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Fine tuning the extracellular environment accelerates the derivation of kidney organoids from human pluripotent stem cells


The generation of organoids is one of the biggest scientific advances in regenerative medicine. Here, by lengthening the time that human pluripotent stem cells (hPSCs) were exposed to a three-dimensional microenvironment, and by applying defined renal inductive signals, we generated kidney organoids that transcriptomically matched second-trimester human fetal kidneys. We validated these results using ex vivo and in vitro assays that model renal development. Furthermore, we developed a transplantation method that utilizes the chick chorioallantoic membrane. This approach created a soft in vivo microenvironment that promoted the growth and differentiation of implanted kidney organoids, as well as providing a vascular component. The stiffness of the in ovo chorioallantoic membrane microenvironment was recapitulated in vitro by fabricating compliant hydrogels. These biomaterials promoted the efficient generation of renal vesicles and nephron structures, demonstrating that a soft environment accelerates the differentiation of hPSC-derived kidney organoids.

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Code availability

MATLAB codes can be requested from the corresponding author.

Data availability

RNA-Seq data are publicly available in Gene Expression Omnibus (GEO, under the accession numbers GSE108349, GSE108350 and GSE108351. All remaining datasets supporting the findings described here are available within the article and its supplementary information files. Additionally, data are available from the corresponding author upon reasonable request.


  1. 1.

    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).

  2. 2.

    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).

  3. 3.

    Morizane, R. et al. Nephron organoids derived from human pluripotent stem cells model kidney development and injury. Nat. Biotechnol. 33, 1193–1200 (2015).

  4. 4.

    Freedman, B. S. et al. Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids. Nat. Commun. 6, 8715 (2015).

  5. 5.

    Toyohara, T. et al. Cell therapy using human induced pluripotent stem cell-derived tenal progenitors ameliorates acute kidney injury in mice. Stem Cells Transl. Med. 4, 980–992 (2015).

  6. 6.

    Imberti, B. 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).

  7. 7.

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

  8. 8.

    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).

  9. 9.

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

  10. 10.

    Taguchi, A., & Nishinakamura, R. Higher-order kidney organogenesis from pluripotent stem cells. Cell Stem Cell 21, 730–746 (2017).

  11. 11.

    Musah, S. et al. Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip.Nat. Biomed. Eng. 1, 0069 (2017).

  12. 12.

    Cruz, N. M. et al. Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease. Nat. Mater. 16, 1112–1119 (2017).

  13. 13.

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

  14. 14.

    Lindström, N. O. et al. Conserved and divergent features of human and mouse kidney organogenesis. J. Am. Soc. Nephrol. 29(3), 785–805 (2018).

  15. 15.

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

  16. 16.

    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).

  17. 17.

    Lindström, N. O. et al. Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron. eLife 3, e04000 (2014).

  18. 18.

    De Bock, K. et al. Role of PFKFB3-driven glycolysis in vessel sprouting. Cell 154, 651–663 (2013).

  19. 19.

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

  20. 20.

    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).

  21. 21.

    Van den Berg, C. W. et al. Renal subcapsular transplantation of PSC-derived kidney organoids induces neo-vasculogenesis and significant glomerular and tubular maturation in vivo. Stem Cell Rep. 10, 751–765 (2018).

  22. 22.

    Ribatti, D. Chick embryo chorioallantoic membrane as a useful tool to study angiogenesis. Int. Rev. Cell Mol. Biol. 270, 181–224 (2008).

  23. 23.

    Cimpean, A. M., Ribatti, D. & Raica, M. The chick embryo chorioallantoic membrane as a model to study tumor metastasis. Angiogenesis 11, 311–319 (2008).

  24. 24.

    Baiguera, S., Macchiarini, P. & Ribatti, D. Chorioallantoic membrane for in vivo investigation of tissue-engineered construct biocompatibility. J. Biomed. Mater. Res. B 100, 1425–1434 (2012).

  25. 25.

    Vining, K. H. & Mooney, D. J. Mechanical forces direct stem cell behaviour in development and regeneration. Nat. Rev. Mol. Cell Biol. 18, 728–742 (2017).

  26. 26.

    Przybyla, L., Lakins, J. N. & Weaver, V. M. Tissue mechanics orchestrate Wnt-dependent human embryonic stem cell differentiation. Cell Stem Cell 19, 462–475 (2016).

  27. 27.

    Ahmed, K. et al. Global chromatin architecture reflects pluripotency and lineage commitment in the early mouse embryo. PLoS One 5, e10531 (2010).

  28. 28.

    Theunissen, T. W. & Jaenisch, R. Mechanisms of gene regulation in human embryos and pluripotent stem cells. Development 144, 4496–4509 (2017).

  29. 29.

    Andrews, S. FastQC: a quality control tool for high throughput sequence data. (2010).

  30. 30.

    Jiang, H., & Lei, R. & Ding, S. W. & Zhu, S. Skewer: A fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinform. 15, 182 (2014).

  31. 31.

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

  32. 32.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

  33. 33.

    Leek, J. T. Svaseq: removing batch effects and other unwanted noise from sequencing data. Nucl. Acids Res. 42, e161 (2014).

  34. 34.

    Kue, C. S., Tan, K. Y., Lam, M. L. & Lee, H. B. Chick embryo chorioallantoic membrane (CAM): an alternative predictive model in acute toxicological studies for anti-cancer drugs. Exp. Anim. 64, 129–138 (2015).

  35. 35.

    Lee, D., Rahman, M. M., Zhou, Y. & Ryu, S. Three-dimensional confocal microscopy indentation method for hydrogel elasticity measurement. Langmuir 31, 9684–9693 (2015).

  36. 36.

