Abstract
Kidney organoids derived from human pluripotent stem cells (hPSCs) are now being used as models of renal disease and nephrotoxicity screening. However, the proximal tubules (PTs), which are responsible for most kidney reabsorption functions, remain immature in kidney organoids with limited expression of critical transporters essential for nephron functionality. Here, we describe a protocol for improved specification of nephron progenitors from hPSCs that results in kidney organoids with elongated proximalized nephrons displaying improved PT maturity compared with those generated using standard kidney organoid protocols. We also describe a methodology for assessing the functionality of the PTs within the organoids and visualizing maturation markers via immunofluorescence. Using these assays, PT-enhanced organoids display increased expression of a range of critical transporters, translating to improved functionality measured by substrate uptake and transport. This protocol consists of an extended (13 d) monolayer differentiation phase, during which time hPSCs are exposed to nephron progenitor maintenance media (CDBLY2), better emulating human metanephric progenitor specification in vivo. Following nephron progenitor specification, the cells are aggregated and cultured as a three-dimensional micromass on an air–liquid interface to facilitate further differentiation and segmentation into proximalized nephrons. Experience in culturing hPSCs is required to conduct this protocol and expertise in kidney organoid generation is advantageous.
Key points
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This protocol generates kidney organoids with elongated proximalized nephrons displaying improved proximal tubule maturity compared with those generated using standard kidney organoid protocols.
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Methods for assessing the functionality of the proximal tubules within the organoids and visualizing maturation markers via immunofluorescence are also described.
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Data availability
The original research relating to this protocol can be accessed in a previous publication6, the manuscript website (https://kidneyregeneration.github.io/Vanslambrouck2022/), and via the Github repository (https://github.com/KidneyRegeneration/Vanslambrouck2022).
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Acknowledgements
This research was supported by the National Health and Medical Research Council (GNT1156440 (M.H.L., J.M.V., K.S.T.)), the National Institutes of Health (UH3DK107344; M.H.L., J.M.V.), the Victorian State Government Department of Jobs Precincts and Regions (DJPR) through the Victorian COVID-19 Research Fund (S.M.)), and the Novo Nordisk Foundation Centre for Stem Cell Research (supported by Novo Nordisk Foundation grant NNF21CC0073729 (M.H.L., J.M.V.)). M.H.L. is a National Health and Medical Research Senior Principal Research Fellow (GNT1136085). We acknowledge the Stafford Fox Medical Research Foundation Murdoch Children’s Research Institute (MCRI) iPSC Derivation & Gene Editing Facility for the generation of all pluripotent stem cell lines. We thank M. Burton and the Murdoch Children’s Research Institute Microscopy Core, as well as S. Howden and the MCRI iPSC Derivation and Gene Editing Core.
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J.M.V. and M.H.L. contributed to the experimental design and planning. J.M.V., K.S.T. and S.M. performed the experiments, developed the reagents and recorded the methodology. J.M.V., K.S.T. and S.M. contributed to the preparation of the manuscript. J.M.V. and M.H.L. wrote the manuscript.
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Vanslambrouck, J. M. et al. Nat. Commun. 13, 5943 (2022): https://doi.org/10.1038/s41467-022-33623-z
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Vanslambrouck, J.M., Tan, K.S., Mah, S. et al. Generation of proximal tubule-enhanced kidney organoids from human pluripotent stem cells. Nat Protoc 18, 3229–3252 (2023). https://doi.org/10.1038/s41596-023-00880-1
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DOI: https://doi.org/10.1038/s41596-023-00880-1
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