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Single-Nucleus and In Situ RNA-Sequencing Reveal Cell Topographies in the Human Pancreas.
BACKGROUND & AIMS: Molecular evidence of cellular heterogeneity in the human exocrine pancreas has not been yet established because of the local concentration and cascade of hydrolytic enzymes that can rapidly degrade cells and RNA upon pancreatic resection. We sought to better understand the heterogeneity and cellular composition of the pancreas in neonates and adults in healthy and diseased conditions using single-cell sequencing approaches. METHODS: We innovated single-nucleus RNA-sequencing protocols and profiled more than 120,000 cells from pancreata of adult and neonatal human donors. We validated the single-nucleus findings using RNA fluorescence in situ hybridization, in situ sequencing, and computational approaches. RESULTS: We created the first comprehensive atlas of human pancreas cells including epithelial and nonepithelial constituents, and uncovered 3 distinct acinar cell types, with possible implications for homeostatic and inflammatory processes of the pancreas. The comparison with neonatal single-nucleus sequencing data showed a different cellular composition of the endocrine tissue, highlighting the tissue dynamics occurring during development. By applying spatial cartography, involving cell proximity mapping through in situ sequencing, we found evidence of specific cell type neighborhoods, dynamic topographies in the endocrine and exocrine pancreas, and principles of morphologic organization of the organ. Furthermore, similar analyses in chronic pancreatitis biopsy samples showed the presence of acinar-REG+ cells, a reciprocal association between macrophages and activated stellate cells, and a new potential role of tuft cells in this disease. CONCLUSIONS: Our human pancreas cell atlas can be interrogated to understand pancreatic cell biology and provides a crucial reference set for comparisons with diseased tissue samples to map the cellular foundations of pancreatic diseases.
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Joint profiling of gene expression and chromatin accessibility during amphioxus development at single-cell resolution.
Vertebrate evolution was accompanied by two rounds of whole-genome duplication followed by functional divergence in terms of regulatory circuits and gene expression patterns. As a basal and slow-evolving chordate species, amphioxus is an ideal paradigm for exploring the origin and evolution of vertebrates. Single-cell sequencing has been widely used to construct the developmental cell atlas of several representative species of vertebrates (human, mouse, zebrafish, and frog) and tunicates (sea squirts). Here, we perform single-nucleus RNA sequencing (snRNA-seq) and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) for different stages of amphioxus (covering embryogenesis and adult tissues). With the datasets generated, we constructed a developmental tree for amphioxus cell fate commitment and lineage specification and characterize the underlying key regulators and genetic regulatory networks. The data are publicly available on the online platform AmphioxusAtlas.
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A single-cell atlas of diffuse large B cell lymphoma.
Diffuse large B cell lymphoma (DLBCL) is one of the most common yet aggressive types of B cell lymphoma and remains incurable in 40% of patients. Herein, we profile the transcriptomes of 94,324 cells from 17 DLBCLs and 3 control samples using single-cell RNA sequencing. Altogether, 73 gene expression programs are identified in malignant cells, demonstrating high intra- and intertumor heterogeneity. Furthermore, 2,754 pairs of suggestive cell-cell interactions are predicted, indicating a complex and highly dynamic tumor microenvironment. Especially for B cell lymphomas, a strong costimulatory CD70-CD27 interaction is predicted between malignant and T cells. Furthermore, coinhibitory signals mediated by TIM3 or TIGIT seem to be the main driving force for T cell exhaustion. Finally, we find that chronic hepatitis B virus infection may have a significant impact on tumor cell survival and immune evasion in DLBCL. Our results provide insights into B cell lymphomagenesis and may facilitate the design of targeted immunotherapy strategies.
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Single-cell transcriptomics identifies chondrocyte differentiation dynamics in vivo and in vitro.
