Biology subjects

Woolf, A. S.

Publications and source records attributed to Woolf, A. S..

6 recordsLinked to original sources

bioRxiv2025

Human pluripotent stem cell-derived macrophages modify development of human kidney organoids

IntroductionThe human fetal kidney contains macrophages, innate immune cells postulated to enhance its development. Macrophages have also been implicated in the pathobiology of human kidney malformations and Wilms tumour. Human pluripotent stem cell (hPSC)-derived kidney organoids contain nephrons differentiated from intermediate mesoderm-like precursors. Endothelia are present between organoid tubules but fail to efficiently populate glomeruli. These organoids lack macrophages, as expected because in vivo kidney macrophages invade from yolk sac and liver. MethodsWe hypothesised that combining hPSC-derived macrophages with hPSC-derived kidney precursors modifies nephrogenesis. Macrophages harvested at early or later maturation stages were added to kidney precursors in numbers of 1%, 5% or 20% compared with constant numbers of nephrogenic cells. ResultsNo macrophages, as assessed by CD68 immunostaining, were detected in organoids without added macrophages. In contrast, composite organoids contained macrophages located between tubules, mimicking native human fetal kidneys. Quantification of tissue macrophages at the end of 18-day organoid culture positively correlated with the numbers of macrophages added. Assessed by CD31/PECAM-1 and CD68 co-immunostaining, some macrophages were near vessels but added macrophages neither increased the proportion of vessels nor endothelial invasion of glomeruli. Early-stage macrophages significantly increased the percentage area occupied by glomeruli, as assessed by synaptopodin immunostaining. The highest macrophage numbers inhibited overall growth, assessed by organoid area, additionally generating dysmorphic tissue when later stage macrophages were added. ConclusionDepending on their maturation stage and quantity, macrophages have beneficial or harmful effects on organoids. This supports the proposition that macrophages play roles in normal and abnormal development of human kidneys.

developmental biology↗

bioRxiv2025

Genetic lineage tracing identifies intermediate mesoderm as a novel contributor to mammalian kidney lymphatics

The lymphatic vasculature is essential for fluid homeostasis, immune regulation and possesses diverse organ-specific functions. During development, lymphatic endothelial cells (LEC) arise from multiple progenitor sources that form organ-specific lymphatic networks. While the origins of LECs in the heart, skin, and mesentery have been studied, those in the kidney remain unresolved. Here, we combined genetic lineage tracing in mouse embryos with optical clearing and high-resolution three-dimensional imaging to identify two distinct progenitor sources of kidney lymphatics. The majority of kidney LECs originate from a Tie2 endothelial lineage previously linked to venous or capillary vessels. Approximately 15% derive from Osr1 intermediate mesoderm, a lineage that generates kidney nephrons and stroma. Osr1-derived LECs were absent from the heart, mesentery, and skin, indicating a kidney-specific contribution, and arose independently of nephron and stromal lineages. Both Tie2 and Osr1 lineages contributed to vessel sprouting and de novo formation of lymphatic clusters. Revealing a novel cellular origin of LECs and identifying a dual origin for kidney lymphatics, we demonstrate that de novo lymphatic formation can occur from both shared and organ-specific progenitors. This work advances our understanding of how lymphatics assemble during development and provides a framework for targeting kidney lymphatics in disease.

developmental biology↗

bioRxiv2024

A unique subset of pericystic endothelium associates with aberrant microvascular remodelling and impaired blood perfusion early in polycystic kidney disease

Hallmarks of autosomal dominant polycystic kidney disease (ADPKD), the most common hereditary kidney anomaly, include expanding fluid-filled epithelial cysts, inflammation, and fibrosis. Despite previous work showing the potential of vascular-based therapies, renal microvascular alterations in ADPKD, and their timing, are poorly understood. Using single-cell transcriptomics of human kidney microvasculature, we identify a population of endothelial cells adjacent to cysts in ADPKD. This pericystic endothelium, distinguishable by its expression of osteopontin (SPP1), has a distinct molecular profile compared to the common endothelial cell injury signature in other kidney diseases. SPP1+ pericystic endothelium was also present in an orthologous mouse model of ADPKD before overt kidney functional decline. By interrogating geometric, topological and fractal properties from three-dimensional imaging of early ADPKD mouse kidneys, we show that pericystic endothelium associates with disorganisation and non-uniformity of the renal cortical microvasculature. Concurrently, we detected region-specific reductions in cortical blood flow within ADPKD murine kidneys using arterial spin labelling. We conclude that ADPKD kidneys contain a unique subset of endothelium manifesting with aberrant remodelling and impaired blood perfusion. Its detection, prior to renal functional decline, advocates the vasculature as a therapeutic target to modulate or preserve renal function in early ADPKD.

pathology↗

bioRxiv2023

Human HPSE2 gene transfer ameliorates bladder pathophysiology in a mutant mouse model of urofacial syndrome.

