Kidney organoids recapitulate human basement membrane assembly in health and disease
- University of Manchester, United Kingdom
- University College London, United Kingdom
- University of São Paulo, Brazil
- 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.
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Single cell RNA-Seq of four human kidney organoidsNCBI Gene Expression Omnibus, GSE114802.
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Pilot Fetal Cell Atlas RNAseqEGA, EGAS00001002553.
Article and author information
Author details
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.
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Kidney organoids recapitulate human basement membrane assembly in health and disease
eLife 11:e73486.
https://doi.org/10.7554/eLife.73486
Further reading
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- Developmental Biology
- Stem Cells and Regenerative Medicine
Niches are often found in specific positions in tissues relative to the stem cells they support. Consistency of niche position suggests that placement is important for niche function. However, the complexity of most niches has precluded a thorough understanding of how their proper placement is established. To address this, we investigated the formation of a genetically tractable niche, the Drosophila Posterior Signaling Center (PSC), the assembly of which had not been previously explored. This niche controls hematopoietic progenitors of the lymph gland (LG). PSC cells were previously shown to be specified laterally in the embryo, but ultimately reside dorsally, at the LG posterior. Here, using live-imaging, we show that PSC cells migrate as a tight collective and associate with multiple tissues during their trajectory to the LG posterior. We find that Slit emanating from two extrinsic sources, visceral mesoderm and cardioblasts, is required for the PSC to remain a collective, and for its attachment to cardioblasts during migration. Without proper Slit-Robo signaling, PSC cells disperse, form aberrant contacts, and ultimately fail to reach their stereotypical position near progenitors. Our work characterizes a novel example of niche formation and identifies an extrinsic signaling relay that controls precise niche positioning.
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- Developmental Biology
- Stem Cells and Regenerative Medicine
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