    Yeung, T. et al. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil. Cytoskelet. 60, 24–34 (2005).

  37. 37.

    Przybyla, L., Lakins, J. N., Sunyer, R., Trepat, X. & Weaver, V. M. Monitoring developmental force distributions in reconstituted embryonic epithelia. Methods 94, 101–113 (2016).

  38. 38.

    Montserrat, N. et al. Generation of induced pluripotent stem cells from human renal proximal tubular cells with only two transcription factors, OCT4 and SOX2. J. Biol. Chem. 287, 24131–24138 (2012).

  39. 39.

    O’Rahilly, R. & Müller, F. Developmental stages in human embryos: revised and new measurements. Cells Tissues Organs 192, 73–84 (2010).

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We are grateful to members of the N. Montserrat laboratory for insightful discussions and critical reading of the manuscript. We thank D. O’Keefe and M. Schwarz for administrative help, L. Bardia, A. Lladó and J. Colombelli from the Advanced Digital Microscopy facility at the Institute for Research in Biomedicine for assistance in confocal microscopy imaging and the Electron Cryo-Microscopy Unit at the Scientific and Technological Centers of the University of Barcelona for their technical assistance. We would particularly like to acknowledge the patients and the Fetal Tissue Bank of Vall d’Hebron University Hospital Biobank (PT13/0010/0021), part of the Spanish National Biobanks Network, for its collaboration. This work has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (StG-2014-640525_REGMAMKID to E.G., P.P., C.T. and N.M. and CoG-616480 to X.T.), the European Commission (project H2020-FETPROACT-01-2016-731957 to X.T. and P.R.-C.), the Spanish Ministry of Economy and Competitiveness/FEDER (BFU2016-77498-P to L.F. and E.M., BFU2015-65074 to X.T., BFU2016-79916-P to P.R.-C., SAF2015-72617-EXP to N.M., SAF2017-89782-R to N.M. and RYC-2014-16242 to N.M.), the Generalitat de Catalunya and CERCA programme (2014-SGR-927 to X.T. and 2017 SGR 1306 to N.M.), Asociación Española contra el Cáncer (AECC CI2016 to L.F. and E.M., LABAE16006 to N.M.). R.O. is supported by an FI fellowship (Generalitat de Catalunya). P.R.-C. is also supported by Obra Social La Caixa. J.C.I.B. is supported by the G. Harold and Leila Y. Mathers Charitable Foundation, the Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002), the Moxie Foundation, the National Institutes of Health (5R21AG055938), the Universidad Católica San Antonio de Murcia and Fundación Dr. Pedro Guillén. C.H.P. is supported by the Bioengineering Excellence of Scientific Training project, cofunded from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 712754 and from the Spanish Ministry of Economy and Competitiveness under the Severo Ochoa grant SEV-2014-0425 (2015–2019). N.M. is also supported by CardioCel (TerCel, Instituto de Salud Carlos III). IBEC is the recipient of a Severo Ochoa Award of Excellence from MINECO.

Author information

E.G. and N.M. conceived and designed the experiments. E.G., P.P., C.T. and C.H.P. performed the experiments. E.G., P.P., C.T. and R.O. characterized the cell lines and contributed to the protocol design. A.G.-N. and C.H.P. carried out the Seahorse analysis. L.C. contributed to the transcriptomic analysis. E.G., P.P., C.T., R.O., L.F., E.M., D.Z., X.T., P.R.-C., J.M.C., J.C.I.B., C.H.P. and N.M. contributed to data interpretation. E.G. and N.M. wrote the manuscript. All authors commented on the manuscript and contributed to it. N.M. oversaw the project.

Correspondence to Nuria Montserrat.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figures 1–17, Supplementary Table 6, Supplementary Video Legends 1–3

Reporting Summary

Supplementary Table 1

Supplementary information related to RNA-seq values across samples at the indicated time frames for Keygenes analysis.

Supplementary Table 2

Supplementary information related to normalized RNA-seq values across samples at the indicated time frames for clustering analysis after correction for batches effect.

Supplementary Table 3

Supplementary information related to genes found to be significantly down or upregulated in hESCs grown for 24 h (day –4) on soft (1 kPa) compared to rigid (60 kPa) PA hydrogels.

Supplementary Table 4

Supplementary information related to genes found to be significantly down or upregulated in hESCs differentiated on soft (1 kPa) compared to rigid (60 kPa) PA hydrogels at day –3 and day –2 of the differentiation process.

Supplementary Table 5

Supplementary information related to the list of primary antibodies used in immunocytochemistry and immunohistochemistry.

Supplementary Table 7

A summary of statistics and reproducibility information.

Supplementary Video 1

Evidence for the circulation of chick blood within an implanted kidney organoid at day 3 of the implantation period. Video recording was performed in n = 3 biologically independent implanted kidney organoids with similar results.

Supplementary Video 2

Evidence for the circulation of chick blood within an implanted kidney organoid at day 5 of the implantation period. White arrow indicates a blood vessel going through the kidney organoid. Video recording was performed in n = 2 biologically independent implanted kidney organoids with similar results.

Supplementary Video 3

Evidence for the circulation of chick blood within an implanted kidney organoid at day 5 of the implantation period after intravital injection of dextran–FITC into the CAM vasculature. Intravital injection of dextran–FITC was performed in n = 3 biologically independent implanted kidney organoids with similar results.

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Further reading

Fig. 1: Efficient generation of kidney organoids in 3D culture.
Fig. 2: Kidney organoids model human kidney organogenesis in vitro.
Fig. 3: In vivo vascularization of kidney organoids using chick CAM.
Fig. 4: Soft hydrogels accelerate the differentiation of kidney organoids.