­­­Kidney organoids recapitulate human basement membrane assembly in health and disease

  1. Mychel RPT Morais
  2. Pinyuan Tian
  3. Craig Lawless
  4. Syed Murtuza-Baker
  5. Louise Hopkinson
  6. Steven Woods
  7. Aleksandr Mironov
  8. David A Long
  9. Daniel P Gale
  10. Telma MT Zorn
  11. Susan J Kimber
  12. Roy Zent
  13. Rachel Lennon  Is a corresponding author
  1. University of Manchester, United Kingdom
  2. University College London, United Kingdom
  3. University of São Paulo, Brazil
  4. Vanderbilt University Medical Center, United States

Abstract

Basement membranes (BMs) are complex macromolecular networks underlying all continuous layers of cells. Essential components include collagen IV and laminins, which are affected by human genetic variants leading to a range of debilitating conditions including kidney, muscle, and cerebrovascular phenotypes. We investigated the dynamics of BM assembly in human pluripotent stem cell-derived kidney organoids. We resolved their global BM composition and discovered a conserved temporal sequence in BM assembly that paralleled mammalian fetal kidneys. We identified the emergence of key BM isoforms, which were altered by a pathogenic variant in COL4A5. Integrating organoid, fetal and adult kidney proteomes we found dynamic regulation of BM composition through development to adulthood, and with single-cell transcriptomic analysis we mapped the cellular origins of BM components. Overall, we define the complex and dynamic nature of kidney BM assembly and provide a platform for understanding its wider relevance in human development and disease.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2019) with the dataset identifiers: PXD025838, PXD025874, PXD025911 and PXD026002.This project also contains the following source data hosted at:https://doi.org/10.6084/m9.figshare.c.5429628Figure 1 Original IF Images: B Whole-mount immunofluorescence for kidney cell types; F Representative whole mount immunofluorescence images of wild-type and Alport kidney organoids; G Immunofluorescence for LAMB2.Figure 1 Original light microscope Images: C Representative photomicrographs of day 18 kidney organoids (left) and human and mouse fetal kidneys (right).Figure 1 Original TEM Images: D Transmission electron micrographs of tubular BM in day 25 kidney organoid and E19 mouse fetal kidney.Figure 1 Original western blotting image: H Immunoblotting for LAMB2 using total lysates from wild-type and Alport organoids.Figure 2 Original IF Images: A Confocal immunofluorescence microscopy of wild-type kidney organoids; B perlecan and nidogen on days 11, 18 and 25 of differentiation.Figure 4 Original IF Images: A Immunofluorescence for key type IV collagen and laminin isoforms showing their emergence and distribution in kidney organoid BM; D Immunofluorescence for specific collagen IV isoforms in maturing glomeruli in E19 mouse kidney and in glomerular structures (indicated by dashed lines) in day 25 organoids.Figure 1-figure supplement 2A Original TEM photomicrographs: A Transmission electron microscopy of day 25 kidney organoids shows advanced differentiation of glomerular structures.Figure 1-figure supplement 2B Original TEM photomicrographs: B Transmission electron microscopy of day 25 kidney organoids shows advanced differentiation of glomerular structures.Figure 1-figure supplement 1C Original IF images: C Immunofluorescence for integrin beta-1 (ITGB1) in day 25 kidney organoid (wild-type). Anti-panlaminin or anti-collagen IV antibodies were used to label basement membranes.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Mychel RPT Morais

    Wellcome Centre for Cell-Matrix Research, University of Manchester, Manchester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Pinyuan Tian

    Wellcome Centre for Cell-Matrix Research, University of Manchester, Manchester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6080-5378
  3. Craig Lawless

    Wellcome Centre for Cell-Matrix Research, University of Manchester, Manchester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Syed Murtuza-Baker

    Division of Informatics, Imaging and Data Sciences, University of Manchester, Manchester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Louise Hopkinson

    Wellcome Centre for Cell-Matrix Research, University of Manchester, Manchester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1758-4201
  6. Steven Woods

    Division of Cell Matrix Biology and Regenerative Medicine, University of Manchester, Manchester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Aleksandr Mironov

    Electron Microscopy Core Facility, University of Manchester, Manchester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  8. David A Long

    Developmental Biology and Cancer Programme, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6580-3435
  9. Daniel P Gale

    Department of Renal Medicine, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9170-1579
  10. Telma MT Zorn

    Department of Cell and Developmental Biology, University of São Paulo, Sao Paulo, Brazil
    Competing interests
    The authors declare that no competing interests exist.
  11. Susan J Kimber

    Division of Cell Matrix Biology and Regenerative Medicine, University of Manchester, Manchester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  12. Roy Zent

    Department of Medicine, Vanderbilt University Medical Center, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Rachel Lennon

    Division of Cell-Matrix Biology and Regenerative Medicine, University of Manchester, Manchester, United Kingdom
    For correspondence
    Rachel.Lennon@manchester.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6400-0227

Funding

Wellcome Trust (Wellcome Senior Fellowship award,202860/Z/16/Z)

