Letter | Published:

Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis

Nature volume 526, pages 564568 (22 October 2015) | Download Citation

  • A Corrigendum to this article was published on 27 April 2016

Abstract

The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells1. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.

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Gene Expression Omnibus

Data deposits

The RNA-seq data have been deposited in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE70101.

References

  1. 1.

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

  2. 2.

    & Patterning of the avian intermediate mesoderm by lateral plate and axial tissues. Dev. Biol. 253, 109–124 (2003)

  3. 3.

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

  4. 4.

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

  5. 5.

    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)

  6. 6.

    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)

  7. 7.

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

  8. 8.

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

  9. 9.

    Retinoic acid synthesis and signaling during early organogenesis. Cell 134, 921–931 (2008)

  10. 10.

    et al. The retinoic acid-inactivating enzyme CYP26 is essential for establishing an uneven distribution of retinoic acid along the anterio-posterior axis within the mouse embryo. Genes Dev. 15, 213–225 (2001)

  11. 11.

    et al. The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures. Genes Dev. 15, 226–240 (2001)

  12. 12.

    , , , & 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)

  13. 13.

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

  14. 14.

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

  15. 15.

    et al. KeyGenes, a tool to probe tissue differentiation using a human fetal transcriptional atlas. Stem Cell Reports 4, 1112–1124 (2015)

  16. 16.

    , , & 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)

  17. 17.

    et al. Endothelial progenitors exist within the kidney and lung mesenchyme. PLoS One 8, e65993 (2013)

  18. 18.

    , , , & Defining the molecular character of the developing and adult kidney podocyte. PLoS One 6, e24640 (2011)

  19. 19.

    et al. Identification of a multipotent self-renewing stromal progenitor population during mammalian kidney organogenesis. Stem Cell Reports 3, 650–662 (2014)

  20. 20.

    et al. Localization of PDGF alpha-receptor in the developing and mature human kidney. Kidney Int. 51, 1140–1150 (1997)

  21. 21.

    , & Effects of oxygen on vascular patterning in Tie1/LacZ metanephric kidneys in vitro. Biochem. Biophys. Res. Commun. 247, 361–366 (1998)

  22. 22.

    et al. Atlas of gene expression in the developing kidney at microanatomic resolution. Dev. Cell 15, 781–791 (2008)

  23. 23.

    et al. Identification of anchor genes during kidney development defines ontological relationships, molecular subcompartments and regulatory pathways. PLoS One 6, e17286 (2011)

  24. 24.

    & Tissue distribution, ontogeny, and hormonal regulation of xenobiotic transporters in mouse kidneys. Drug Metab. Dispos. 37, 2178–2185 (2009)

  25. 25.

    , , , & The role of caspase family protease, caspase-3 on cisplatin-induced apoptosis in cisplatin-resistant A431 cell line. Cancer Chemother. Pharmacol. 46, 241–245 (2000)

  26. 26.

    & Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways. J. Pharmacol. Exp. Ther. 302, 8–17 (2002)

  27. 27.

    et al. Global quantification of tissue dynamics in the developing mouse kidney. Dev. Cell 29, 188–202 (2014)

  28. 28.

    et al. Integration-free induced pluripotent stem cells model genetic and neural developmental features of down syndrome etiology. Stem Cells 31, 467–478 (2013)

  29. 29.

    , , & Generation of kidney organoids from human pluripotent stem cells. Protoc. Exch. (2015)

  30. 30.

    et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013)

  31. 31.

    , & Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014)

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Acknowledgements

This research was supported by National Health and Medical Research Council (NHMRC) of Australia (APP1041277, APP1037320), Australian Research Council (ARC) (SRI110001002, CE140100036), Bontius Stiching and Organovo Inc. M.H.L. and R.G.P. are NHMRC Senior Principal Research Fellows. B.M. is a Rosamond Siemon Postgraduate Scholar. We thank A. Christ and T. Bruxner at the IMB Sequencing Facility for providing NGS service. We also acknowledge the IMB ACRF Imaging Facility and the Australian Microscopy & Microanalysis Research Facility at the Center for Microscopy and Microanalysis at The University of Queensland.

