Gut Network

Gut Network - Overview

We performed single-cell RNA-sequencing to create a cell census of the human thymus during development, early childhood and adult life. We sampled 15 embryonic and fetal thymi spanning thymic developmental stages between 7 post-conception weeks (PCW) to 17 PCW, and 9 postnatal thymi from paediatric and adult samples. We compared the cellular composition and T cell differentiation within human and mouse thymus using newly generated and repository mouse datasets. Finally, we investigated the bias in the recombination and selection of human versus mouse TCR repertoires.

A cell atlas of human thymic development defines T cell repertoire formation

The gene expression program underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of gene expression and chromatin accessibility in fetal tissues. For gene expression, we applied three-level combinatorial indexing to >110 samples representing 15 organs, ultimately profiling ~4 million single cells. We leveraged the literature and other atlases to identify and annotate hundreds of cell types and subtypes, both within and across tissues. Our analyses focused on organ-specific specializations of broadly distributed cell types (such as blood, endothelial, and epithelial), sites of fetal erythropoiesis (which notably included the adrenal gland), and integration with mouse developmental atlases (such as conserved specification of blood cells). These data represent a rich resource for the exploration of in vivo human gene expression in diverse tissues and cell types.

A human cell atlas of fetal gene expression.

Background and Aims: Single-cell transcriptomics offer unprecedented resolution of tissue function at the cellular level, yet studies analyzing healthy adult human small intestine and colon are sparse. Here, we present single-cell transcriptomics covering the duodenum, jejunum, ileum, and ascending, transverse, and descending colon from 3 humans. Methods: 12,590 single epithelial cells from three independently processed organ donors were evaluated for organ-specific lineage biomarkers, differentially regulated genes, receptors, and drug targets. Analyses focused on intrinsic cell properties and capacity for response to extrinsic signals along the gut axis across different humans. Results: Cells were assigned to 25 epithelial lineage clusters. Human intestinal stem cells (ISCs) are not specifically marked by many murine ISC markers. Lysozyme expression is not unique to human Paneth cells (PCs), and PCs lack expression of expected niche-factors. BEST4+ cells express NPY and show maturational differences between SI and colon. Tuft cells possess a broad ability to interact with the innate and adaptive immune systems through previously unreported receptors. Some classes of mucins, hormones, cell-junction, and nutrient absorption genes show unappreciated regional expression differences across lineages. Differential expression of receptors and drug targets across lineages reveals biological variation and potential for variegated responses. Conclusions: Our study identifies novel lineage marker genes; covers regional differences; shows important differences between mouse and human gut epithelium; and reveals insight into how the epithelium responds to the environment and drugs. This comprehensive cell atlas of the healthy adult human intestinal epithelium resolves likely functional differences across anatomical regions along the gastrointestinal tract and advances our understanding of human intestinal physiology. Overall design: Single cell RNA sequencing libraries from epithelium isolated from Duodenum, Jejunum, Ileum, Ascending Colon, Transverse Colon, and Descending Colon from each of 3 human organ donors were compiled

A proximal-to-distal survey of healthy adult human small intestine and colon epithelium by single-cell transcriptomics

Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of gene regulatory elements in diverse cell types and tissues in the human body, we applied single-cell chromatin accessibility assays to 30 adult human tissue types from multiple donors. We integrated these datasets with single-cell chromatin accessibility data from 15 fetal tissue types to reveal the status of open chromatin for approximately 1.2 million candidate cis-regulatory elements (cCREs) in 222 distinct cell types comprised of >1.3 million nuclei. We used these chromatin accessibility maps to delineate cell type-specificity of fetal and adult human cCREs and to systematically interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues, life stages, and organ systems. Overall design: Applying single cell chromatin accessibility assays to diverse human tissue types from four donors.

A single-cell atlas of chromatin accessibility in the human genome

Human intestinal organoids (HIOs) generated from pluripotent stem cells provide extraordinary opportunities to explore development and disease. Here, we generate a single-cell transcriptome reference atlas from HIOs and from multiple developing human organs to quantify the specificity of HIO cell fate acquisition, and to explore alternative fates. We identify epithelium-mesenchyme interactions, transcriptional regulators involved in cell fate specification, and stem cell maturation features in the primary tissue that are recapitulated in HIOs. We use an HIO time course to reconstruct the molecular dynamics of intestinal stem cell emergence, as well as the specification of multiple mesenchyme subtypes. We find that the intestinal master regulator CDX2 correlates with distinct phases of epithelial and mesenchymal development, and CDX2 deletion perturbs the differentiation of both intestinal epithelium and mesenchyme. Collectively our data provides a comprehensive and quantitative assessment of HIO development, and illuminates the molecular machinery underlying endodermal and mesodermal cell fate specification.

