Protocol | Published:

The generation of kidney organoids by differentiation of human pluripotent cells to ureteric bud progenitor–like cells

Nature Protocols volume 9, pages 26932704 (2014) | Download Citation

Abstract

This protocol presents recently developed methodologies for the differentiation of human pluripotent stem cells (hPSCs) into ureteric bud (UB) progenitor–like cells. Differentiation of human PSCs to UB progenitor–like cells allows for the generation of chimeric kidney cultures in which the human cells can self-assemble into chimeric 3D structures in combination with embryonic mouse kidney cells over a period of 18 d. UB progenitor–like cells are generated by a two-step process that combines in vitro commitment of human PSCs, whether embryonic stem cells (ESCs) or induced PSCs (iPSCs), under chemically defined culture conditions, with ex vivo cultures for the induction of 3D organogenesis. The models described here provide new opportunities for investigating human kidney development, modeling disease, evaluating regenerative medicine strategies, as well as for toxicology studies.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    et al. Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant. Nat. Protoc. 9, 396–409 (2014).

  2. 2.

    et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).

  3. 3.

    et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 10, 771–785 (2012).

  4. 4.

    et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011).

  5. 5.

    et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods 11, 399–402 (2014).

  6. 6.

    et al. Targeted gene correction of laminopathy-associated LMNA mutations in patient-specific iPSCs. Cell Stem Cell 8, 688–694 (2011).

  7. 7.

    et al. Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells. Nat. Cell Biol. 15, 1507–1515 (2013).

  8. 8.

    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).

  9. 9.

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

  10. 10.

    et al. Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature 460, 53–59 (2009).

  11. 11.

    et al. Primary cultures of glomerular parietal epithelial cells or podocytes with proven origin. PLoS ONE 7, e34907 (2012).

  12. 12.

    , , & Podocytes in culture: past, present, and future. Kidney Int. 72, 26–36 (2007).

  13. 13.

    & Mammalian kidney development: principles, progress, and projections. Cold Spring Harb. Perspect. Biol. 4, pii: a008300 (2012).

  14. 14.

    , , , & Renal ontogeny in the rhesus monkey (Macaca mulatta) and directed differentiation of human embryonic stem cells towards kidney precursors. Differentiation 78, 45–56 (2009).

  15. 15.

    et al. Mouse embryonic stem cell-derived embryoid bodies generate progenitors that integrate long term into renal proximal tubules in vivo. J. Am. Soc. Nephrol. 18, 1709–1720 (2007).

  16. 16.

    et al. In vitro differentiation of murine embryonic stem cells toward a renal lineage. Differentiation 75, 337–349 (2007).

  17. 17.

    & Nephrogenic factors promote differentiation of mouse embryonic stem cells into renal epithelia. J. Am. Soc. Nephrol. 16, 3527–3534 (2005).

  18. 18.

    , , , & Dissociation of embryonic kidney followed by re-aggregation as a method for chimeric analysis. Methods Mol. Biol. 886, 135–146 (2012).

  19. 19.

    & Dissociation of embryonic kidneys followed by reaggregation allows the formation of renal tissues. Kidney Int. 77, 407–416 (2010).

  20. 20.

    & An improved method of renal tissue engineering, by combining renal dissociation and reaggregation with a low-volume culture technique, results in development of engineered kidneys complete with loops of Henle. Nephron Exp. Nephrol. 121, e79–85 (2012).

  21. 21.

    et al. A novel, low-volume method for organ culture of embryonic kidneys that allows development of cortico-medullary anatomical organization. PLoS ONE 5, e10550 (2010).

  22. 22.

    & Atlas of Human Pluripotent Stem Cells (Humana Press, 2012).

  23. 23.

    , , , & Dissection of embryonic mouse kidney, culture in vitro, and imaging of the developing organ. Cold Spring Harb. Protoc. 2011 10.1101/pdb.prot5613 (2011).

  24. 24.

    et al. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637–646 (2006).

Download references

Acknowledgements

We thank M. Schwarz for administrative support. We thank J. Kasuboski from the Waitt Advanced Biophotonics Core at the Salk Institute for Biological Studies for help with imaging processing. Y.X. was partially supported by the California Institute for Regenerative Medicine (CIRM) through a CIRM Training grant. I.S.-M. was partially supported by a Nomis Foundation postdoctoral fellowship. Work in the laboratory of J.C.I.B. was supported by grants from the G. Harold and Leila Y. Mathers Charitable Foundation and The Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002).

Author information

Author notes

    • Yun Xia
    • , Ignacio Sancho-Martinez
    •  & Emmanuel Nivet

    These authors contributed equally to this work.

    • Emmanuel Nivet

    Present address: Aix Marseille Université, Centre National de la Recherche Scientifique (CNRS), Neurobiologie des Interactions Cellulaires et Neurophysiopathologie (NICN) Unité Mixte de Recherche (UMR) 7259, Marseille, France.

Affiliations

  1. Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California, USA.

    • Yun Xia
    • , Ignacio Sancho-Martinez
    • , Emmanuel Nivet
    • , Concepcion Rodriguez Esteban
    •  & Juan Carlos Izpisua Belmonte
  2. Renal Division, Hospital Clinic, University of Barcelona, Institut d'investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.

    • Josep Maria Campistol

Authors

  1. Search for Yun Xia in:

  2. Search for Ignacio Sancho-Martinez in:

  3. Search for Emmanuel Nivet in:

  4. Search for Concepcion Rodriguez Esteban in:

  5. Search for Josep Maria Campistol in:

  6. Search for Juan Carlos Izpisua Belmonte in:

Contributions

Y.X., I.S.-M., E.N., C.R.E., J.M.C. and J.C.I.B. designed all experiments and developed the methodologies presented here. Y.X., I.S.-M., E.N. and J.C.I.B. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Juan Carlos Izpisua Belmonte.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2014.182

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.