Developing in vitro chondrocytes that replicate in vivo development would benefit musculoskeletal disease modeling and regenerative medicine. Although current methodologies have made progress, challenges such as off-target differentiation can result in heterogeneous cell states. Furthermore, the lack of comparison with human embryonic tissue precludes detailed evaluation of in vitro cells. Here, we perform single-cell RNA sequencing (scRNA-seq) of embryonic long bones and combine this with public data to form an atlas of endochondral ossification. We use this to evaluate published in vitro chondrogenesis protocols that use human cell lines, finding variability in cells produced by each. We apply single-nuclear RNA sequencing (snRNA-seq) to our human embryonic stem cell chondrogenesis protocol and perform trajectory alignment with in vivo data to shed light on off-target differentiation in vitro. Using this information, we inhibit FOXO1, a transcription factor active in embryonic osteoblasts and in vitro cells, to increase chondrocyte transcripts in vitro. This work offers a framework for improving in vitro chondrogenesis using developmental data.
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Single-cell activation screen identifies hepatic maturation regulators with zonal resolution.
The maturation of lineage-committed embryonic hepatocytes requires both the timed activation of metabolic gene regulatory networks (GRNs) and silencing of embryonic programs to achieve adult hepatic functions. However, in vitro derivation of mature hepatocytes remains imperfect, and key transcriptional regulators governing GRN rewiring during late development are still insufficiently defined. To address this, we generated a developmental reference atlas and employed a dCas9 activation screen with single-cell transcriptomics on primary mouse embryonic hepatocytes, enabling effect ranking among late-onset transcription regulators. We identify Nr1i3 as a potent inducer of pericentrally expressed metabolic genes and Nfix as a critical suppressor of embryonic and periportal signatures. Supplementing liver zonation patterning signals with these regulators further enhanced the expression of pericentrally zonated metabolic genes, emphasizing the importance of a microenvironment-targeted approach. Our screening and analysis therefore highlight regulatory mechanisms underlying organ maturation and offer general strategies for improving the functionality of in vitro-derived cells.
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Single-cell transcriptomics reveals cellular heterogeneity and macrophage-to-mesenchymal transition in bicuspid calcific aortic valve disease.
BACKGROUND: Bicuspid aortic valve (BAV) is the most prevalent congenital valvular heart defect, and around 50% of severe isolated calcific aortic valve disease (CAVD) cases are associated with BAV. Although previous studies have demonstrated the cellular heterogeneity of aortic valves, the cellular composition of specific BAV at the single-cell level remains unclear. METHODS: Four BAV specimens from aortic valve stenosis patients were collected to conduct single-cell RNA sequencing (scRNA-seq). In vitro experiments were performed to further validate some phenotypes. RESULTS: The heterogeneity of stromal cells and immune cells were revealed based on comprehensive analysis. We identified twelve subclusters of VICs, four subclusters of ECs, six subclusters of lymphocytes, six subclusters of monocytic cells and one cluster of mast cells. Based on the detailed cell atlas, we constructed a cellular interaction network. Several novel cell types were identified, and we provided evidence for established mechanisms on valvular calcification. Furthermore, when exploring the monocytic lineage, a special population, macrophage derived stromal cells (MDSC), was revealed to be originated from MRC1+ (CD206) macrophages (Macrophage-to-Mesenchymal transition, MMT). FOXC1 and PI3K-AKT pathway were identified as potential regulators of MMT through scRNA analysis and in vitro experiments. CONCLUSIONS: With an unbiased scRNA-seq approach, we identified a full spectrum of cell populations and a cellular interaction network in stenotic BAVs, which may provide insights for further research on CAVD. Notably, the exploration on mechanism of MMT might provide potential therapeutic targets for bicuspid CAVD.
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Northstar enables automatic classification of known and novel cell types from tumor samples.
Single cell transcriptomics is revolutionising our understanding of tissue and disease heterogeneity, yet cell type identification remains a partially manual task. Published algorithms for automatic cell annotation are limited to known cell types and fail to capture novel populations, especially cancer cells. We developed northstar, a computational approach to classify thousands of cells based on published data within seconds while simultaneously identifying and highlighting new cell states such as malignancies. We tested northstar on data from glioblastoma, melanoma, and seven different healthy tissues and obtained high accuracy and robustness. We collected eleven pancreatic tumors and identified three shared and five private neoplastic cell populations, offering insight into the origins of neuroendocrine and exocrine tumors. Northstar is a useful tool to assign known and novel cell type and states in the age of cell atlases.
