Human pluripotent stem cell (hPSC)-derived kidney organoids reproduce early nephrogenesis but remain transcriptionally and structurally fetal-like, produce a finite wave of nephrons, and their functional properties have never been quantitatively benchmarked against native human kidney tissue. A 2019 reporter-toolbox paper in the Journal of the American Society of Nephrology (JASN) posed a composite agenda for the field: use fluorescent reporter iPSC lines to drive and validate compartment maturation, establish self-renewing nephron progenitor niches, generate contiguous collecting duct epithelium connected to distal nephrons, and control off-target cells. Later literature has substantially resolved several strands: human organoids with an integrated collecting system now exist (2025), long-term expandable nephron progenitor cells were established (2024), and off-target states have been comprehensively classified (2026). Drive organoid tissue from its fetal-like state to adult-like functional maturity — including sustained nephrogenesis from a maintained progenitor niche — and quantify the recovered function (transporter expression, kidney-specific physiological responses) of defined compartments against native human kidney tissue, after in vitro maturation or in vivo engraftment.

Background

The human kidney contains roughly one million nephrons, each a segmented epithelial tube (glomerulus, proximal tubule, loop of Henle, distal nephron) that drains into a branched collecting duct system. During embryogenesis, nephrons arise from nephron progenitors — a self-renewing pool of SIX2-positive cap mesenchyme cells — whose recruitment into new nephrons is induced by the ureteric bud, the branching epithelial tree that becomes the collecting ducts and ureter. Human induced pluripotent stem cells (iPSCs), reprogrammed adult cells with embryonic-like potential, can be directed through intermediate mesoderm into these kidney lineages, yielding kidney organoids: self-organizing three-dimensional cultures that form nephron-like structures. A widely used validation strategy is fluorescent reporter lines, generated by CRISPR/Cas9 knock-in of a fluorescent protein at an endogenous locus (for example SIX2 for nephron progenitors, MAFB or NPHS1 for podocytes, LRP2 or HNF4A for proximal tubule, GATA3 for ureteric epithelium), so that a given compartment can be visualized, filmed, and purified by fluorescence-activated cell sorting (FACS). Vanslambrouck et al. (Journal of the American Society of Nephrology, 2019) generated and validated ten such reporter iPSC lines and used them for time-lapse imaging, lineage tracing, and transplantation into immunodeficient mice. Their assessment, echoing the field at the time, was that organoid profiles resemble the early fetal kidney; nephrogenesis in vitro ceases rather than continuing from a maintained progenitor niche; nephron-containing organoids lack a genuine ureteric/collecting duct system contiguous with distal nephrons (a debate then current about whether ureteric bud progenitors and nephron progenitors can arise in one differentiation protocol); nonrenal off-target cells appear; and the functional competence of organoid-derived populations had not been quantified against native kidney. Single-cell transcriptomic work (Wu et al., Cell Stem Cell 2018) had independently documented fetal-like maturity and off-target populations, and Howden et al. (Cell Stem Cell 2021) later confirmed that standard nephron-containing organoids contain distal nephron epithelium but no ureteric epithelium, which can however be induced from the distal nephron by defined culture conditions. Subsequent milestones — organoids with collecting systems (Uchimura et al. 2020), expandable ureteric bud organoids (Zeng et al. 2021), mouse organotypic kidneys with stroma (Tanigawa et al. 2022), long-term expandable induced nephron progenitor cells (Huang et al. 2024), and human nephron–collecting duct integration by distal-tubule-to-ureteric-bud fusion and spatially patterned assembloids (Shi et al. 2025; Huang et al. 2025; Wilson et al. 2025) — have rebuilt the platform, but every one of these studies reports only partial, compartment-level maturation, and field assessments as late as 2026 still list lack of functional maturity as a central limitation.

Problem Statement

Can human pluripotent stem cell-derived kidney organoids be driven from their fetal-like state to functionally mature, adult-like kidney tissue in vitro or after engraftment? The question, posed by Vanslambrouck et al. (2019) and still open in the 2026 literature, has three linked components that together define a functionally mature organoid. (1) Sustained nephrogenesis: establish culture or assembly conditions in which a self-renewing, induction-competent nephron progenitor population is maintained within the organoid, so that new nephron formation continues beyond the single finite wave characteristic of standard protocols, supporting long-term organoid growth over extended culture. Expandable, self-renewing nephron progenitor sources are already available (Huang et al. 2024); what remains open in this component is their maintenance as a self-renewing, induction-competent niche inside the maturing organoid, not the establishment of the progenitor source itself. (2) Compartment maturation: drive the individual epithelial compartments, including the now-achievable integrated nephron-collecting duct system, from their early-fetal state toward adult-like maturity, validated with compartment-resolved markers and functional readouts (for example using reporter-based isolation of defined populations). (3) Quantitative functional benchmarking: measure, on defined differentiated compartments after in vitro maturation or in vivo engraftment, the recovery of adult-like functional properties such as expression and polarized activity of transporters, protein reabsorption, organic ion transport, and hormone-dependent water and electrolyte handling, and compare these quantitatively against native adult human kidney tissue matched to the compartment assayed (nephron segment or collecting duct region) and, where relevant, to donor variables such as age and kidney region. Because native adult kidney contains no ongoing nephron progenitor niche, the benchmarking in (3) applies to differentiated epithelial compartments, while component (1) is assessed by continued nephron formation from the maintained progenitor population.