An organoid and multi-organ developmental cell atlas reveals multilineage fate specification in the human intestine

Crohn’s disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. We used single cell RNA sequencing (scRNAseq) to define the cellular and transcriptional activity of the mucosa at different stages of disease progression in CD. Overall design: Terminal ileal mucosal derived single cells was processed and analyzed using scRNAseq

Assessing Cellular and Transcriptional Diversity of Ileal Mucosa Among Treatment-Naïve and Treated Crohn’s Disease

Single-cell RNA sequencing (scRNA-seq) is the leading technique for charting the molecular properties of individual cells. The latest methods are scalable to thousands of cells, enabling in- depth characterization of sample composition without prior knowledge. However, there are important differences between scRNA-seq techniques, and it remains unclear which are the most suitable protocols for drawing cell atlases of tissues, organs and organisms. We have generated benchmark datasets to systematically evaluate techniques in terms of their power to comprehensively describe cell types and states. We performed a multi-center study comparing 13 commonly used single-cell and single-nucleus RNA-seq protocols using a highly heterogeneous reference sample resource. Comparative and integrative analysis at cell type and state level revealed marked differences in protocol performance, highlighting a series of key features for cell atlas projects. These should be considered when defining guidelines and standards for international consortia, such as the Human Cell Atlas project. Overall design: The samples consists of two complex tissues (human PBMC and mouse colon) and three cell lines (HEK293-RFP, NIH3T3-GFP, MDCK-Turbo650). The primary PBMC and the colon sample constitute 90% and the cell lines 10%. The sample preparation aims to be standardized for all methods to allow a comparison of library preparation performance (6% HEK293T-RFP, 3% NIH3T3-GFP, 1% MDCK-Turbo650) of the sample content.

Benchmarking Single-Cell RNA Sequencing Protocols for Cell Atlas Projects

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. To comprehensively map cell lineages in the healthy developing, pediatric and adult human gut from ten distinct anatomical regions, as well as draining lymph nodes, we used single cell RNA-seq and VDJ analysis of roughly one third of a million cells. This reveals the presence of BEST4+ absorptive cells throughout the human intestinal tract, demonstrating the existence of this cell type beyond the colon for the first time. Furthermore, we implicate IgG sensing as a novel function of intestinal tuft cells, and link these cells to the pathogenesis of inflammatory bowel disease. We define novel glial and neuronal cell populations in the developing enteric nervous system, and predict cell-type specific expression of Hirschsprung’s disease-associated genes. Finally, using a systems approach, we identify key cell players across multiple cell lineages driving secondary lymphoid tissue formation in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. These data provide an unprecedented catalogue of intestinal cells, and new insights into cellular programs in development, homeostasis and disease.

Cells of the human intestinal tract mapped across space and time

Three single cell RNA-sequencing (10x, Chromium) experiments were performed on healthy and inflamed human colonic samples, including from patients with Ulcerative Colitis. The authors characterised the composition of the intestinal epithelial cells in these samples.

Colonic single epithelial cell census reveals goblet cell drivers of barrier breakdown in inflammatory bowel disease.

Single-cell RNA sequencing was used to determine the cell type composition of all major human organs and construct a basic scheme for the human cell landscape (HCL).

Construction of A Human Cell Landscape by Single-cell mRNA-seq

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. Here, we surveyed the immune compartment of 15 tissues of six deceased adult donors by single-cell RNA sequencing and paired VDJ sequencing. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of 45 finely phenotyped immune cell types and states, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. In summary, our multi-tissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis and antigen receptor sequencing.

Cross-tissue immune cell analysis reveals tissue-specific features in humans

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps

Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) lack some cellular populations found in the native organ, including vasculature. Using single-cell RNA sequencing (scRNA-seq), we have identified a population of endothelial cells (ECs) present early in HIO differentiation that declines over time in culture. Here, we developed a method to expand and maintain this endogenous population of ECs within HIOs (vHIOs). Given that ECs possess organ-specific gene expression, morphology, and function, we used bulk RNA-seq and scRNA-seq to interrogate the developing human intestine, lung, and kidney in order to identify organ-enriched EC gene signatures. By comparing these gene signatures and validated markers to HIO ECs, we find that HIO ECs grown in vitro share the highest similarity with native intestinal ECs relative to kidney and lung. Together, these data demonstrate that HIOs can co-differentiate a native EC population that is properly patterned with an intestine-specific EC transcriptional signature in vitro.

