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Aims Specific fibroblast markers and in-depth heterogeneity analysis are currently lacking, hindering functional studies in cardiovascular diseases (CVDs). Here, we established cell-type markers and heterogeneity in murine and human arteries and studied the adventitial fibroblast response to CVD and its risk factors hypercholesterolaemia and ageing. Methods and results Murine aorta single-cell RNA-sequencing analysis of adventitial mesenchymal cells identified fibroblast-specific markers. Immunohistochemistry and flow cytometry validated platelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, whereas traditional markers such as the cluster of differentiation (CD)90 and vimentin also marked transgelin+ vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiating along trajectories. Three trajectories, marked by CD55 (Cd55+), Cxcl chemokine 14 (Cxcl14+), and lysyl oxidase (Lox+), were reproduced in an independent RNA-seq dataset. Gene ontology (GO) analysis showed divergent functional profiles of the three trajectories, related to vascular development, antigen presentation, and/or collagen fibril organization, respectively. Trajectory-specific genes included significantly more genes with known genome-wide associations (GWAS) to CVD than expected by chance, implying a role in CVD. Indeed, differential regulation of fibroblast clusters by CVD risk factors was shown in the adventitia of aged C57BL/6J mice, and mildly hypercholesterolaemic LDLR KO mice on chow by flow cytometry. The expansion of collagen-related CXCL14+ and LOX+ fibroblasts in aged and hypercholesterolaemic aortic adventitia, respectively, coincided with increased adventitial collagen. Immunohistochemistry, bulk, and single-cell transcriptomics of human carotid and aorta specimens emphasized translational value as CD55+, CXCL14+ and LOX+ fibroblasts were observed in healthy and atherosclerotic specimens. Also, trajectory-specific gene sets are differentially correlated with human atherosclerotic plaque traits. Conclusion We provide two adventitial fibroblast-specific markers, PDGFRA and DPEP1, and demonstrate fibroblast heterogeneity in health and CVD in humans and mice. Biological relevance is evident from the regulation of fibroblast clusters by age and hypercholesterolaemia in vivo, associations with human atherosclerotic plaque traits, and enrichment of genes with a GWAS for CVD.

Kidney fibrosis is the hallmark of chronic kidney disease progression; however, at present no antifibrotic therapies exist1–3. The origin, functional heterogeneity and regulation of scar-forming cells that occur during human kidney fibrosis remain poorly understood1,2,4. Here, using single-cell RNA sequencing, we profiled the transcriptomes of cells from the proximal and non-proximal tubules of healthy and fibrotic human kidneys to map the entire human kidney. This analysis enabled us to map all matrix-producing cells at high resolution, and to identify distinct subpopulations of pericytes and fibroblasts as the main cellular sources of scar-forming myofibroblasts during human kidney fibrosis. We used genetic fate-tracing, time-course single-cell RNA sequencing and ATAC–seq (assay for transposase-accessible chromatin using sequencing) experiments in mice, and spatial transcriptomics in human kidney fibrosis, to shed light on the cellular origins and differentiation of human kidney myofibroblasts and their precursors at high resolution. Finally, we used this strategy to detect potential therapeutic targets, and identified NKD2 as a myofibroblast-specific target in human kidney fibrosis.

Liver disease is a major global health- care problem, affecting an estimated 844 million people worldwide. Despite this substantial burden, therapeutic options for liver disease remain limited, in part owing to a paucity of detailed analyses defining the cellular and molecular mechanisms that drive these conditions in humans. Single- cell transcriptomic technologies are transforming our understanding of cellular diversity and function in health and disease. In this Review, we discuss how these technologies have been applied in hepatology, advancing our understanding of cellular heterogeneity and providing novel insights into fundamental liver biology such as the metabolic zonation of hepatocytes, endothelial cells and hepatic stellate cells, and the cellular mechanisms underpinning liver regeneration. Application of these methodologies is also uncovering critical pathophysiological changes driving disease states such as hepatic fibrosis, where distinct populations of macrophages, endothelial cells and mesenchymal cells reside within a spatially distinct fibrotic niche and interact to promote scar formation. In addition, single- cell approaches are starting to dissect key cellular and molecular functions in liver cancer. In the near future, new techniques such as spatial transcriptomics and multiomic approaches will further deepen our understanding of disease pathogenesis, enabling the identification of novel therapeutic targets for patients across the spectrum of liver diseases

Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis. Single-cell RNA sequencing is used to characterize and compare the functional diversity of cells from liver biopsies of human scarred and normal liver, and identifies markers for scar-associated macrophages and endothelial cells.

