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      3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening

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          Abstract

          The podocytes within the glomeruli of the kidney maintain the filtration barrier by forming interdigitating foot processes with intervening slit diaphragms, disruption in which results in proteinuria. Studies into human podocytopathies to date have employed primary or immortalised podocyte cell lines cultured in 2D. Here we compare 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids with conditionally immortalised human podocyte cell lines, revealing improved podocyte-specific gene expression, maintenance in vitro of polarised protein localisation and an improved glomerular basement membrane matrisome compared to 2D cultures. Organoid-derived glomeruli retain marker expression in culture for 96 h, proving amenable to toxicity screening. In addition, 3D organoid glomeruli from a congenital nephrotic syndrome patient with compound heterozygous NPHS1 mutations reveal reduced protein levels of both NEPHRIN and PODOCIN. Hence, human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modelling of human podocytopathies and screening for podocyte toxicity.

          Abstract

          Studies examining human podocytopathies have utilised 2D cultured primary or immortalised podocyte cell lines. Here, the authors demonstrate that 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids retain an improved podocyte identity in vitro facilitating disease modelling and toxicity testing.

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          Most cited references60

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          Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.

          The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.
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            Nephron organoids derived from human pluripotent stem cells model kidney development and injury

            Kidney cells and tissues derived from human pluripotent stem cells (hPSCs) would enable organ regeneration, disease modeling, and drug screening in vitro. We established an efficient, chemically defined protocol for differentiating hPSCs into multipotent nephron progenitor cells (NPCs) that can form nephron-like structures. By recapitulating metanephric kidney development in vitro, we generate SIX2+SALL1+WT1+PAX2+ NPCs with 90% efficiency within 9 days of differentiation. The NPCs possess the developmental potential of their in vivo counterparts and form PAX8+LHX1+ renal vesicles that self-pattern into nephron structures. In both 2D and 3D culture, NPCs form kidney organoids containing epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle, and distal tubules in an organized, continuous arrangement that resembles the nephron in vivo. We also show that this organoid culture system can be used to study mechanisms of human kidney development and toxicity.
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              Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells.

              Recapitulating three-dimensional (3D) structures of complex organs, such as the kidney, from pluripotent stem cells (PSCs) is a major challenge. Here, we define the developmental origins of the metanephric mesenchyme (MM), which generates most kidney components. Unexpectedly, we find that posteriorly located T(+) MM precursors are developmentally distinct from Osr1(+) ureteric bud progenitors during the postgastrulation stage, and we identify phasic Wnt stimulation and stage-specific growth factor addition as molecular cues that promote their development into the MM. We then use this information to derive MM from PSCs. These progenitors reconstitute the 3D structures of the kidney in vitro, including glomeruli with podocytes and renal tubules with proximal and distal regions and clear lumina. Furthermore, the glomeruli are efficiently vascularized upon transplantation. Thus, by reevaluating the developmental origins of metanephric progenitors, we have provided key insights into kidney specification in vivo and taken important steps toward kidney organogenesis in vitro. Copyright © 2014 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                +61 3 9936 6206 , melissa.little@mcri.edu.au
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                4 December 2018
                4 December 2018
                2018
                : 9
                : 5167
                Affiliations
                [1 ]ISNI 0000 0000 9442 535X, GRID grid.1058.c, Murdoch Children’s Research Institute, ; Flemington Rd, Melbourne, 3052 VIC Australia
                [2 ]ISNI 0000 0001 2179 088X, GRID grid.1008.9, Department of Paediatrics and Department of Anatomy and Neuroscience, , The University of Melbourne, ; Melbourne, 3010 VIC Australia
                [3 ]ISNI 0000000121662407, GRID grid.5379.8, Wellcome Trust Centre for Cell Matrix Research, , University of Manchester, ; Manchester, M13 9PT UK
                [4 ]ISNI 0000 0001 2179 088X, GRID grid.1008.9, School of BioScience, , The University of Melbourne, ; Melbourne, 3010 VIC Australia
                [5 ]ISNI 0000 0004 0614 0346, GRID grid.416107.5, Department of Nephrology, , Royal Children’s Hospital, ; Melbourne, 3052 VIC Australia
                Author information
                http://orcid.org/0000-0002-1711-7454
                http://orcid.org/0000-0002-8994-0781
                http://orcid.org/0000-0001-9788-5690
                http://orcid.org/0000-0001-6400-0227
                http://orcid.org/0000-0003-0380-2263
                Article
                7594
                10.1038/s41467-018-07594-z
                6279764
                30514835
                8a9ca904-4864-4598-880c-c71329f57c42
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 10 June 2017
                : 5 November 2018
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