Differentiation of Human Intestinal Organoids with Endogenous Vascular Endothelial Cells

Gastrointestinal microbiota and immune cells interact closely and display regional specificity, but little is known about how these communities differ with location. Here, we simultaneously assess microbiota and single immune cells across the healthy, adult human colon, with paired characterisation of immune cells in the mesenteric lymph nodes, to delineate colonic immune niches at steady-state. We describe distinct T helper 1 cell activation and migration profiles along the colon and characterise the transcriptional adaptation trajectory of T regulatory cells between lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal expansion and mutational frequency from caecum to sigmoid colon, and link this to the increasing number of reactive bacterial species

Distinct microbial and immune niches of the human colon

Unbiased whole-genome methylome was studied in the wound-edge (WE) tissue of chronic wound patients. Methylation status of proximal promoter (1Kb) was calculated using MethylCap-Seq and PrEMeR-CG data analyses. A total of 4689 differentially methylated regions (DMRs) were identified in chronic WE compared to unwounded (UW) human skin. Hypermethylation was more frequently observed (3661 DMRs) in the chronic WE compared to hypomethylation (1028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial to mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. Whole genome RNA sequencing analysis was performed to qualify findings from methylome analysis. Hierarchical clustering analyses identified a large set of genes, the expression of which were significantly downregulated in chronic WE compared to UW skin. Analysis of the downregulated genes identified the TP53 signaling pathway, including P63, P73, FOXO3, SIRT1 and HDAC1, as being significantly silenced. Direct comparison of hypermethylation and downregulated gene expression identified four genes, ADAM17, NOTCH, TWIST1 and SMURF1, that were common to both data sets and functionally represented the EMT pathway. Single cell RNA sequencing studies identified that these effects on gene expression were limited to the keratinocyte cell compartment. Overall design: In this work, scRNA-seq analyses of human chronic WE characterized its heterogenous cellular composition as compared to unwounded skin.

Genome-Wide DNA Hypermethylation in the Wound-Edge of Chronic Wound Patients Opposes Closure by Impairing Epithelial to Mesenchymal Transition

Inflammatory bowel disease (IBD) encompasses a spectrum of gastrointestinal disorders driven by dysregulated immune responses against gut microbiota. We integrated single-cell RNA and antigen receptor sequencing to elucidate key components, cellular states, and clonal relationships of the peripheral and gastrointestinal mucosal immune systems in health and ulcerative colitis (UC). UC was associated with an increase in IgG1+ plasma cells in colonic tissue, increased colonic regulatory T cells characterized by elevated expression of the transcription factor ZEB2, and an enrichment of a γδ T cell subset in the peripheral blood. Moreover, we observed heterogeneity in CD8+ tissue-resident memory T (TRM) cells in colonic tissue, with four transcriptionally distinct states of differentiation observed across health and disease. In the setting of UC, there was a marked shift of clonally related CD8+ TRM cells toward an inflammatory state, mediated, in part, by increased expression of the T-box transcription factor Eomesodermin. Together, these results provide a detailed atlas of transcriptional changes occurring in adaptive immune cells in the context of UC and suggest a role for CD8+ TRM cells in IBD.

Heterogeneity and clonal relationships of adaptive immune cells in ulcerative colitis revealed by single-cell analyses

The impact of tissue-resident memory T cells (Trm) on the pathogenesis of inflammatory bowel disease remains controversial due to their heterogeneity. In particular, the pathological function of CD4+Trm, as opposed to their CD8+counterparts, is largely unknown. Here, using a comprehensive analytical approach, we found that CD103+CD4+Trm with an inflammatory phenotype were increased in the gut of Crohn’s disease (CD) patients but not in ulcerative colitis patients. Further profiling revealed that a subset of CD103+CD4+Trm, expressing CD161 and CCR5, were specific to CD patients and that this subset exerted cytotoxic activity. CD103+CD161+CCR5+CD4+Trm were predominant producers of proinflammatory cytokines, highlighting the importance of this specific subset in the pathogenesis of CD.

