Abstract
A variety of protocols have been developed that demonstrate the capability to differentiate human pluripotent stem cells (hPSCs) into kidney structures. Our goal was to develop a high-efficiency protocol to generate nephron progenitor cells (NPCs) and kidney organoids to facilitate applications for tissue engineering, disease modeling and chemical screening. Here, we describe a detailed protocol resulting in high-efficiency production (80–90%) of NPCs from hPSCs within 9 d of differentiation. Kidney organoids were generated from NPCs within 12 d with high reproducibility using 96-well plates suitable for chemical screening. The protocol requires skills for culturing hPSCs and careful attention to morphological changes indicative of differentiation. This kidney organoid system provides a platform for studies of human kidney development, modeling of kidney diseases, nephrotoxicity and kidney regeneration. The system provides a model for in vitro study of kidney intracellular and intercompartmental interactions using differentiated human cells in an appropriate nephron and stromal context.
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Acknowledgements
The authors thank N. Gupta for providing the immunohistochemistry images of kidney organoids in Supplementary Figure 1. This study was supported by National Institutes of Health grants R37 DK039773 and R01 DK072381 (to J.V.B.), a Grant-in-Aid for a Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowship for Research Abroad (to R.M.), a ReproCell Stem Cell Research grant (to R.M.), a Brigham and Women's Hospital Research Excellence Award (to R.M.), a Brigham and Women's Hospital Faculty Career Development Award (to R.M.) and a Harvard Stem Cell Institute Seed grant (to R.M.).
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R.M. and J.V.B. formulated the strategy for this study. R.M. designed and performed experiments. R.M. and J.V.B. wrote the manuscript. J.V.B. helped to design the experiments and to interpret the results.
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J.V.B. is a co-inventor on KIM-1 patents that have been licensed by Partners Healthcare to several companies. He has received royalty income from Partners Healthcare. J.V.B. and R.M. are co-inventors on patents (PCT/US16/52350) on organoid technologies that are assigned to Partners Healthcare. J.V.B. or his family has received income for consulting from companies interested in biomarkers: Sekisui, Millennium, Johnson & Johnson and Novartis. J.V.B. is a consultant to Goldfinch Bio. J.V.B. owns equity in Goldfinch Bio.
Integrated supplementary information
Supplementary Figure 1 Immunostaining for interstitial cells and connecting tubules and collecting ducts.
(a) Immunostaining for PDGFRb (platelet derived growth factor receptor beta) and endomucin in 3D kidney organoids. PDGFRβ was assessed by immunohistochemistry in 3D kidney organoids on day 65. Endomucin was evaluated by whole mount staining in 3D kidney organoids on day 24. Arrows indicate endomucin+ endothelia in a glomerular structure. (b) Immunohistochemistry for α-SMA (smooth muscle alpha-actin) in 3D kidney organoids on day 65. There was a very small population of α-SMA+ interstitial cells in kidney organoids. (c) Immunohistochemistry for aquaporin-2 (AQP2) in CDH1+ tubule in 3D kidney organoids on day 35. AQP2+ tubules were found in only CDH1+ tubules, indicating presence of connecting tubules/collecting ducts in kidney organoids. Scale bars: 50 μm.
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Morizane, R., Bonventre, J. Generation of nephron progenitor cells and kidney organoids from human pluripotent stem cells. Nat Protoc 12, 195–207 (2017). https://doi.org/10.1038/nprot.2016.170
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DOI: https://doi.org/10.1038/nprot.2016.170
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