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Directed differentiation of ureteric bud and collecting duct organoids from human pluripotent stem cells

Nature Protocols volume 18, pages 2485–2508 (2023)Cite this article

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Abstract

Developing models of human kidney tissue in vitro is an important challenge in regenerative nephrology research, given the paucity of novel and effective therapies in kidney disease. However, the de novo generation of kidney tissues from human pluripotent stem cells (hPSCs) is challenging owing to the structural and functional complexity of the organ, as well its developmental origin from two distinct embryologic populations: the metanephric mesenchyme and the ureteric bud (UB). Directed differentiation strategies have been developed to generate kidney organoids containing nephron-like structures; we recently reported an efficient and practical method to generate UB tissues. Here, we describe a detailed step-by-step protocol for differentiation of hPSCs into three-dimensonal UB organoids that exhibit complex morphological development and the capacity to differentiate into functional collecting duct tissues. Over 3 d, hPSCs are induced into PAX2+GATA3+ pronephric (anterior) intermediate mesoderm fates in monolayer cultures at high efficiency. The cells are aggregated into three-dimensional spheroids, which then assemble and organize into nephric duct-like tissue over 4 d. When embedded into an extracellular matrix, the spheroids grow into UB organoids that recapitulate fetal branching morphogenesis for 1 week of culture. When switched to permissive conditions, the UB organoids spontaneously differentiate to form collecting duct principal cells. This approach provides robust and reproducible methods that can be readily adopted by users with basic experience in hPSC and organoid differentiation to generate UB tissues, which may be used to investigate human kidney development, model disease processes and catalyze further efforts in engineering functional kidney tissue.

Key points

  • Human pluripotent stem cells are differentiated into three-dimensional ureteric bud organoids that exhibit complex morphological development and the capacity to differentiate into functional collecting duct tissues.

  • The procedure accurately recapitulates early developmental stages and produces uretic bud structures showing branching via terminal bifurcation and stalk elongation. It can also be easily scaled as there is a high degree of inter- and intra-experimental consistency.

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Fig. 1: A schematized depiction of the protocol for directed differentiation of UB organoids.
Fig. 2: Morphologic appearance of starting cultures.
Fig. 3: The duration of the mesendoderm induction step is critical for efficient formation of pronephric IM cells.
Fig. 4: Formation of 3D ND spheroids.
Fig. 5: Morphogenesis and differentiation of UB organoids in 3D culture.
Fig. 6: Optimization of UB medium for branching in iPSC72-3-derived organoids.
Fig. 7: Expression of TBXT on day 1.

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Data availability

The datasets containing single-cell RNA sequencing results to characterize ND spheroids (day 7) and CD organoids (day 18) were deposited at the Gene Expression Omnibus (GSE206153) as part of a previous publication18. No additional datasets were generated as part of this study.

References

  1. Ransick, A. et al. Single-cell profiling reveals sex, lineage, and regional diversity in the mouse kidney. Dev. Cell 51, 399–413 e397 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Park, J. et al. Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease. Science 360, 758–763 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. England, A. R. et al. Identification and characterization of cellular heterogeneity within the developing renal interstitium. Development 147, dev190108 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Daniel, E. et al. Spatiotemporal heterogeneity and patterning of developing renal blood vessels. Angiogenesis 21, 617–634 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Munro, D. A. D., Hohenstein, P. & Davies, J. A. Cycles of vascular plexus formation within the nephrogenic zone of the developing mouse kidney. Sci. Rep. 7, 3273 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Barak, H., Rosenfelder, L., Schultheiss, T. M. & Reshef, R. Cell fate specification along the anterior–posterior axis of the intermediate mesoderm. Dev. Dyn. 232, 901–914 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. 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 

  8. Costantini, F. Genetic controls and cellular behaviors in branching morphogenesis of the renal collecting system. Wiley Interdiscip. Rev. Dev. Biol. 1, 693–713 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kobayashi, A. et al. Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development. Cell Stem Cell 3, 169–181 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 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 

  11. 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 

  12. Tsujimoto, H. et al. A modular differentiation system maps multiple human kidney lineages from pluripotent stem cells. Cell Rep. 31, 107476 (2020).

    Article  CAS  PubMed  Google Scholar 

  13. Howden, S. E. et al. Plasticity of distal nephron epithelia from human kidney organoids enables the induction of ureteric tip and stalk. Cell Stem Cell 28, 671–684 e676 (2021).

    Article  CAS  PubMed  Google Scholar 

  14. Mae, S. I. et al. Generation of branching ureteric bud tissues from human pluripotent stem cells. Biochem. Biophys. Res. Commun. 495, 954–961 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. Mae, S. I. et al. Expansion of human iPSC-derived ureteric bud organoids with repeated branching potential. Cell Rep. 32, 107963 (2020).

    Article  CAS  PubMed  Google Scholar 

  16. 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 

  17. Zeng, Z. et al. Generation of patterned kidney organoids that recapitulate the adult kidney collecting duct system from expandable ureteric bud progenitors. Nat. Commun. 12, 3641 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Shi, M. et al. Human ureteric bud organoids recapitulate branching morphogenesis and differentiate into functional collecting duct cell types. Nat. Biotechnol. 41, 252–261 (2023).

    Article  CAS  PubMed  Google Scholar 

  19. Sawada, K. & Aoyama, H. Fate maps of the primitive streak in chick and quail embryo: ingression timing of progenitor cells of each rostro-caudal axial level of somites. Int. J. Dev. Biol. 43, 809–815 (1999).

