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.
-
Single cell RNA-Seq of four human kidney organoidsNCBI Gene Expression Omnibus, GSE114802.
-
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.
Metrics
-
- 4,224
- views
-
- 758
- downloads
-
- 28
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Kidney organoids recapitulate human basement membrane assembly in health and disease
eLife 11:e73486.
https://doi.org/10.7554/eLife.73486
Further reading
-
- Stem Cells and Regenerative Medicine
The lateral wall of the mouse subventricular zone harbors neural stem cells (NSC, B cells) which generate proliferating transient-amplifying progenitors (TAP, C cells) that ultimately give rise to neuroblasts (NB, A cells). Molecular profiling at the single-cell level struggles to distinguish these different cell types. Here, we combined transcriptome analyses of FACS-sorted cells and single-cell RNAseq to demonstrate the existence of an abundant, clonogenic and multipotent population of immature neuroblasts (iNB cells) at the transition between TAP and migrating NB (mNB). iNB are reversibly engaged in neuronal differentiation. Indeed, they keep molecular features of both undifferentiated progenitors, plasticity and unexpected regenerative properties. Strikingly, they undergo important progressive molecular switches, including changes in the expression of splicing regulators leading to their differentiation in mNB subdividing them into two subtypes, iNB1 and iNB2. Due to their plastic properties, iNB could represent a new target for regenerative therapy of brain damage.
-
- Biochemistry and Chemical Biology
- Stem Cells and Regenerative Medicine
Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.