  • Rachel Lennon

Kidney Research UK (Kidney Research UK grant (RP52/2014)

  • Pinyuan Tian
  • Rachel Lennon

São Paulo Research Foundation (Fellowship grants 2015/02535-2 and 2017/26785-5)

  • Mychel RPT Morais

Global Challenges Research Fund

  • Mychel RPT Morais

Veterans Affairs (Veterans Affairs Merit Awards 1I01BX002196-01 and DK069221)

  • Roy Zent

NIHR Biomedical Research Centre, Royal Marsden NHS Foundation Trust/Institute of Cancer Research

  • David A Long

Wellcome Trust (Investigator Award,220895/Z/20/Z)

  • David A Long

Medical Research Council (Project grants MR/P018629/1 and MR/J003638/1)

  • David A Long

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All mouse handling and experimental procedures were approved by the Animal EthicsCommittee of the Institute of Biomedical Sciences (University of São Paulo, Brazil; reference 019/2015). This was performed in accordance with recommendations from the current Brazilian legislation. All surgery was performed under avertin anaesthesia.

Human subjects: Human fetal kidney sections were provided by the Joint MRC/Wellcome Trust HumanDevelopmental Biology Resource (HDBR) (http://hdbr.org). The HDBR obtains written consent from the donors and has ethics approval (REC reference: 08/H0712/34+5) to collect, store and distribute human material sampled between 4 and 21 weeks post conception. All experimental protocols were approved by the Institute's Ethical Committee (reference 010/H0713/6) and were performed in accordance with institutional ethical and regulatory guidelines.

Copyright

© 2022, Morais et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 4,456
    views
  • 790
    downloads
  • 32
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

Share this article

https://doi.org/10.7554/eLife.73486

Further reading

    1. Stem Cells and Regenerative Medicine
    Sujeethkumar Prithiviraj, Alejandro Garcia Garcia ... Paul E Bourgine
    Research Article

    Tissue engineering strategies predominantly rely on the production of living substitutes, whereby implanted cells actively participate in the regenerative process. Beyond cost and delayed graft availability, the patient-specific performance of engineered tissues poses serious concerns on their clinical translation ability. A more exciting paradigm consists in exploiting cell-laid, engineered extracellular matrices (eECMs), which can be used as off-the-shelf materials. Here, the regenerative capacity solely relies on the preservation of the eECM structure and embedded signals to instruct an endogenous repair. We recently described the possibility to exploit custom human stem cell lines for eECM manufacturing. In addition to the conferred standardization, the availability of such cell lines opened avenues for the design of tailored eECMs by applying dedicated genetic tools. In this study, we demonstrated the exploitation of CRISPR/Cas9 as a high precision system for editing the composition and function of eECMs. Human mesenchymal stromal/stem cell (hMSC) lines were modified to knock out vascular endothelial growth factor (VEGF) and Runt-related transcription factor 2 (RUNX2) and assessed for their capacity to generate osteoinductive cartilage matrices. We report the successful editing of hMSCs, subsequently leading to targeted VEGF and RUNX2-knockout cartilage eECMs. Despite the absence of VEGF, eECMs retained full capacity to instruct ectopic endochondral ossification. Conversely, RUNX2-edited eECMs exhibited impaired hypertrophy, reduced ectopic ossification, and superior cartilage repair in a rat osteochondral defect. In summary, our approach can be harnessed to identify the necessary eECM factors driving endogenous repair. Our work paves the road toward the compositional eECMs editing and their exploitation in broad regenerative contexts.

    1. Developmental Biology
    2. Stem Cells and Regenerative Medicine
    Joshua G Medina-Feliciano, Griselle Valentín-Tirado ... José E Garcia-Arraras
    Research Article

    In holothurians, the regenerative process following evisceration involves the development of a ‘rudiment’ or ‘anlage’ at the injured end of the mesentery. This regenerating anlage plays a pivotal role in the formation of a new intestine. Despite its significance, our understanding of the molecular characteristics inherent to the constituent cells of this structure has remained limited. To address this gap, we employed state-of-the-art scRNA-seq and hybridization chain reaction fluorescent in situ hybridization analyses to discern the distinct cellular populations associated with the regeneration anlage. Through this approach, we successfully identified 13 distinct cell clusters. Among these, two clusters exhibit characteristics consistent with putative mesenchymal cells, while another four show features akin to coelomocyte cell populations. The remaining seven cell clusters collectively form a large group encompassing the coelomic epithelium of the regenerating anlage and mesentery. Within this large group of clusters, we recognized previously documented cell populations such as muscle precursors, neuroepithelial cells, and actively proliferating cells. Strikingly, our analysis provides data for identifying at least four other cellular populations that we define as the precursor cells of the growing anlage. Consequently, our findings strengthen the hypothesis that the coelomic epithelium of the anlage is a pluripotent tissue that gives rise to diverse cell types of the regenerating intestinal organ. Moreover, our results provide the initial view into the transcriptomic analysis of cell populations responsible for the amazing regenerative capabilities of echinoderms.