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.

  • Protocol
  • Published:

Generation of kidney organoids from human pluripotent stem cells

Abstract

The human kidney develops from four progenitor populations—nephron progenitors, ureteric epithelial progenitors, renal interstitial progenitors and endothelial progenitors—resulting in the formation of maximally 2 million nephrons. Until recently, the reported methods differentiated human pluripotent stem cells (hPSCs) into either nephron progenitor or ureteric epithelial progenitor cells, consequently forming only nephrons or collecting ducts, respectively. Here we detail a protocol that simultaneously induces all four progenitors to generate kidney organoids within which segmented nephrons are connected to collecting ducts and surrounded by renal interstitial cells and an endothelial network. As evidence of functional maturity, proximal tubules within organoids display megalin-mediated and cubilin-mediated endocytosis, and they respond to a nephrotoxicant to undergo apoptosis. This protocol consists of 7 d of monolayer culture for intermediate mesoderm induction, followed by 18 d of 3D culture to facilitate self-organizing renogenic events leading to organoid formation. Personnel experienced in culturing hPSCs are required to conduct this protocol.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic diagram of the time line for generating kidney organoids from hPSCs.
Figure 2: Bright-field images of hPSC differentiation to kidney organoids.
Figure 3: Immunological characterization of structures within kidney organoids.

Similar content being viewed by others

References

  1. Tesar, P.J. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 (2007).

    Article  CAS  Google Scholar 

  2. Takasato, M., Maier, B. & Little, M.H. Recreating kidney progenitors from pluripotent cells. Pediatr. Nephrol. 29, 543–552 (2014).

    Article  Google Scholar 

  3. Davis, R.P. et al. Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood 111, 1876–1884 (2008).

    Article  CAS  Google Scholar 

  4. Yang, L. et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524–528 (2008).

    Article  CAS  Google Scholar 

  5. Wang, D., Haviland, D.L., Burns, A.R., Zsigmond, E. & Wetsel, R.A. A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 104, 4449–4454 (2007).

    Article  CAS  Google Scholar 

  6. Pagliuca, F.W. et al. Generation of functional human pancreatic β cells in vitro. Cell 159, 428–439 (2014).

    Article  CAS  Google Scholar 

  7. 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  Google Scholar 

  8. Xia, X. & Zhang, S. Differentiation of neuroepithelia from human embryonic stem cells. Methods Mol. Biol. 549, 51–58 (2009).

    Article  CAS  Google Scholar 

  9. 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  Google Scholar 

  10. Lancaster, M.A. & Knoblich, J.A. Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125 (2014).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  15. Takebe, T. et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Takasato, M., Er, P.X., Chiu, H.S. & Little, M.H. Generation of kidney organoids from human pluripotent stem cells. Protoc. Exch. doi:10.1038/protex.2015.087 (2015).

  18. Iimura, T. & Pourquié, O. Collinear activation of Hoxb genes during gastrulation is linked to mesoderm cell ingression. Nature 442, 568–571 (2006).

    Article  CAS  Google Scholar 

  19. Sweetman, D., Wagstaff, L., Cooper, O., Weijer, C. & MĂĽnsterberg, A. The migration of paraxial and lateral plate mesoderm cells emerging from the late primitive streak is controlled by different Wnt signals. BMC Dev. Biol. 8, 63 (2008).

    Article  Google Scholar 

  20. Saxen, L. Organogenesis of the Kidney (Cambridge University Press, 1987).

  21. Mendelsohn, C. Using mouse models to understand normal and abnormal urogenital tract development. Organogenesis 5, 306–314 (2009).

    Article  Google Scholar 

  22. 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  Google Scholar 

  23. Mugford, J.W., Sipilä, P., McMahon, J.A. & McMahon, A.P. Osr1 expression demarcates a multi-potent population of intermediate mesoderm that undergoes progressive restriction to an Osr1-dependent nephron progenitor compartment within the mammalian kidney. Dev. Biol. 324, 88–98 (2008).

    Article  CAS  Google Scholar 

  24. Gadue, P., Huber, T.L., Paddison, P.J. & Keller, G.M. Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells. Proc. Natl. Acad. Sci. 103, 16806–16811 (2006).

    Article  CAS  Google Scholar 

  25. Tam, P.P.L. & Loebel, D.A.F. Gene function in mouse embryogenesis: get set for gastrulation. Nat. Rev. Genet. 8, 368–381 (2007).

    Article  CAS  Google Scholar 

  26. Sumi, T., Tsuneyoshi, N., Nakatsuji, N. & Suemori, H. Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/β-catenin, Activin/Nodal and BMP signaling. Development 135, 2969–2979 (2008).

    Article  CAS  Google Scholar 

  27. James, R.G. & Schultheiss, T.M. Bmp signaling promotes intermediate mesoderm gene expression in a dose-dependent, cell-autonomous and translation-dependent manner. Dev. Biol. 288, 113–125 (2005).

    Article  CAS  Google Scholar 

  28. Colvin, J.S., Feldman, B., Nadeau, J.H., Goldfarb, M. & Ornitz, D.M. Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene. Dev. Dyn. 216, 72–88 (1999).

    Article  CAS  Google Scholar 

  29. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  31. 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  Google Scholar 

  32. Kang, M. & Han, Y. Differentiation of human pluripotent stem cells into nephron progenitor cells in a serum and feeder free system. PLoS One 9, e94888 (2014).

    Article  Google Scholar 

  33. 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  Google Scholar 

  34. 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  Google Scholar 

  35. Sharmin, S. et al. Human induced pluripotent stem cell-derived podocytes mature into vascularized glomeruli upon experimental transplantation. J. Am. Soc. Nephrol. 27, 1778–1791 (2016).

    Article  CAS  Google Scholar 

  36. 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  Google Scholar 

  37. 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  Google Scholar 

  38. Takasato, M. & Little, M.H. The origin of the mammalian kidney: implications for recreating the kidney in vitro. Development 142, 1937–1947 (2015).

    Article  CAS  Google Scholar 

  39. Ciarimboli, G. et al. Cisplatin nephrotoxicity is critically mediated via the human organic cation transporter 2. Am. J. Pathol. 167, 1477–1484 (2005).

    Article  CAS  Google Scholar 

  40. Ishida, S., Lee, J., Thiele, D.J. & Herskowitz, I. Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals. Proc. Natl. Acad. Sci. USA 99, 14298–14302 (2002).

    Article  CAS  Google Scholar 

  41. Hildebrandt, F. Genetic kidney diseases. Lancet 375, 1287–1295 (2010).

    Article  CAS  Google Scholar 

  42. Cho, E.A. et al. Differential expression and function of cadherin-6 during renal epithelium development. Development 125, 803–812 (1998).

    CAS  PubMed  Google Scholar 

  43. Combes, A.N., Davies, J.A. & Little, M.H. Cell-cell interactions driving kidney morphogenesis. Curr. Top. Dev. Biol. 112, 467–508 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  45. Nouwen, E.J., Dauwe, S., van der Biest, I. & De Broe, M.E. Stage- and segment-specific expression of cell-adhesion molecules N-CAM, A-CAM, and L-CAM in the kidney. Kidney Int. 44, 147–158 (1993).

    Article  CAS  Google Scholar 

  46. Briggs, J.A. et al. Integration-free induced pluripotent stem cells model genetic and neural developmental features of down syndrome etiology. Stem Cells 31, 467–78 (2013).

    Article  CAS  Google Scholar 

  47. Xu, J. et al. Eya1 interacts with Six2 and Myc to regulate expansion of the nephron progenitor pool during nephrogenesis. Dev. Cell 31, 434–447 (2014).

    Article  CAS  Google Scholar 

  48. Mugford, J.W., Sipilä, 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  Google Scholar 

  49. Osafune, K. et al. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat. Biotechnol. 26, 313–315 (2008).

    Article  CAS  Google Scholar 

  50. Ng, E.S., Davis, R., Stanley, E.G. & Elefanty, A.G. A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies. Nat. Protoc. 3, 768–776 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to F. Froemling for experimental support and to E.J. Wolvetang (The University of Queensland, St Lucia, Australia) for providing the CRL1502 clone C32 iPSC line. This research was supported by the National Health and Medical Research Council (NHMRC) of Australia (APP1041277), the Australian Research Council (Stem Cells Australia, SRI110001002) and Organovo. M.H.L. is an NHMRC senior principal research fellow. We also acknowledge the use of the Australian Cancer Research Foundation's (ACRF's) Cancer Biology Imaging Facility at The University of Queensland.

Author information

Authors and Affiliations

Authors

Contributions

M.T. and M.H.L. wrote the manuscript. M.T., P.X.E. and H.S.C. performed the experiments.

Corresponding authors

Correspondence to Minoru Takasato or Melissa H Little.

Ethics declarations

Competing interests

M.T. and M.H.L. are named inventors on a patent relating to this methodology.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Takasato, M., Er, P., Chiu, H. et al. Generation of kidney organoids from human pluripotent stem cells. Nat Protoc 11, 1681–1692 (2016). https://doi.org/10.1038/nprot.2016.098

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.098

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.

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