Abstract
Our understanding in the inherent properties of human pluripotent stem cells (hPSCs) have made possible the development of differentiation procedures to generate three-dimensional tissue-like cultures, so-called organoids. Here we detail a stepwise methodology to generate kidney organoids from hPSCs. This is achieved through direct differentiation of hPSCs in two-dimensional monolayer culture toward the posterior primitive streak fate, followed by induction of intermediate mesoderm-committed cells, which are further aggregated and cultured in three-dimensions to generate kidney organoids containing segmented nephron-like structures in a process that lasts 20 days. We also provide a concise description on how to assess renal commitment during the time course of kidney organoid generation. This includes the use of flow cytometry and immunocytochemistry analyses for the detection of specific renal differentiation markers.
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Acknowledgments
I.L.S. has received financial support through the “la Caixa” INPhINIT Fellowship Grant for Doctoral studies at Spanish Research Centres of Excellence, “la Caixa” Banking Foundation, Barcelona, Spain. The fellowship code is LCF/BQ/IN17/11620003. E.G. and N.M. were funded by the EFSD/Boehringer Ingelheim European Research Programme in Microvascular Complications of Diabetes. This research has been supported by EIT Health under grant ID 20366 (R2U‐Tox‐Assay) to E.G. and N.M.
This work has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (StG-2014-640525_REGMAMKID to E.G. and N.M.), the Spanish Ministry of Economy and Competitiveness/FEDER (SAF2015-72617-EXP to N.M., SAF2017-89782-R to M.G. and N.M., and RYC-2014-16242 to N.M.), the Generalitat de Catalunya and CERCA program (2017 SGR 1306 to N.M.), la Asociación Española contra el Cáncer (LABAE16006 to N.M.). N.M. is also supported Instituto de Salud Carlos III (Cardiocel and ACE2ORG)). IBEC is the recipient of a Severo Ochoa Award of Excellence from MINECO.
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Selfa, I.L., Gallo, M., Montserrat, N., Garreta, E. (2021). Directed Differentiation of Human Pluripotent Stem Cells for the Generation of High-Order Kidney Organoids. In: Ebrahimkhani, M.R., Hislop, J. (eds) Programmed Morphogenesis. Methods in Molecular Biology, vol 2258. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1174-6_12
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DOI: https://doi.org/10.1007/978-1-0716-1174-6_12
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