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Cellular atlases of ovarian microenvironment alterations by diet and genetically-induced obesity.
Obesity, which can arise from genetic or environmental factors, has been shown to cause serious damages to the reproductive system. The ovary, as one of the primary regulators of female fertility, is a complex organ comprised of heterogeneous cell types that work together to maintain a normal ovarian microenvironment (OME). Despite its importance, the effect of obesity on the entire ovary remains poorly documented. In this study, we performed ovary single-cell and nanoscale spatial RNA sequencing to investigate how the OME changed under different kinds of obesity, including high-fat diet (HFD) induced obesity and Leptin ablation induced obesity (OB). Our results demonstrate that OB, but not HFD, dramatically altered the proportion of ovarian granulosa cells, theca-interstitial cells, luteal cells, and endothelial cells. Furthermore, based on the spatial dynamics of follicular development, we defined four subpopulations of granulosa cell and found that obesity drastically disrupted the differentiation of mural granulosa cells from small to large antral follicles. Functionally, HFD enhanced follicle-stimulating hormone (FSH) sensitivity and hormone conversion, while OB caused decreased sensitivity, inadequate steroid hormone conversion, and impaired follicular development. These differences can be explained by the differential expression pattern of the transcription factor Foxo1. Overall, our study provides a powerful and high-resolution resource for profiling obesity-induced OME and offers insights into the diverse effects of obesity on female reproductive disorders.
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Insights gained from single-cell RNA analysis of murine endothelial cells in aging hearts.
Aging is the strongest risk factor for cardiovascular disease, with progressive decline in the function of vascular endothelial cells (ECs) with age. Systematic analyses of the effects of aging on different cardiac EC types remain limited. Here, we constructed a scRNA atlas of EC transcriptomes in young and old mouse hearts. We identified 10 EC subclusters. The multidimensionally differential genes (DEGs) analysis across different EC clusters shows molecular changes with aging, showing the increase in the overall inflammatory microenvironment and the decrease in angiogenesis and cytoskeletal support capacity of aged ECs. And we performed an in-depth analysis of 3 special ECs, Immunology, Proliferating and Angiogenic. The Immunology EC seems highly associated with some immune regulatory functions, which decline with aging at different degrees. Analysis of two types of neovascular ECs, Proliferating, Angiogenic, implied that Angiogenic ECs can differentiate into multiple EC directions after initially originating from proliferating ECs. And aging leads to a decrease in the ability of vascular angiogenesis and differentiation. Finally, we summarized the effects of aging on cell signaling communication between different EC clusters. This cardiac EC atlas offers comprehensive insights into the molecular regulations of cardiovascular aging, and provides new directions for the prevention and treatment of age-related cardiovascular disease.
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Comprehensive mapping of Neurofibromin (NF1) expression in developing mouse brain.
Neurofibromin (NF1) is a critical negative regulator of the RAS-RAF-ERK pathway, mutations in which have been clinically implicated in various neurodevelopmental disorders. However, the lack of a high-resolution spatiotemporal map has obscured the understanding of why specific cell populations and developmental processes are uniquely vulnerable to NF1 loss. In this study, we present a comprehensive atlas of NF1 expression in the developing mouse brain. Using in situ hybridization and immunohistochemistry, we characterized the spatial distribution of NF1 expression from early embryonic stages through postnatal maturation. We further integrated these findings with reanalysis of single-nuclei RNA-sequencing (snRNA-seq) datasets from adult mouse brain to achieve higher resolution. Our results reveal a previously undocumented graded expression pattern of NF1 across various brain regions and cell lineages. This comprehensive study will not only help us understand the fundamental role of NF1 in brain development but will also be pivotal in providing a framework for studying NF1-associated brain disorders.