubule cell culture models. J. Am. Soc. Nephrol. 32, 86–97 (2021).

    Article  CAS  PubMed  Google Scholar 

  35. Duan, Y. et al. Shear-induced reorganization of renal proximal tubule cell actin cytoskeleton and apical junctional complexes. Proc. Natl Acad. Sci. USA 105, 11418–11423 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Frohlich, E. M., Zhang, X. & Charest, J. L. The use of controlled surface topography and flow-induced shear stress to influence renal epithelial cell function. Integr. Biol. 4, 75–83 (2012).

    Article  CAS  Google Scholar 

  37. Jang, K. K. et al. Variable susceptibility of intestinal organoid-derived monolayers to SARS-CoV-2 infection. PLoS Biol. 20, e3001592 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ng, C. P., Zhuang, Y., Lin, A. W. H. & Teo, J. C. M. A fibrin-based tissue-engineered renal proximal tubule for bioartificial kidney devices: development, characterization and in vitro transport study. Int. J. Tissue Eng. 2013, 319476 (2013).

    Article  Google Scholar 

  39. Secker, P. F., Luks, L., Schlichenmaier, N. & Dietrich, D. R. RPTEC/TERT1 cells form highly differentiated tubules when cultured in a 3D matrix. ALTEX 35, 223–234 (2018).

    Article  PubMed  Google Scholar 

  40. Xu, Y. et al. Adult human kidney organoids originate from CD24+ cells and represent an advanced model for adult polycystic kidney disease. Nat. Genet. 54, 1690–1701 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Aceves, J. O. et al. 3D proximal tubule-on-chip model derived from kidney organoids with improved drug uptake. Sci. Rep. 12, 14997 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Homan, K. A. et al. Bioprinting of 3D convoluted renal proximal tubules on perfusable chips. Sci. Rep. 6, 34845 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Schutgens, F. et al. Tubuloids derived from human adult kidney and urine for personalized disease modeling. Nat. Biotechnol. 37, 303–313 (2019).

    Article  CAS  PubMed  Google Scholar 

  44. Weber, E. J. et al. Development of a microphysiological model of human kidney proximal tubule function. Kidney Int. 90, 627–637 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Lin, N. Y. C. et al. Renal reabsorption in 3D vascularized proximal tubule models. Proc. Natl Acad. Sci. USA 116, 5399–5404 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kumar, S. V. et al. Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells. Development https://doi.org/10.1242/dev.172361 (2019).

  47. Lam, A. Q. et al. Rapid and efficient differentiation of human pluripotent stem cells into intermediate mesoderm that forms tubules expressing kidney proximal tubular markers. J. Am. Soc. Nephrol. 25, 1211–1225 (2014).

    Article  CAS  PubMed  Google Scholar 

  48. Taguchi, A. et al. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells. Cell Stem Cell 14, 53–67 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Wilson, S. B. et al. DevKidCC allows for robust classification and direct comparisons of kidney organoid datasets. Genome Med. 14, 19 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Wu, H. et al. Comparative analysis and refinement of human PSC-derived kidney organoid differentiation with single-cell transcriptomics. Cell Stem Cell 23, 869–881 e868 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Combes, A. N., Zappia, L., Er, P. X., Oshlack, A. & Little, M. H. Single-cell analysis reveals congruence between kidney organoids and human fetal kidney. Genome Med. 11, 3 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Subramanian, A. et al. Single cell census of human kidney organoids shows reproducibility and diminished off-target cells after transplantation. Nat. Commun. 10, 5462 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Das, A. et al. Stromal-epithelial crosstalk regulates kidney progenitor cell differentiation. Nat. Cell Biol. 15, 1035–1044 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. England, A. R. et al. Identification and characterization of cellular heterogeneity within the developing renal interstitium. Development https://doi.org/10.1242/dev.190108 (2020).

  55. Lindstrom, N. O. et al. Integrated beta-catenin, BMP, PTEN, and Notch signalling patterns the nephron. eLife 3, e04000 (2015).

    Article  PubMed  Google Scholar 

  56. Nielsen, R., Christensen, E. I. & Birn, H. Megalin and cubilin in proximal tubule protein reabsorption: from experimental models to human disease. Kidney Int. 89, 58–67 (2016).

    Article  CAS  PubMed  Google Scholar 

  57. Yasujima, T., Ohta, K. Y., Inoue, K., Ishimaru, M. & Yuasa, H. Evaluation of 4′,6-diamidino-2-phenylindole as a fluorescent probe substrate for rapid assays of the functionality of human multidrug and toxin extrusion proteins. Drug Metab. Dispos. 38, 715–721 (2010).

    Article  CAS  PubMed  Google Scholar 

  58. Vanslambrouck, J. M. et al. A toolbox to characterize human induced pluripotent stem cell-derived kidney cell types and organoids. J. Am. Soc. Nephrol. 30, 1811–1823 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Carcamo-Orive, I. et al. Analysis of transcriptional variability in a large human iPSC library reveals genetic and non-genetic determinants of heterogeneity. Cell Stem Cell 20, 518–532 e519 (2017).

    Article  CAS  PubMed  Google Scholar 

  60. Volpato, V. & Webber, C. Addressing variability in iPSC-derived models of human disease: guidelines to promote reproducibility. Dis. Model Mech. https://doi.org/10.1242/dmm.042317 (2020).

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Acknowledgements

This research was supported by the National Health and Medical Research Council (GNT1156440 (M.H.L., J.M.V., K.S.T.)), the National Institutes of Health (UH3DK107344; M.H.L., J.M.V.), the Victorian State Government Department of Jobs Precincts and Regions (DJPR) through the Victorian COVID-19 Research Fund (S.M.)), and the Novo Nordisk Foundation Centre for Stem Cell Research (supported by Novo Nordisk Foundation grant NNF21CC0073729 (M.H.L., J.M.V.)). M.H.L. is a National Health and Medical Research Senior Principal Research Fellow (GNT1136085). We acknowledge the Stafford Fox Medical Research Foundation Murdoch Children’s Research Institute (MCRI) iPSC Derivation & Gene Editing Facility for the generation of all pluripotent stem cell lines. We thank M. Burton and the Murdoch Children’s Research Institute Microscopy Core, as well as S. Howden and the MCRI iPSC Derivation and Gene Editing Core.

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Authors and Affiliations

  1. Novo Nordisk Foundation Centre for Stem Cell Medicine (reNEW), Murdoch Children’s Research Institute, Melbourne, Victoria, Australia

    Jessica M. Vanslambrouck, Ker Sin Tan, Sophia Mah & Melissa H. Little

  2. Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Melbourne, Victoria, Australia

    Jessica M. Vanslambrouck & Melissa H. Little

  3. Novo Nordisk Foundation Centre for Stem Cell Medicine (reNEW), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Melissa H. Little

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Contributions

J.M.V. and M.H.L. contributed to the experimental design and planning. J.M.V., K.S.T. and S.M. performed the experiments, developed the reagents and recorded the methodology. J.M.V., K.S.T. and S.M. contributed to the preparation of the manuscript. J.M.V. and M.H.L. wrote the manuscript.

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Correspondence to Melissa H. Little.

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Vanslambrouck, J. M. et al. Nat. Commun. 13, 5943 (2022): https://doi.org/10.1038/s41467-022-33623-z

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Vanslambrouck, J.M., Tan, K.S., Mah, S. et al. Generation of proximal tubule-enhanced kidney organoids from human pluripotent stem cells. Nat Protoc 18, 3229–3252 (2023). https://doi.org/10.1038/s41596-023-00880-1

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