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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MATLAB codes can be requested from the corresponding author.
RNA-Seq data are publicly available in Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/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.
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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.
The authors declare no competing interests.
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Supplementary Figures 1–17, Supplementary Table 6, Supplementary Video Legends 1–3
Supplementary information related to RNA-seq values across samples at the indicated time frames for Keygenes analysis.
Supplementary information related to normalized RNA-seq values across samples at the indicated time frames for clustering analysis after correction for batches effect.
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 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 information related to the list of primary antibodies used in immunocytochemistry and immunohistochemistry.
A summary of statistics and reproducibility information.
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.
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.
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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Garreta, E., Prado, P., Tarantino, C. et al. Fine tuning the extracellular environment accelerates the derivation of kidney organoids from human pluripotent stem cells. Nat. Mater. 18, 397–405 (2019). https://doi.org/10.1038/s41563-019-0287-6
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