92
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Human-pluripotent-stem-cell-derived kidney cells (hPSC-KCs) have important potential for disease modelling and regeneration. Whether the hPSC-KCs can reconstitute tissue-specific phenotypes is currently unknown. Here we show that hPSC-KCs self-organize into kidney organoids that functionally recapitulate tissue-specific epithelial physiology, including disease phenotypes after genome editing. In three-dimensional cultures, epiblast-stage hPSCs form spheroids surrounding hollow, amniotic-like cavities. GSK3β inhibition differentiates spheroids into segmented, nephron-like kidney organoids containing cell populations with characteristics of proximal tubules, podocytes and endothelium. Tubules accumulate dextran and methotrexate transport cargoes, and express kidney injury molecule-1 after nephrotoxic chemical injury. CRISPR/Cas9 knockout of podocalyxin causes junctional organization defects in podocyte-like cells. Knockout of the polycystic kidney disease genes PKD1 or PKD2 induces cyst formation from kidney tubules. All of these functional phenotypes are distinct from effects in epiblast spheroids, indicating that they are tissue specific. Our findings establish a reproducible, versatile three-dimensional framework for human epithelial disease modelling and regenerative medicine applications.

          Abstract

          Generating organized kidney tissues from human pluripotent stem cell is a major challenge. Here, Freedman et al. describe a differentiation system forming spheroids and tubular structures, characteristic of these kidney structures, and using CRISPR/Cas9, delete PKD1/2, to model polycystic kidney disease.

          Related collections

          Most cited references37

          • Record: found
          • Abstract: found
          • Article: not found

          Kidney injury molecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury.

          We report the identification of rat and human cDNAs for a type 1 membrane protein that contains a novel six-cysteine immunoglobulin-like domain and a mucin domain; it is named kidney injury molecule-1 (KIM-1). Structurally, KIM-1 is a member of the immunoglobulin gene superfamily most reminiscent of mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Human KIM-1 exhibits homology to a monkey gene, hepatitis A virus cell receptor 1 (HAVcr-1), which was identified recently as a receptor for the hepatitis A virus. KIM-1 mRNA and protein are expressed at a low level in normal kidney but are increased dramatically in postischemic kidney. In situ hybridization and immunohistochemistry revealed that KIM-1 is expressed in proliferating bromodeoxyuridine-positive and dedifferentiated vimentin-positive epithelial cells in regenerating proximal tubules. Structure and expression data suggest that KIM-1 is an epithelial cell adhesion molecule up-regulated in the cells, which are dedifferentiated and undergoing replication. KIM-1 may play an important role in the restoration of the morphological integrity and function to postischemic kidney.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            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.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Feeder-free growth of undifferentiated human embryonic stem cells.

              Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.
                Bookmark

                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Pub. Group
                2041-1723
                23 October 2015
                2015
                : 6
                : 8715
                Affiliations
                [1 ]Division of Renal Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Harvard Institutes of Medicine Suite 550, 4 Blackfan Circle, Boston, Massachusetts 02115, USA
                [2 ]Division of Nephrology, Department of Medicine, University of Washington School of Medicine , 850 Republican Street, Room S565, Seattle, Washington 98109, USA
                [3 ]Kidney Research Institute, Department of Medicine, University of Washington , 325 Ninth Avenue, Box 359606, Seattle, Washington 98104, USA
                [4 ]Institute for Stem Cell and Regenerative Medicine, University of Washington , Seattle, Washington 98109, USA
                [5 ]Harvard Stem Cell Institute, Harvard University , 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA
                [6 ]Department of Biological Chemistry and Pharmacology, Boston Children's Hospital, Center for Life Sciences, Harvard Medical School , Room 3103, 3 Blackfan Circle, Boston, Massachusetts 02115, USA
                [7 ]Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Harvard Institutes of Medicine Suite 823, 4 Blackfan Circle, Boston, Massachusetts 02115, USA
                [8 ]Department of Pediatric Newborn Medicine, Brigham and Women's Hospital, Harvard Medical School , Boston, Massachusetts 02115, USA
                [9 ]Department of Stem Cell and Regenerative Biology, Harvard University , Sherman Fairchild Biochemistry Building 160, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA
                [10 ]The Biomedical Research Centre, University of British Columbia , 2222 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3
                [11 ]Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School , Boston, Massachusetts 02115, USA
                [12 ]Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School , 330 Brookline Avenue DA517, Boston, Massachusetts 02115, USA
                Author notes
                [*]

                Present address: Division of Nephrology, Department of Medicine, University of Washington School of Medicine, 850 Republican Street, PO Box 358056, Seattle, Washington 98109, USA

                Article
                ncomms9715
                10.1038/ncomms9715
                4620584
                26493500
                58b7b5ff-7b2d-496e-871a-bb7ed7e47717
                Copyright © 2015, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 28 May 2015
                : 24 September 2015
                Categories
                Article

                Uncategorized
                Uncategorized

                Comments

                Comment on this article