Author information

Affiliations

  1. Murdoch Childrens Research Institute, The Royal Children's Hospital Melbourne, Parkville, Victoria 3052, Australia

    • Minoru Takasato
    • , Pei X. Er
    •  & Melissa H. Little
  2. Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia

    • Minoru Takasato
    • , Han S. Chiu
    • , Barbara Maier
    • , Gregory J. Baillie
    • , Charles Ferguson
    • , Robert G. Parton
    •  & Melissa H. Little
  3. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland 4072, Australia

    • Ernst J. Wolvetang
  4. Department of Anatomy and Embryology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands

    • Matthias S. Roost
    •  & Susana M. Chuva de Sousa Lopes
  5. Department of Paediatrics, The University of Melbourne, Parkville, Victoria 3010, Australia

    • Melissa H. Little

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Contributions

M.T. and M.H.L. planned the project, designed experiments, analysed and interpreted data and wrote the manuscript. M.T. performed experiments. P.X.E. maintained hES/iPS cells. P.X.E. and H.S.C. performed experiments under the supervision of M.T and M.H.L.; B.M. generated organoids for TEM. G.J.B. analysed bioinformatic data. C.F. performed TEM. R.G.P. captured and interpreted TEM images. E.J.W. provided the iPS cell line and advised on general iPS cell quality control. M.S.R. and S.M.C.d.S.L. developed NGS analytical tools and analysed data for RNA-seq profiling.

Competing interests

M.T. and M.H.L. are named inventors on a patent relating to this methodology. Some research funding was provided by Organovo Inc.

Corresponding authors

Correspondence to Minoru Takasato or Melissa H. Little.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    This table contains a list of primer sequences used for qPCR.

  2. 2.

    Supplementary Table 2

    This table shows RNA-seq profiling of organoid differentiation timecourse (day 0, 3, 11, 18). Data presented is normalized read counts including average counts for each timepoint.

  3. 3.

    Supplementary Table 3

    This file contains a table of 85 Classifer genes15 used for the unbiased comparative analysis of RNAseq profiling data generated from trimester 1 (1T) and trimester 2 (2T) human fetal tissues compared to kidney organoids day 0, 3, 11 and 18 after commencement of 3D culture (Figure 2g, Extended Data Figure 6).

Videos

  1. 1.

    Z-stacks within developing kidney organoids at day 11 of culture

    Confocal microscopic Z-stack images from the bottom to the top of kidney organoids after 11 days in 3D culture. GATA3+ECAD+ CD, yellow with green nucleus; ECAD+ DT, yellow; LTL+ PT, red; NPHS1+ glomerulus, green membrane; DAPI, blue. Fields of view: 354.25 μm x 354.25 μm.

  2. 2.

    Z-stacks within developing kidney organoids at day 11 of culture

    Confocal microscopic Z-stack images from the bottom to the top of kidney organoids after 11 days in 3D culture. GATA3+ECAD+ CD, yellow with green nucleus; ECAD+ DT, yellow; LTL+ PT, red; NPHS1+ glomerulus, green membrane; DAPI, blue. Fields of view: 354.25 μm x 354.25 μm.

  3. 3.

    Z-stacks of vascularizing glomeruli within a kidney organoid

    Both videos represent confocal microscopic Z-stack images within a day 18 organoid scanning through vascularizing glomeruli. NPHS1+ podocytes of the glomerulus, green; CD31+ endothelium, pink; DAPI, blue. Fields of view: 212.55 μm x 212.55 μm.

  4. 4.

    Z-stacks of vascularizing glomeruli within a kidney organoid

    Both videos represent confocal microscopic Z-stack images within a day 18 organoid scanning through vascularizing glomeruli. NPHS1+ podocytes of the glomerulus, green; CD31+ endothelium, pink; DAPI, blue. Fields of view: 212.55 μm x 212.55 μm.

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DOI

https://doi.org/10.1038/nature15695

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