    CAS  PubMed  Google Scholar 

  20. Parameswaran, M. & Tam, P. P. Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation. Dev. Genet 17, 16–28 (1995).

    Article  CAS  PubMed  Google Scholar 

  21. Attia, L., Yelin, R. & Schultheiss, T. M. Analysis of nephric duct specification in the avian embryo. Development 139, 4143–4151 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Przepiorski, A. et al. A simple bioreactor-based method to generate kidney organoids from pluripotent stem cells. Stem Cell Rep. 11, 470–484 (2018).

    Article  CAS  Google Scholar 

  23. Morizane, R. & Bonventre, J. V. Generation of nephron progenitor cells and kidney organoids from human pluripotent stem cells. Nat. Protoc. 12, 195–207 (2017).

    Article  CAS  PubMed  Google Scholar 

  24. Sanchez-Ferras, O. et al. A coordinated progression of progenitor cell states initiates urinary tract development. Nat. Commun. 12, 2627 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Pepicelli, C. V., Kispert, A., Rowitch, D. H. & McMahon, A. P. GDNF induces branching and increased cell proliferation in the ureter of the mouse. Dev. Biol. 192, 193–198 (1997).

    Article  CAS  PubMed  Google Scholar 

  26. Vega, Q. C., Worby, C. A., Lechner, M. S., Dixon, J. E. & Dressler, G. R. Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis. Proc. Natl Acad. Sci. USA 93, 10657–10661 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Michos, O. et al. Kidney development in the absence of Gdnf and Spry1 requires Fgf10. PLoS Genet. 6, e1000809 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Qiao, J., Sakurai, H. & Nigam, S. K. Branching morphogenesis independent of mesenchymal-epithelial contact in the developing kidney. Proc. Natl Acad. Sci. USA 96, 7330–7335 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yuri, S., Nishikawa, M., Yanagawa, N., Jo, O. D. & Yanagawa, N. In vitro propagation and branching morphogenesis from single ureteric bud cells. Stem Cell Rep. 8, 401–416 (2017).

    Article  CAS  Google Scholar 

  30. Toka, H. R., Toka, O., Hariri, A. & Nguyen, H. T. Congenital anomalies of kidney and urinary tract. Semin. Nephrol. 30, 374–386 (2010).

    Article  PubMed  Google Scholar 

  31. Kuure, S. & Sariola, H. Mouse models of congenital kidney anomalies. Adv. Exp. Med. Biol. 1236, 109–136 (2020).

    Article  CAS  PubMed  Google Scholar 

  32. Grobstein, C. Inductive epitheliomesenchymal interaction in cultured organ rudiments of the mouse. Science 118, 52–55 (1953).

    Article  CAS  PubMed  Google Scholar 

  33. Broda, T. R., McCracken, K. W. & Wells, J. M. Generation of human antral and fundic gastric organoids from pluripotent stem cells. Nat. Protoc. 14, 28–50 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. McCracken, K. W., Howell, J. C., Wells, J. M. & Spence, J. R. Generating human intestinal tissue from pluripotent stem cells in vitro. Nat. Protoc. 6, 1920–1928 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Spence, J. R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011).

    Article  PubMed  Google Scholar 

  36. McCracken, K. W. et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516, 400–404 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This study was supported by funding from the National Institutes of Health National Institute for Diabetes and Digestive and Kidney Diseases and National Center for Advancing Translational Sciences (UH3 TR002155, R37 DK39773), and the Pediatric Center of Excellence at the University of Virginia (P50 DK096373). K.W.M. was supported by the National Institutes of Health training grant (T32 DK00772) and the Ben J. Lipps Fellowship from the American Society of Nephrology. M.S. was supported by National Natural Science Foundation of China (32100909) and Post-Doctor Research Project, West China Hospital, Sichuan University (2021HXBH016). Studies involving hPSCs were reviewed and approved by MGB Institutional Biosafety Committee (2011B000287). No new hESC lines were generated as part of this study.

Author information

Author notes

  1. Kyle W. McCracken

    Present address: Division of Nephrology, Center for Stem Cell and Organoid Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

Authors and Affiliations

  1. Division of Nephrology, Kidney Research Institute, West China Hospital of Sichuan University, Chengdu, China

    Min Shi & Ping Fu

  2. Division of Renal Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Min Shi, Joseph V. Bonventre & Kyle W. McCracken

  3. Harvard Stem Cell Institute, Cambridge and Boston, MA, USA

    Joseph V. Bonventre

  4. Division of Engineering in Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Joseph V. Bonventre

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Contributions

M.S., J.V.B. and K.W.M. conceived of this study and formulated experiments. M.S., J.V.B. and K.W.M. performed the experiments and wrote the manuscript. P.F. performed experiments and contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Joseph V. Bonventre or Kyle W. McCracken.

Ethics declarations

Competing interests

J.V.B. is an inventor on kidney organoid patents assigned to MGB, and is cofounder and holds equity in Goldfinch Bio. K.W.M. has kidney organoid patents pending. J.V.B.’s interests were reviewed and are managed by BWH and MGB in accordance with their conflict-of-interest policies. The remaining authors declare no competing interests.

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Nature Protocols thanks Melissa Little, Zhongwei Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Shi, M. et al. Nat. Biotechnol. 41, 252–261 (2023): https://doi.org/10.1038/s41587-022-01429-5

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Shi, M., Fu, P., Bonventre, J.V. et al. Directed differentiation of ureteric bud and collecting duct organoids from human pluripotent stem cells. Nat Protoc 18, 2485–2508 (2023). https://doi.org/10.1038/s41596-023-00847-2

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