Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells

Abstract

Diseases affecting the kidney constitute a major health issue worldwide. Their incidence and poor prognosis affirm the urgent need for the development of new therapeutic strategies. Recently, differentiation of pluripotent cells to somatic lineages has emerged as a promising approach for disease modelling and cell transplantation. Unfortunately, differentiation of pluripotent cells into renal lineages has demonstrated limited success. Here we report on the differentiation of human pluripotent cells into ureteric-bud-committed renal progenitor-like cells. The generated cells demonstrated rapid and specific expression of renal progenitor markers on 4-day exposure to defined media conditions. Further maturation into ureteric bud structures was accomplished on establishment of a three-dimensional culture system in which differentiated human cells assembled and integrated alongside murine cells for the formation of chimeric ureteric buds. Altogether, our results provide a new platform for the study of kidney diseases and lineage commitment, and open new avenues for the future application of regenerative strategies in the clinic.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Differentiation of human PSCs towards intermediate mesoderm-like cells.
Figure 2: Differentiation of human PSCs into intermediate mesoderm ureteric bud progenitor-like cells.
Figure 3: Maturation and integration of differentiated human PSCs into ureteric bud on 3D organ culture.
Figure 4: Differentiated human PSCs demonstrate a ureteric-bud-committed fate.
Figure 5: Differentiation and integration of polycystic kidney disease patient iPSCs into ureteric bud structures in 3D organ cultures.

References

  1. Robinton, D. A. & Daley, G. Q. The promise of induced pluripotent stem cells in research and therapy. Nature 481, 295–305 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 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 

  3. Lian, X. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions. Nat. Protoc. 8, 162–175 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Osakada, F., Ikeda, H., Sasai, Y. & Takahashi, M. Stepwise differentiation of pluripotent stem cells into retinal cells. Nat. Protoc. 4, 811–824 (2009).

    Article  CAS  PubMed  Google Scholar 

  5. Wong, A. P. et al. Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat. Biotechnol. 30, 876–882 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Eiraku, M. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Dressler, G. R. The cellular basis of kidney development. Annu. Rev. Cell Dev. Biol. 22, 509–529 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Costantini, F. & Kopan, R. Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development. Dev. Cell 18, 698–712 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Little, M. H. & McMahon, A. P. Mammalian kidney development: principles, progress, and projections. Cold Spring Harb Perspect Biol. 4, a008300 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Bouchard, M., Souabni, A., Mandler, M., Neubuser, A. & Busslinger, M. Nephric lineage specification by Pax2 and Pax8. Genes Dev. 16, 2958–2970 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Carroll, T. J. & Vize, P. D. Synergism between Pax-8 and lim-1 in embryonic kidney development. Dev. Biol. 214, 46–59 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Grote, D., Souabni, A., Busslinger, M. & Bouchard, M. Pax 2/8-regulated Gata 3 expression is necessary for morphogenesis and guidance of the nephric duct in the developing kidney. Development 133, 53–61 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Boyle, S. et al. Fate mapping using Cited1-CreERT2 mice demonstrates that the cap mesenchyme contains self-renewing progenitor cells and gives rise exclusively to nephronic epithelia. Dev. Biol. 313, 234–245 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. 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 

  16. Batchelder, C. A., Lee, C. C., Matsell, D. G., Yoder, M. C. & Tarantal, A. F. Renal ontogeny in the rhesus monkey (Macaca mulatta) and directed differentiation of human embryonic stem cells towards kidney precursors. Differentiation 78, 45–56 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  19. Mae, S. et al. Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells. Nature Commun. 4, 1367 (2013).

    Article  Google Scholar 

  20. 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 

  21. Song, B. et al. The directed differentiation of human iPS cells into kidney podocytes. PLoS ONE 7, e46453 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Vigneau, C. 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).

    Article  CAS  PubMed  Google Scholar 

  23. Hendry, C. E. et al. Direct transcriptional reprogramming of adult cells to embryonic nephron progenitors. J. Am. Soc. Nephrol. 24, 1424–1434 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Okita, K. et al. A more efficient method to generate integration-free human iPS cells. Nature Methods 8, 409–412 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Hendry, C., Rumballe, B., Moritz, K. & Little, M. H. Defining and redefining the nephron progenitor population. Pediatr. Nephrol. 26, 1395–1406 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Kuure, S. et al. Crosstalk between Jagged1 and GDNF/Ret/GFRalpha1 signalling regulates ureteric budding and branching. Mech. Dev. 122, 765–780 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Shakya, R., Watanabe, T. & Costantini, F. The role of GDNF/Ret signaling in ureteric bud cell fate and branching morphogenesis. Dev. Cell 8, 65–74 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Davies, J. A., Unbekandt, M., Ineson, J., Lusis, M. & Little, M. H. Dissociation of embryonic kidney followed by re-aggregation as a method for chimeric analysis. Methods Mol. Biol. 886, 135–146 (2012).