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality, develops almost exclusively in patients with chronic liver disease (CLD) and advanced fibrosis(1,2). Here we interrogated functions of hepatic stellate cells (HSC), the main source of liver fibroblasts(3), during hepatocarcinogenesis. Genetic depletion, activation or inhibition established HSC as tumour-promoting in mouse models of HCC. HSC were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analysis of mouse and human HSC subpopulations and their associated mediators by single cell RNA-sequencing in conjunction with genetic ablation revealed dual functions of HSC in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSC (cyHSC), protected from hepatocyte death and HCC development. In contrast, type I collagen, enriched in activated myofibroblastic HSC (myHSC), promoted proliferation and tumour development via increased stiffness and TAZ activation in pretumoural hepatocytes and via activation of discoidin domain receptor 1 in established tumours. An increasing HSC dysbalance between cyHSC and myHSC during liver disease progression was associated with elevated HCC risk in patients. In summary, the dynamic shift of HSC subpopulations and their mediators during CLD is associated with a switch from HCC protection to HCC promotion.

In the homeostatic liver, ductal cells intermingle with a microenvironment of endothelial and mesenchymal cells to form the functional unit of the portal tract. Ductal cells proliferate rarely in homeostasis but do so transiently after tissue injury to replenish any lost epithelium. We have shown that liver ductal cells can be expanded as liver organoids that recapitulate several of the cell-autonomous mechanisms of regeneration, but lack the stromal cell milieu of the biliary tract in vivo. Here, we describe a subpopulation of SCA1+ periportal mesenchymal cells that closely surrounds ductal cells in vivo and exerts a dual control on their proliferative capacity. Mesenchymal-secreted mitogens support liver organoid formation and expansion from differentiated ductal cells. However, direct mesenchymal-to-ductal cell-cell contact, established following a microfluidic co-encapsulation that enables the cells to self-organize into chimeric organoid structures, abolishes ductal cell proliferation in a mesenchyme-dose dependent manner. We found that it is the ratio between mesenchymal and epithelial cell contacts that determines the net outcome of ductal cell proliferation both in vitro, and in vivo, during damage-regeneration. SCA1+ mesenchymal cells control ductal cell proliferation dynamics by a mechanism involving, at least in part, Notch signalling activation. Our findings underscore how the relative abundance of cell-cell contacts between the epithelium and its mesenchymal microenvironment are key regulatory cues involved in the control of tissue regeneration.SummaryIn the homeostatic liver, the ductal epithelium intermingles with a microenvironment of stromal cells to form the functional unit of the portal tract. Ductal cells proliferate rarely in homeostasis but do so transiently after tissue injury. We have shown that these cells can be expanded as liver organoids that recapitulate several of the cell-autonomous mechanisms of regeneration, but lack the stromal cell milieu of the portal tract in vivo. Here, we describe a subpopulation of SCA1+ periportal mesenchymal niche cells that closely surrounds ductal cells in vivo and exerts a dual control on their proliferative capacity. Mesenchymal-secreted mitogens support liver organoid formation and expansion from differentiated ductal cells. However, direct mesenchymal-to-ductal cell-cell contact, established through a microfluidic co-encapsulation method that enables the cells to self-organize into chimeric organoid structures, abolishes ductal cell proliferation in a mesenchyme-dose dependent manner. We found that it is the ratio between mesenchymal and epithelial cell contacts that determines the net outcome of ductal cell proliferation both in vitro, and in vivo, during damage-regeneration. SCA1+ mesenchymal cells control ductal cell proliferation dynamics by a mechanism involving, at least in part, Notch signalling activation. Our findings re-evaluate the concept of the cellular niche, whereby the proportions of cell-cell contacts between the epithelium and its mesenchymal niche, and not the absolute cell numbers, are the key regulatory cues involved in the control of tissue regeneration.

