Biology subjects

Combes, A. N.

Publications and source records attributed to Combes, A. N..

6 recordsLinked to original sources

bioRxiv2024

Comparative analysis of iPSC-derived human kidney organoid and tubuloid models

BackgroundEpithelial kidney organoids (tubuloids) made from kidney biopsies, urine, or iPSC-derived kidney organoids offer new opportunities in experimental and clinical nephrology. Yet, we have limited knowledge of how tubuloid models differ from each other, from iPSC-derived kidney organoids and from the human kidney. New insight is required to guide model selection for studies in kidney physiology and disease. MethodsTubuloids were generated from adult nephrectomy samples (adult tubuloids n=3), iPSC-derived kidney organoids (iTubuloids n=3), and for the first time, from human fetal kidneys (fetal tubuloids n=3). Kidney organoid and tubuloid models were compared to each other and to adult human kidney using bulk RNA sequencing. Tubuloids were subject to hypoxic insults over three passages and profiled with RNA sequencing to assess cumulative injury and repair. ResultsExpression signatures of proximal and distal tubules were stronger in adult kidneys than any organoid or tubuloid model. Comparative analysis between the models revealed that iPSC-derived kidney organoids had the highest expression of proximal tubule markers, even though adult tubuloids were derived from mature kidneys. Collecting duct signatures were enriched in adult and fetal tubuloids. Adult tubuloids showed stronger signatures of ageing and inflammation, while fetal tubuloids had enhanced ureteric tip progenitor signatures. Over 80 genes linked to inherited disorders were expressed in all tubuloid cultures, while a further 54 were expressed at higher levels in either adult, fetal or iTubuloids. Tubuloids subject to a single hypoxic injury effectively recovered by the end of the passage, while iTubuloids exposed to hypoxia over three passages expressed markers of maladaptive repair. ConclusionsThis study provides new transcriptome-wide reference data to aid in the selection and optimization of disease modelling for the human kidney. It defines common and unique opportunities to model inherited disorders in adult, fetal and iTubuloid models and illustrates new potential to model repetitive injury in long-lived tubuloid cultures.

molecular biology↗

bioRxiv2023

Forming nephrons promote nephron progenitor maintenance and branching morphogenesis via paracrine BMP4 signalling under the control of Wnt4

Kidney development is known to be driven by interactions between stromal, nephron and ureteric epithelium progenitors in the nephrogenic niche. In contrast, the epithelial nephrons generated in this environment have largely been considered a product of niche rather than an active participant in the signalling interactions that maintain it. However, knockout of Wnt4, a gene required for nephron formation and stromal development, results in hypoplastic kidneys. We hypothesised that the forming nephron may play a role in maintaining the nephrogenic niche. In support of this hypothesis, conditional deletion of Wnt4 from the nephron lineage resulted in nephron progenitor dispersal and death, reduced branching morphogenesis and nephron progenitor cell number. Bulk and single cell transcriptional profiling of Wnt4 mutant kidneys revealed a downregulation of BMP signalling effectors Id1, and Id3 in nephron progenitor cells, implicating Wnt4 target BMP4 as a paracrine signal mediating feedback from the committing nephron. Recombinant BMP4 restored nephron progenitor compaction in cultured Wnt4 mutant kidneys and blocked differentiation in wildtype controls mirroring the role of BMP7-MAPK signalling in progenitor self-renewal. Our data supports a revised model of the nephrogenic niche in which forming nephrons promote progenitor maintenance and branching morphogenesis, in part via paracrine BMP4 signalling under the control of Wnt4. This requirement for nephron-derived signals for maintenance of the nephrogenic niche provides new mechanistic insight into kidney morphogenesis and human renal hypodysplasia phenotypes associated with deleterious WNT4 mutations.