References

  1. Vanslambrouck JM, Wilson SB, Tan KS, Soo JY-C, Scurr M, Spijker HS, Starks LT, Neilson A, Cui X, Jain S, Little MH, Howden SE. A Toolbox to Characterize Human Induced Pluripotent Stem Cell-Derived Kidney Cell Types and Organoids. Journal of the American Society of Nephrology 30(10):1811-1823 (2019). DOI: 10.1681/ASN.2019030303. https://doi.org/10.1681/ASN.2019030303
  2. Wu H, Uchimura K, Donnelly EL, Kirita Y, Morris SA, Humphreys BD. Comparative Analysis and Refinement of Human PSC-Derived Kidney Organoid Differentiation with Single-Cell Transcriptomics. Cell Stem Cell 23(6):869-881.e8 (2018). DOI: 10.1016/j.stem.2018.10.010. https://doi.org/10.1016/j.stem.2018.10.010
  3. Howden SE, Wilson SB, Groenewegen E, Starks L, Forbes TA, Tan KS, Vanslambrouck JM, Holloway EM, Chen Y-H, Jain S, Spence JR, Little MH. Plasticity of distal nephron epithelia from human kidney organoids enables the induction of ureteric tip and stalk. Cell Stem Cell 28(4):671-684.e6 (2021). DOI: 10.1016/j.stem.2020.12.001. https://doi.org/10.1016/j.stem.2020.12.001
  4. Uchimura K, Wu H, Yoshimura Y, Humphreys BD. Human Pluripotent Stem Cell-Derived Kidney Organoids with Improved Collecting Duct Maturation and Injury Modeling. Cell Reports 33(11):108514 (2020). DOI: 10.1016/j.celrep.2020.108514. https://doi.org/10.1016/j.celrep.2020.108514
  5. Zeng Z, Huang B, Parvez RK, Li Y, Chen J, Vonk AC, Thornton ME, Patel T, Rutledge EA, Kim AD, Yu J, Grubbs BH, McMahon JA, Pastor-Soler NM, Hallows KR, McMahon AP, Li Z. Generation of patterned kidney organoids that recapitulate the adult kidney collecting duct system from expandable ureteric bud progenitors. Nature Communications 12:3641 (2021). DOI: 10.1038/s41467-021-23911-5. https://doi.org/10.1038/s41467-021-23911-5
  6. Tanigawa S, Tanaka E, Miike K, Ohmori T, Inoue D, Cai C-L, Taguchi A, Kobayashi A, Nishinakamura R. Generation of the organotypic kidney structure by integrating pluripotent stem cell-derived renal stroma. Nature Communications 13:611 (2022). DOI: 10.1038/s41467-022-28226-7. https://doi.org/10.1038/s41467-022-28226-7
  7. Li Z, Araoka T, Wu J, Liao H-K, Li M, Lazo M, Zhou B, Sui Y, Wu M-Z, Tamura I, Xia Y, Beyret E, Matsusaka T, Pastan I, Rodriguez Esteban C, Guillen I, Guillen P, Campistol JM, Izpisua Belmonte JC. 3D Culture Supports Long-Term Expansion of Mouse and Human Nephrogenic Progenitors. Cell Stem Cell 19(4):516-529 (2016). DOI: 10.1016/j.stem.2016.07.016. https://doi.org/10.1016/j.stem.2016.07.016
  8. Huang B, Zeng Z, Kim S, Fausto CC, Koppitch K, Li H, Li Z, Chen X, Guo J, Zhang CC, Ma T, Medina P, Schreiber ME, Xia MW, Vonk AC, Xiang T, Patel T, Li Y, Parvez RK, Der B, Chen JH, Liu Z, Thornton ME, Grubbs BH, Diao Y, Dou Y, Gnedeva K, Ying Q, Pastor-Soler NM, Fei T, Hallows KR, Lindstrom NO, McMahon AP, Li Z. Long-term expandable mouse and human-induced nephron progenitor cells enable kidney organoid maturation and modeling of plasticity and disease. Cell Stem Cell 31(6):921-939.e17 (2024). DOI: 10.1016/j.stem.2024.04.002. https://doi.org/10.1016/j.stem.2024.04.002
  9. Huang B, Medina P, Ma T, Schreiber ME, Li Z. Expansion of human pluripotent stem cell-induced nephron progenitor cells (iNPCs) and the generation of nephron organoids from iNPCs. Nature Protocols 21(3):1167-1191 (2025). DOI: 10.1038/s41596-025-01236-7. https://doi.org/10.1038/s41596-025-01236-7