The endometrium is a dynamic tissue that exhibits remarkable resilience to repeated episodes of differentiation, breakdown, regeneration, and remodeling. Endometrial physiology relies on a complex interplay between the stromal and epithelial compartments with the former containing a mixture of fibroblasts, vascular, and immune cells. There is evidence for rare populations of putative mesenchymal progenitor cells located in the perivascular niche of human endometrium, but the existence of an equivalent cell population in mouse is unclear. We used the Pdgfrb-BAC-eGFP transgenic reporter mouse in combination with bulk and single-cell RNA sequencing to redefine the endometrial mesenchyme. In contrast to previous reports we show that CD146 is expressed in both PDGFRβ + perivascular cells and CD31 + endothelial cells. Bulk RNAseq revealed cells in the perivascular niche which express the high levels of Pdgfrb as well as genes previously identified in pericytes and/or vascular smooth muscle cells (Acta2, Myh11, Olfr78, Cspg4, Rgs4, Rgs5, Kcnj8, and Abcc9). scRNA-seq identified five subpopulations of cells including closely related pericytes/vascular smooth muscle cells and three subpopulations of fibroblasts. All three fibroblast populations were PDGFRα+/CD34 + but were distinct in their expression of Ngfr/Spon2/Angptl7 (F1), Cxcl14/Smoc2/Rgs2 (F2), and Clec3b/Col14a1/Mmp3 (F3), with potential functions in the regulation of immune responses, response to wounding, and organization of extracellular matrix, respectively. Immunohistochemistry was used to investigate the spatial distribution of these populations revealing F1/NGFR + cells in most abundance beside epithelial cells. We provide the first definitive analysis of mesenchymal cells in the adult mouse endometrium identifying five subpopulations providing a platform for comparisons between mesenchymal cells in endometrium and other adult tissues which are prone to fibrosis.

The endometrium is a dynamic tissue that exhibits remarkable resilience to repeated episodes of differentiation, breakdown, regeneration and remodelling. Endometrial physiology relies on a complex interplay between the stromal and epithelial compartments with the former containing a mixture of fibroblasts, vascular and immune cells. There is evidence for rare populations of putative mesenchymal progenitor cells located in the perivascular niche of human endometrium, but the existence of an equivalent cell population in mouse is unclear.In the current study we used the Pdgfrb-BAC-eGFP transgenic reporter mouse in combination with bulk and single cell RNA sequencing (scRNAseq) to redefine the endometrial mesenchyme. Contrary to previous reports we show that CD146 is expressed in both PDGFRβ+ perivascular cells as well as CD31+ endothelial cells. Bulk RNAseq revealed cells in the perivascular niche which express high levels of Pdgfrb as well as genes previously identified in pericytes and/or vascular smooth muscle cells (Acta2, Myh11, Olfr78, Cspg4, Rgs4, Rgs5, Kcnj8, Abcc9). scRNAseq identified five subpopulations of cells including closely related pericytes/vascular smooth muscle cells and three subpopulations of fibroblasts. All three fibroblast populations were PDGFRα+/CD34+ but were distinct in their expression of Spon2/Angptl7 (fibroblast 1), Smoc2/Rgs2 (fibroblast 2) and Clec3b/Col14a1/Mmp3 (fibroblast 3), with potential functions in regulation of immune responses, response to wounding and organisation of extracellular matrix respectively.In conclusion, these data are the first to provide a single cell atlas of the mesenchymal cell landscape in mouse endometrium. By identifying novel markers for subpopulations of mesenchymal cells we can use mouse models investigate their contribution to endometrial function, compare with other tissues and apply these findings to further our understanding of human endometrium.HighlightsGFP expression in the mouse endometrium, under the control of the Pdgfrb promoter, is restricted to two cell populations based on the intensity of GFP with GFPbright cells close to the vasculatureSingle cell RNAseq identified five subpopulations of GFP+ mesenchymal cells: pericytes, vascular smooth muscle cells (vSMC) and three closely related but distinct populations of fibroblastsBioinformatics revealed that pericytes and vSMC share functions associated with the circulatory system, actin-filament process and cell adhesion, and an apparent role for pericytes in smooth muscle cell migration and response to interferonsComparisons between the fibroblast subpopulations suggest distinct roles in regulation of immune response, response to wound healing and collagen organisation.Graphical Abstract