developmental biology↗

bioRxiv2023

Hypoxic injury triggers maladaptive repair in human kidney organoids

Acute kidney injury (AKI) is a common clinical disorder linked to high rates of illness and death. Ischemia is a leading cause of AKI, which can result in chronic kidney disease (CKD) through maladaptive repair marked by impaired epithelial regeneration, inflammation, and metabolic dysregulation. There are no targeted therapies for AKI or to prevent progression to CKD and insight into human disease mechanisms remains limited. Here we show that human kidney organoids recapitulate key molecular and metabolic signatures of AKI and maladaptive repair in response to hypoxic injury. Transcriptional, proteomic, and metabolomic profiling revealed tubular injury, cell death, cell cycle arrest and metabolic reprogramming in organoids exposed to hypoxia. Following return to normoxic conditions, injured organoids had increased signatures of TNF and NF-{kappa}B signalling pathways and S100A8/9, associated with maladaptive repair. Single cell RNA sequencing localized AKI and maladaptive repair markers including GDF15, MMP7, ICAM1, IL32, SPP1, C3 and CCN1 to injured tubules. Metabolic phenotypes linked to CKD were also evident, including dysregulated gluconeogenesis, altered amino acid metabolism and lipid peroxidation. iPSC-derived macrophages incorporated into organoids displayed a robust activation and inflammatory response to hypoxia. Spatial transcriptomics revealed a shift from a tissue resident-like to inflammatory macrophage states and localized effects on tubular injury and inflammation. This multi-omic analysis defines conserved mechanisms of human ischemic AKI and maladaptive repair, highlighting new opportunities to test therapeutics and model immune-mediated interactions.

systems biology↗

bioRxiv2020

Spatially resolved transcriptome profiles of mammalian kidneys illustrate the molecular complexity of functional nephron segments, cell-to-cell interactions and genetic variants.

Understanding the molecular mechanisms underlying mammalian kidney function requires transcriptome profiling of the interplay between cells comprising nephron segments. Traditional transcriptomics requires cell dissociation, resulting in loss of the spatial context of gene expression within native tissue. To address this problem, we performed spatial transcriptomics (ST) to retain the spatial context of the transcriptome in human and mouse kidneys. The generated ST data allowed spatially resolved differential gene expression analysis, spatial identification of functional nephron segments, cell-to-cell interaction analysis, and chronic kidney disease-associated genetic variant calling. Novel ST thus provides an opportunity to enhance kidney diagnostics and knowledge, by retaining the spatial context of gene expression within intact tissue.

molecular biology↗

bioRxiv2018

Bioprinted pluripotent stem cell-derived kidney organoids provide opportunities for high content screening.

Recent advances in the directed differentiation of human pluripotent stem cells to kidney brings with it the prospect of drug screening and disease modelling using patient-derived stem cell lines. Development of such an approach for high content screening will require substantial quality control and improvements in throughput. Here we demonstrate the use of the NovoGen MMX 3D bioprinter for the generation of highly reproducible kidney organoids from as few as 4,000 cells. Histological and immunohistochemical analyses confirmed the presence of renal epithelium, glomeruli, stroma and endothelium, while single cell RNAseq revealed equivalence to the cell clusters present within previously described organoids. The process is highly reproducible, rapid and transferable between cell lines, including genetically engineered reporter lines. We also demonstrate the capacity to bioprint organoids in a 96-well format and screen for response to doxorubicin toxicity as a proof of concept for high content compound screening.

cell biology↗

bioRxiv2017

High throughput single cell RNA-seq of developing mouse kidney and human kidney organoids reveals a roadmap for recreating the kidney

Recent advances in our capacity to differentiate human pluripotent stem cells to human kidney tissue are moving the field closer to novel approaches for renal replacement. Such protocols have relied upon our current understanding of the molecular basis of mammalian kidney morphogenesis. To date this has depended upon population based-profiling of non-homogenous cellular compartments. In order to improve our resolution of individual cell transcriptional profiles during kidney morphogenesis, we have performed 10x Chromium single cell RNA-seq on over 6000 cells from the E18.5 developing mouse kidney, as well as more than 7000 cells from human iPSC-derived kidney organoids. We identified 16 clusters of cells representing all major cell lineages in the E18.5 mouse kidney. The differentially expressed genes from individual murine clusters were then used to guide the classification of 16 cell clusters within human kidney organoids, revealing the presence of distinguishable stromal, endothelial, nephron, podocyte and nephron progenitor populations. Despite the congruence between developing mouse and human organoid, our analysis suggested limited nephron maturation and the presence of off target populations in human kidney organoids, including unidentified stromal populations and evidence of neural clusters. This may reflect unique human kidney populations, mixed cultures or aberrant differentiation in vitro. Analysis of clusters within the mouse data revealed novel insights into progenitor maintenance and cellular maturation in the major renal lineages and will serve as a roadmap to refine directed differentiation approaches in human iPSC-derived kidney organoids.

developmental biology↗