Rare early onset lower urinary tract disorders include defects of functional maturation of the bladder. Current treatments do not target the primary pathobiology of these diseases. Some have a monogenic basis, such as urofacial, or Ochoa, syndrome (UFS). Here, the bladder does not empty fully because of incomplete relaxation of its outflow tract, and subsequent urosepsis can cause kidney failure. UFS is associated with biallelic variants of HPSE2, encoding heparanase-2. This protein is detected in pelvic ganglia, autonomic relay stations that innervate the bladder and control voiding. Bladder outflow tracts of Hpse2 mutant mice display impaired neurogenic relaxation. We hypothesized that HPSE2 gene transfer soon after birth would ameliorate this defect and explored an adeno-associated viral (AAV) vector-based approach. AAV9/HPSE2, carrying human HPSE2 driven by CAG, was administered intravenously into neonatal mice. In the third postnatal week, transgene transduction and expression were sought, and ex vivo myography was undertaken to measure bladder function. In mice administered AAV9/HPSE2, the viral genome was detected in pelvic ganglia. Human HPSE2 was expressed and heparanase-2 became detectable in pelvic ganglia of treated mutant mice. On autopsy, wild-type mice had empty bladders whereas bladders were uniformly distended in mutant mice, a defect ameliorated by AAV9/HPSE2 treatment. Therapeutically, AAV9/HPSE2 significantly ameliorated impaired neurogenic relaxation of Hpse2 mutant bladder outflow tracts. Impaired neurogenic contractility of mutant detrusor smooth muscle was also significantly improved. These results constitute first steps towards curing UFS, a clinically devastating genetic disease featuring a bladder autonomic neuropathy. SummaryIn the first gene therapy for genetic bladder disease, we cured autonomic neurons using AAV-mediated gene delivery in a mouse model of urofacial syndrome.

neuroscience↗

bioRxiv2023

Human pluripotent stem cell-derived kidney organoids reveal tubular epithelial pathobiology of heterozygous HNF1B-associated dysplastic kidney malformations

Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human ESCs and iPSCs reprogrammed from a family with HNF1B-asscociated DKMs. Mutant organoids contained enlarged malformed tubules and displayed deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cAMP-mediated dilatation seen in controls. Bioinformatic analyses indicated abnormal WNT, calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 was upregulated in mutant organoids and was detected in their tubules and in fetal human DKM dysplastic epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule morphogenesis and functional differentiation. They additionally suggest druggable targets to ameliorate disease.

developmental biology↗

bioRxiv2022

Exploration of single-cell transcriptomic landscape identifies aberrant glomerular crosstalk in a murine model of WT1 kidney disease

The glomerulus mediates kidney ultrafiltration through specialised epithelial cells called podocytes which line a basement membrane shared with blood capillary endothelium. Cell-cell crosstalk is critical for glomerular function, but its investigation in childhood glomerular diseases has received little attention. WT1 encodes a transcription factor expressed in podocytes, whose heterozygous variants cause devastating kidney disease in childhood. We used single-cell RNA sequencing and ligand-receptor interaction analysis to resolve the glomerular transcriptional landscape of mice that carry an orthologous human mutation in WT1 (Wt1R394W/+). Podocytes were the most dysregulated cell type in early disease, with disrupted angiogenic signalling preceding glomerular capillary loss. Comparative analyses with additional murine and human glomerular disease datasets identified unique transcriptional changes in WT1 glomerular disease, reflecting a non-immunological pathology, whilst revealing a common injury signature across multiple glomerular diseases. Collectively, this work advocates vascular-based therapies over immunosuppressive drugs in the treatment of WT1 glomerular disease.

cell biology↗