    Article  CAS  PubMed  Google Scholar 

  29. Moretti, A. et al. Patient-specific induced pluripotent stem-cell models for long-QT syndrome. New Engl. J. Med. 363, 1397–1409 (2010).

    Article  CAS  PubMed  Google Scholar 

  30. Itzhaki, I. et al. Modelling the long QT syndrome with induced pluripotent stem cells. Nature 471, 225–229 (2011).

    Article  CAS  PubMed  Google Scholar 

  31. Nagao, S., Kugita, M., Yoshihara, D. & Yamaguchi, T. Animal models for human polycystic kidney disease. Exp. Anim. 61, 477–488 (2012).

    Article  CAS  PubMed  Google Scholar 

  32. Nagao, S. et al. Polycystic kidney disease in Han:SPRD Cy rats is associated with elevated expression and mislocalization of SamCystin. Am. J. Physiol. Renal. Physiol. 299, F1078–F1086 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ren, X. et al. Differentiation of murine embryonic stem cells toward renal lineages by conditioned medium from ureteric bud cells in vitro. Acta Biochim. Biophys. Sin. 42, 464–471 (2010).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Schwarz for administrative support. We thank J. Kasuboski from Waitt Advanced Biophotonics Core at Salk for help with imaging processing. We thank B. C. Lu for his suggestions regarding kidney dissection and organ culture. Y.X. and K.S. were partially supported by the California Institute for Regenerative Medicine. 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 Fundacion Cellex, the G. Harold and L. Y. Mathers Charitable Foundation, The Leona M. and Harry B. Helmsley Charitable Trust, IPSEN Foundation, Fundació La Marató de TV3 (121330), CIBER BBN and ISCIII-TERCEL-MINECO.

Author information

Authors and Affiliations

Authors

Contributions

Y.X., I.S-M., E.N. and J.C.I.B. designed all experiments. Y.X., I.S-M., E.N. and J.C.I.B. wrote the manuscript. Y.X., I.S-M., T.G. and E.N. performed and analysed all experiments. D.O., I.D., C.R.E. and Y.X. performed in vivo experiments. N.M. was responsible for cell culture and maintenance of the patient samples related to this work. K.S. and M-Z.W. provided reagents. J.M.C. contributed to the overall design of the project.

Corresponding author

Correspondence to Juan Carlos Izpisua Belmonte.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 iPS cells derived by non-integrative approaches demonstrated the typical hallmarks of pluripotency.

a) Immunofluorescence analysis demonstrating expression of pluripotency markers. b) Karyotype of iPSCs (Fibro-Epi). (c) mRNA fold change of pluripotency markers and different lineage markers during EB differentiation of iPSC/ESCs. (d) Copy numbers of episomal vectors in iPSCs (Fibro-Epi), human ESCs and fibroblasts infected with episomal vectors. Data are represented as mean +/− SD. *p<0.05, (c,d) n = 6 (3 dishes-containing cells per experiment x 2 independent experiments). Scale bars: 100 μm (a).

Supplementary Figure 2 Differentiation of hPSCs into intermediate mesodermlike cells does not induce other lineages.

a,b) mRNA fold change of endodermal and ectodermal-related genes during the course of differentiation of human iPSCs (a) and ESCs (b). Data are represented as mean +/− SD. *p<0.05, n = 9 (3 dishes-containing cells per experiment×3 independent experiments).

Supplementary Figure 3 Differentiation of hPSCs into intermediate mesoderm UB progenitor-like cells.

a) Immunofluorescence analysis demonstrating specificity of the Human Nuclear antigen antibody used in the presence of pure murine cultures. b,c) Immunofluorescence analysis demonstrating specificity of the in vitro differentiation protocol of human PSCs. Non-renal epithelial cells of human origin failed to induce any chimeric UB-like structure. n = >3. Scale bars: 50 μm (a,b,c).

Supplementary Table 1 List of primers used for real-time PCR experiments.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Xia, Y., Nivet, E., Sancho-Martinez, I. et al. Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells. Nat Cell Biol 15, 1507–1515 (2013). https://doi.org/10.1038/ncb2872

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2872

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing