Article | Published:

Nephron organoids derived from human pluripotent stem cells model kidney development and injury

Nature Biotechnology volume 33, pages 11931200 (2015) | Download Citation

Abstract

Kidney cells and tissues derived from human pluripotent stem cells (hPSCs) may enable organ regeneration, disease modeling and drug screening. We report an efficient, chemically defined protocol for differentiating hPSCs into multipotent nephron progenitor cells (NPCs) that can form nephron-like structures. By recapitulating metanephric kidney development in vitro, we generate SIX2+SALL1+WT1+PAX2+ NPCs with 90% efficiency within 9 days of differentiation. The NPCs possess the developmental potential of their in vivo counterparts and form PAX8+LHX1+ renal vesicles that self-organize into nephron structures. In both two- and three-dimensional culture, NPCs form kidney organoids containing epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle and distal tubules in an organized, continuous arrangement that resembles the nephron in vivo. We also show that this organoid culture system can be used to study mechanisms of human kidney development and toxicity.

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. Prevalence of chronic kidney disease in the United States. J. Am. Med. Assoc. 298, 2038–2047 (2007).

  2. 2.

    , , & Metanephric mesenchyme contains multipotent stem cells whose fate is restricted after induction. Development 114, 565–572 (1992).

  3. 3.

    et al. Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney. EMBO J. 25, 5214–5228 (2006).

  4. 4.

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

  5. 5.

    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).

  6. 6.

    , , , & Human intrauterine renal growth expressed in absolute number of glomeruli assessed by the disector method and Cavalieri principle. Lab. Invest. 64, 777–784 (1991).

  7. 7.

    et al. Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell 2, 284–291 (2008).

  8. 8.

    , , , & Expression of metanephric nephron-patterning genes in differentiating mesonephric tubules. Dev. Dyn. 240, 1600–1612 (2011).

  9. 9.

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

  10. 10.

    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).

  11. 11.

    , & Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro. Biochem. Biophys. Res. Commun. 390, 1334–1339 (2009).

  12. 12.

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

  13. 13.

    et al. Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells. PLoS One 8, e64843 (2014).

  14. 14.

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

  15. 15.

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

  16. 16.

    , & Directed differentiation of pluripotent stem cells to kidney cells. Semin. Nephrol. 34, 445–461 (2014).

  17. 17.

    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).

  18. 18.

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

  19. 19.

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

  20. 20.

    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).

  21. 21.

    et al. Renal progenitors derived from human iPSCs engraft and restore function in a mouse model of acute kidney injury. Sci. Rep. 5, 8826 (2015).

  22. 22.

    , , & 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).

  23. 23.

    , & Analysis of nephric duct specification in the avian embryo. Development 139, 4143–4151 (2012).

  24. 24.

    et al. BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway. Cell Stem Cell 2, 72–82 (2008).

  25. 25.

    et al. Requirement for Wnt3 in vertebrate axis formation. Nat. Genet. 22, 361–365 (1999).

  26. 26.

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

  27. 27.

    & Developmental regulation of the Hox genes during axial morphogenesis in the mouse. Development 132, 2931–2942 (2005).

  28. 28.

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

  29. 29.

    et al. Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell 144, 439–452 (2011).

  30. 30.

    , , & The forkhead transcription factor Foxf1 is required for differentiation of extra-embryonic and lateral plate mesoderm. Development 128, 155–166 (2001).

  31. 31.

    , & The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86, 599–606 (1996).

  32. 32.

    et al. FGF9 and FGF20 maintain the stemness of nephron progenitors in mice and man. Dev. Cell 22, 1191–1207 (2012).

  33. 33.

    , , , & Pax2 and pax8 regulate branching morphogenesis and nephron differentiation in the developing kidney. J. Am. Soc. Nephrol. 18, 1121–1129 (2007).

  34. 34.

    , & Wnt-4 is a mesenchymal signal for epithelial transformation of metanephric mesenchyme in the developing kidney. Development 125, 4225–4234 (1998).

  35. 35.

    , , & A ureteric bud cell line induces nephrogenesis in two steps by two distinct signals. Am. J. Physiol. 271, F50–F61 (1996).

  36. 36.

    , , , & Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system. Dev. Cell 9, 283–292 (2005).

  37. 37.

    , , , & Laser capture-microarray analysis of Lim1 mutant kidney development. Genesis 45, 432–439 (2007).

  38. 38.

    et al. Six2 and Wnt regulate self-renewal and commitment of nephron progenitors through shared gene regulatory networks. Dev. Cell 23, 637–651 (2012).

  39. 39.

    , , & 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).

  40. 40.

    et al. Crucial roles of Brn1 in distal tubule formation and function in mouse kidney. Development 130, 4751–4759 (2003).

  41. 41.

    et al. Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney. Histochem. Cell Biol. 130, 927–942 (2008).

  42. 42.

    et al. Analysis of early nephron patterning reveals a role for distal RV proliferation in fusion to the ureteric tip via a cap mesenchyme-derived connecting segment. Dev. Biol. 332, 273–286 (2009).

  43. 43.

    et al. Gamma-secretase activity is dispensable for mesenchyme-to-epithelium transition but required for podocyte and proximal tubule formation in developing mouse kidney. Development 130, 5031–5042 (2003).

  44. 44.

    et al. Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron. Development 134, 801–811 (2007).

  45. 45.

    et al. Acute renal failure in critically ill patients: a multinational, multicenter study. J. Am. Med. Assoc. 294, 813–818 (2005).

  46. 46.

    & The relationship between enzymuria and kidney enzyme activities in experimental gentamicin nephrotoxicity. Ren. Fail. 18, 899–909 (1996).

  47. 47.

    et al. Kidney injury molecule-1 outperforms traditional biomarkers of kidney injury in preclinical biomarker qualification studies. Nat. Biotechnol. 28, 478–485 (2010).

  48. 48.

    & Nephron reconstitution from pluripotent stem cells. Kidney Int. 87, 894–900 (2015).

  49. 49.

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

  50. 50.

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

  51. 51.

    et al. Reduced ciliary polycystin-2 in induced pluripotent stem cells from polycystic kidney disease patients with PKD1 mutations. J. Am. Soc. Nephrol. 24, 1571–1586 (2013).

Download references

Acknowledgements

The authors thank V. Bijol for providing electron microscopy images of normal human kidneys, L. Racusen (Johns Hopkins Hospital) for HKC-8, J. Barasch (Columbia University) for a mouse ureteric bud cell line, and A.P. McMahon (University of Southern California) for NIH3T3-Wnt4. This study was supported by US National Institutes of Health (NIH) grants R37 DK039773 and R01 DK072381 (J.V.B.); Grant-in-Aid for JSPS (Japan Society for the Promotion of Science); Postdoctoral Fellowship for Research Abroad (R.M.); American Heart Association grant 11FTF7320023 (A.Q.L.); Harvard Stem Cell Institute (A.Q.L., J.V.B. and M.T.V.); and NIH DK102826 and National Kidney Foundation Young Investigator Grant (B.S.F.).

Author information

Affiliations

  1. Renal Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.

    • Ryuji Morizane
    • , Albert Q Lam
    • , Benjamin S Freedman
    • , Seiji Kishi
    • , M Todd Valerius
    •  & Joseph V Bonventre
  2. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.

    • Ryuji Morizane
    • , Albert Q Lam
    • , Benjamin S Freedman
    • , Seiji Kishi
    • , M Todd Valerius
    •  & Joseph V Bonventre
  3. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.

    • Albert Q Lam
    • , M Todd Valerius
    •  & Joseph V Bonventre

Authors

  1. Search for Ryuji Morizane in:

  2. Search for Albert Q Lam in:

  3. Search for Benjamin S Freedman in:

  4. Search for Seiji Kishi in:

  5. Search for M Todd Valerius in:

  6. Search for Joseph V Bonventre in:

Contributions

R.M. and J.V.B. formulated the strategy for this study. R.M. designed and performed experiments. R.M., A.Q.L. and J.V.B. wrote the manuscript. A.Q.L. and B.S.F. performed nephrotoxicity assays. S.K. performed real-time PCR. M.T.V. and J.V.B. helped to design experiments. All authors helped to interpret the results.

Competing interests

J.V.B. is a co-inventor on KIM-1 patents, which have been licensed by Partners Healthcare to several companies. He has received royalty income from Partners Healthcare. J.V.B. or his family has received income for consulting from companies interested in biomarkers: Sekisui, Millennium, Johnson & Johnson and Novartis.

Corresponding authors

Correspondence to Ryuji Morizane or Joseph V Bonventre.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Figures and Tables

    Supplementary Figures 1–7 and Supplementary Tables 1–3

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt.3392