  10. Shi M, Crouse B, Sundaram N, Pode Shakked N, Thorner K, King NM, Dutta P, Ester L, Zhang W, Govindarajah V, Kopan R, Cebrian C, Mayhew CN, Helmrath MA, Bonventre JV, McCracken KW. Integrating collecting systems in human kidney organoids through fusion of distal nephron to ureteric bud. Cell Stem Cell 32(7):1055-1070.e8 (2025). DOI: 10.1016/j.stem.2025.04.008. https://doi.org/10.1016/j.stem.2025.04.008
  11. Huang B, Medina P, He J, Zeng Z, Kim S, Romo J, Koppitch K, Zhang CC, Gyarmati G, Park Y, Bohovyk R, N'Guetta PEY, Guo J, Ma T, Schreiber ME, Xu C, Pham J, Parvez RK, Su J, Xia MW, Liu Z, Perin L, El-Nachef D, Murry CE, Kumar S, O'Brien L, Zimmerman KA, Ichida J, Lindstrom NO, Pastor-Soler NM, Hallows KR, Staruschenko A, Peti-Peterdi J, Jiang C, McMahon AP, Li Z. Spatially patterned kidney assembloids recapitulate progenitor self-assembly and enable high-fidelity in vivo disease modeling. Cell Stem Cell 32(10):1614-1633.e13 (2025). DOI: 10.1016/j.stem.2025.08.013. https://doi.org/10.1016/j.stem.2025.08.013
  12. Wilson SB, Santos IP, Wildfang L, Imsa K, Little MH. Generation of multi-lineage kidney assembloids with integration between nephrons and a single exiting collecting duct. bioRxiv (2025). DOI: 10.1101/2025.02.27.640561. https://doi.org/10.1101/2025.02.27.640561
  13. Wilson SB, Vanslambrouck JM, Murphy A, Neavin DR, Powell JE, Howden SE, Little MH. Classification of indeterminate and off-target cell types within human kidney organoid differentiation. iScience 29(6):115752 (2026). DOI: 10.1016/j.isci.2026.115752. https://doi.org/10.1016/j.isci.2026.115752
  14. Kairath P, Er PX, Wilson S, Ghobrial I, Vanslambrouck JM, Chen Y-H, Pruett-Miller SM, Jain S, Little MH. In vitro plasticity between ureteric epithelial and distal nephron identity and maturity is controlled by extracellular signals. Developmental Biology 533:75-88 (2026). DOI: 10.1016/j.ydbio.2026.02.009. https://doi.org/10.1016/j.ydbio.2026.02.009
  15. Maggiore JC, LeGraw R, Przepiorski A, Velazquez J, Chaney C, Vanichapol T, Streeter E, Almuallim Z, Oda A, Chiba T, Silva-Barbosa A, Franks J, Hislop J, Hill A, Wu H, Pfister K, Howden SE, Watkins SC, Little MH, Humphreys BD, Kiani S, Watson A, Stolz DB, Davidson AJ, Carroll T, Cleaver O, Sims-Lucas S, Ebrahimkhani MR, Hukriede NA. A genetically inducible endothelial niche enables vascularization of human kidney organoids with multilineage maturation and emergence of renin expressing cells. Kidney International 106(6):1086-1100 (2024). DOI: 10.1016/j.kint.2024.05.026. https://doi.org/10.1016/j.kint.2024.05.026
  16. Achieng MA, Schnell J, Fausto CC, Csipan RL, Koppitch K, Thornton ME, Grubbs BH, Lindstrom NO. Axial nephron fate switching demonstrates a plastic system tunable on demand. Nature Communications 16:7912 (2025). DOI: 10.1038/s41467-025-63290-9. https://doi.org/10.1038/s41467-025-63290-9
  17. Warrner E, Huang AZ, Hughes AJ. Developmentally inspired synthetic kidney engineering. Nature Biotechnology 44(5):726-739 (2026). DOI: 10.1038/s41587-026-03011-9. https://doi.org/10.1038/s41587-026-03011-9
  18. Zhang C, Liu M, Xia Y. Multilineage assembly brings spatial order to human kidney organoids. Kidney International 109(6):1070-1074 (2026). DOI: 10.1016/j.kint.2025.11.020. https://doi.org/10.1016/j.kint.2025.11.020