Identification of a unique subset of tissue-resident memory CD4+ T cells in Crohn’s disease

Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 cells from the colon mucosa of 18 UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4+ enterocytes, microfold-like cells, and IL13RA2+IL11+ inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and T cells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways.

Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis.

Immunotherapy for metastatic colorectal cancer is effective only for mismatch repair-deficient tumors with high microsatellite instability that demonstrate immune infiltration, suggesting that tumor cells can determine their immune microenvironment. To understand this cross-talk, we analyzed the transcriptome of 91,103 unsorted single cells from 23 Korean and 6 Belgian patients. Cancer cells displayed transcriptional features reminiscent of normal differentiation programs, and genetic alterations that apparently fostered immunosuppressive microenvironments directed by regulatory T cells, myofibroblasts and myeloid cells. Intercellular network reconstruction supported the association between cancer cell signatures and specific stromal or immune cell populations. Our collective view of the cellular landscape and intercellular interactions in colorectal cancer provide mechanistic information for the design of efficient immuno-oncology treatment strategies.

Lineage-dependent gene expression programs influence the immune landscape of colorectal cancer.

The human intestinal stem cell niche supports self-renewal and epithelial function, but little is known about its development. We used single-cell mRNA sequencing with in situ validation approaches to interrogate human intestinal development from 7–21 weeks post conception, assigning molecular identities and spatial loca- tions to cells and factors that comprise the niche. Smooth muscle cells of the muscularis mucosa, in close proximity to proliferative crypts, are a source of WNT and RSPONDIN ligands, whereas EGF is expressed far from crypts in the villus epithelium. Instead, an PDGFRAHI/F3HI/DLL1HI mesenchymal population lines the crypt-villus axis and is the source of the epidermal growth factor (EGF) family member NEUREGULIN1 (NRG1). In developing intestine enteroid cultures, NRG1, but not EGF, permitted increased cellular diversity via differentiation of secretory lineages. This work highlights the complexities of intestinal EGF/ERBB signaling and delineates key niche cells and signals of the developing intestine.

Mapping Development of the Human Intestinal Niche at Single-Cell Resolution

Recent advances in single cell genomics technologies have facilitated studies on the developing immune system at unprecedented scale and resolution. However, these studies have focused on one or a few organs and were thus limited in understanding the developing immune system as a distributed network across tissues. Here, we profiled prenatal haematopoietic organs, lymphoid organs and non-lymphoid tissues using a combination of single-cell RNA sequencing, paired antigen-receptor sequencing and spatial transcriptomics to reconstruct the developing human immune system. Our analysis revealed the acquisition of immune effector transcriptome profiles in macrophages, mast cells and NK cells from the second trimester, and the transcriptomic changes accompanying the late-stage maturation of developing monocytes and T cells that extended from their organ of origin to peripheral tissues. We uncovered system-wide blood and immune cell development beyond the conventional primary haematopoietic organs. We further identified, extensively characterised and functionally validated the human prenatal B1 cells. Finally, we provide evidence for thymocyte-thymocyte selection origin for αβTCR- expressing unconventional T cells based on TCR gene usage and an in vitro artificial thymic organoid culture model. Our comprehensive atlas of the developing human immune system provides both valuable data resources and biological insights that will facilitate cell engineering, regenerative medicine and disease understanding. 

One-Sentence Summary 
By performing a comprehensive single-cell RNA sequencing atlas of human developing immune system together with antigen-receptor sequencing and spatial transcriptomics, we explored the cross-gestation and cross-organ variability in immune cells, discovered system-wide blood and immune cell development, identified, characterised and functionally validated the properties of human prenatal B1 cells and the origin of unconventional T cells.

Mapping the developing human immune system across organs

Checkpoint blockade with antibodies specific for the PD-1 and CTLA-4 inhibitory receptors can induce durable responses in a wide range of human cancers. However, the immunological mechanisms responsible for severe inflammatory side effects remain poorly understood. Here we report a comprehensive single cell analysis of immune cell populations in colitis, a common and severe side effect of checkpoint blockade. We observed strong enrichment of several T cell clusters in colitis lesions compared to control cases, in particular a striking accumulation of CD8 T cells with signatures reflecting highly cytotoxic and proliferative states. T cell receptor (TCR) sequence analysis demonstrated that a substantial fraction of colitis-associated CD8 T cells originated from tissue-resident populations, explaining the frequently early onset of colitis symptoms following treatment initiation. Our analysis also identified cytokines, chemokines and surface receptors that could serve as therapeutic targets for colitis and potentially other inflammatory side effects of checkpoint blockade. Overall design: Samples from 22 patients from 3 different cohorts (Normal control, +CPI no colitis, +CPI colitis)

Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy

Pediatric-onset colitis and inflammatory bowel disease (IBD) have significant effects on the growth of infants and children, but the etiopathogenesis underlying disease subtypes remains incompletely understood. Here, we report single-cell clustering, immune phenotyping, and risk gene analysis for children with undifferentiated colitis, Crohn's disease, and ulcerative colitis. We demonstrate disease-specific characteristics, as well as common pathogenesis marked by impaired cyclic AMP (cAMP)-response signaling. Specifically, infiltration of PDE4B- and TNF-expressing macrophages, decreased abundance of CD39-expressing intraepithelial T cells, and platelet aggregation and release of 5-hydroxytryptamine at the colonic mucosae were common in colitis and IBD patients. Targeting these pathways by using the phosphodiesterase inhibitor dipyridamole restored immune homeostasis and improved colitis symptoms in a pilot study. In summary, comprehensive analysis of the colonic mucosae has uncovered common pathogenesis and therapeutic targets for children with colitis and IBD.

Mucosal Profiling of Pediatric-Onset Colitis and IBD Reveals Common Pathogenics and Therapeutic Pathways

During early human pregnancy the uterine mucosa transforms into the decidua, into which the fetal placenta implants and where placental trophoblast cells intermingle and communicate with maternal cells. Trophoblast–decidual interactions underlie common diseases of pregnancy, including pre-eclampsia and stillbirth. Here we profile the transcriptomes of about 70,000 single cells from first-trimester placentas with matched maternal blood and decidual cells. The cellular composition of human decidua reveals subsets of perivascular and stromal cells that are located in distinct decidual layers. There are three major subsets of decidual natural killer cells that have distinctive immunomodulatory and chemokine profiles. We develop a repository of ligand–receptor complexes and a statistical tool to predict the cell-type specificity of cell–cell communication via these molecular interactions. Our data identify many regulatory interactions that prevent harmful innate or adaptive immune responses in this environment. Our single-cell atlas of the maternal–fetal interface reveals the cellular organization of the decidua and placenta, and the interactions that are critical for placentation and reproductive success.

Reconstructing the human first trimester fetal-maternal interface using single cell transcriptomics

To determine and dissect the extent of tumor, immunological, and stromal heterogeneity in CRC patients, we performed droplet-based scRNA-seq on 16 racially diverse, treatment naïve CRC patient tissue samples and seven adjacent normal colonic tissue samples (totaling 23 samples), yielded 49,589 single cells enabling high-resolution depiction of the cellular diversity and heterogeneity within the tumor and microenvironmental cells.

Refining Colorectal Cancer Classification and Clinical Stratification Through a Single-Cell Atlas

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) which causes the disease COVID-19. SARS-CoV-2 spike (S)-protein binds ACE2, and in concert with host proteases, principally TMPRSS2, promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues, and the factors that regulate ACE2 expression, remain unknown. Here, we leverage human and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 amongst tissue-resident cell subsets. Overall design: Samples from 8 datasets are included: single-cell RNA-Seq studies: 2 human adult donors of inferior turbinate scrapings with two replicates each. Four mouse donors of nasal epithelial cells, two stimulated with IFNA and two controls with two replicates from each animal. 13 human ileal small intestine donors. 8 human lung tissue donors with mTB infection, mTB infection and HIV, or no mTB infection. Bulk RNA-seq samples: Two human donors stimulated with three replicates of each stimulation with IFNA, IFNG, IL17A, or IL4, each at increasing doses (0, 0.1 ,0.5, 1, 2, 5, 10 ng/mL) and 12 replicates of unstimulated samples. One mouse donor of cells from the trachea with three replicates of each stimulation with IFNA, IFNB, or IFNG, each at increasing doses (0, 0.1 ,0.5, 1, 2, 5, 10 ng/mL) and 12 replicates of unstimulated samples. One human cell line stimulated with three replicates of each stimulation with IFNA, IFNG, IL17A, or IL4, each at increasing doses (0, 0.1 ,0.5, 1, 2, 5, 10 ng/mL) and 12 replicates of unstimulated samples.

SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues.

Endometriosis is characterized by growth of endometrial-like tissue outside of the uterus affecting many women during their reproductive age, causing years of pelvic pain and potential infertility. Its pathophysiology remains largely unknown, limiting early diagnosis and treatment. We characterized peritoneal and ovarian lesions at single-cell transcriptome resolution and compared to matched eutopic endometrium, control endometrium, and organoids derived from these tissues, generating data on over 122,000 cells across 14 individuals. We spatially localized many of the cell types using imaging mass cytometry. We identify a perivascular mural cell unique to the peritoneal lesions with dual roles in angiogenesis promotion and immune cell trafficking. We define an immunotolerant peritoneal niche, fundamental differences in eutopic endometrium and between lesions microenvironments, and an unreported progenitor-like epithelial cell subpopulation. Altogether, this study provides a holistic view of the endometriosis microenvironment representing a comprehensive cell atlas of the disease in hormonally treated patients, essential information for advancing therapeutics and diagnostics. Overall design: Biopsies were collected from a total of 27 patients, 19 with confirmed endometriosis and 8 patients control. Biopsies were subjected to single cell experiments, bulk sequencing and cell hashing on patient-derived organoid. Single cells experiement were conducted on 31 biopsies consisting of 3 control and 9 eutopic endometrium, 8 ectopic peritoneal, 6 ectopic peritoneal adjacent, 4 ectopic ovary of endometriosis patient. A total of 24 biopsies were subjected to bulk sequencing, consist of 5 control and 7 eutopic endometrium, 6 ectopic peritoneal, 6 ectopic ovary of endometriosis patient. Single-cell cell hashing experiment were conducted on patient-derived organoid from 2 control and 4 eutopic endometrium, 4 ectopic peritoneum, 1 ectopic peritoneal adjacent of endometriosis patient. All single cell experiments were performed on 10x Genomics platform.

Single cell analysis of endometriosis reveals a coordinated transcriptional program driving immunotolerance and angiogenesis across eutopic and ectopic tissues

Celiac disease (CeD) is an autoimmune disorder in which ingestion of dietary gluten triggers an immune reaction in the small intestine 1,2 . The CeD lesion is characterized by crypt hyperplasia, villous atrophy and chronic inflammation with accumulation of leukocytes both in the lamina propria (LP) and in the epithelium 3 , which eventually leads to destruction of the intestinal epithelium 1 and subsequent digestive complications and higher risk of non-hodgkin lymphoma 4 . A lifetime gluten-free diet is currently the only available treatment 5 . Gluten-specific LP CD4 T cells and cytotoxic intraepithelial CD8+ T cells are thought to be central in disease pathology 1,6-8 , however, CeD is a complex immune-mediated disorder and to date the findings are mostly based on analysis of heterogeneous cell populations and on animal models. Here, we comprehensively explore the cellular heterogeneity of CD45+ immune cells in human small intestine using index-sorting single-cell RNA-sequencing 9,10 . We find that myeloid and mast cell transcriptomes are reshaped in CeD. We observe extensive changes in the proportion and transcriptomes of CD4+ and CD8+ T cells and define a CD3zeta expressing NK-T-like cell population present in the control LP and epithelial layers that is absent and replaced in CeD. Our findings show that the immune landscape is dramatically changed in active CeD which provide new insights and considerably extend the current knowledge of CeD immunopathology.

Single cell transcriptomic analysis of the immune cell compartment in the human small intestine and in Celiac disease

Gastric cancer heterogeneity represents a barrier to disease management. We generated a comprehensive single-cell atlas of gastric cancer (>200,000 cells) comprising 48 samples from 31 patients across clinical stages and histologic subtypes. We identified 34 distinct cell-lineage states including novel rare cell populations. Many lineage states exhibited distinct cancer-associated expression profiles, individually contributing to a combined tumor-wide molecular collage. We observed increased plasma cell proportions in diffuse-type tumors associated with epithelial-resident KLF2 and stage-wise accrual of cancer-associated fibroblast subpopulations marked by high INHBA and FAP coexpression. Single-cell comparisons between patient-derived organoids (PDO) and primary tumors highlighted inter- and intralineage similarities and differences, demarcating molecular boundaries of PDOs as experimental models. We complemented these findings by spatial transcriptomics, orthogonal validation in independent bulk RNA-sequencing cohorts, and functional demonstration using in vitro and in vivo models. Our results provide a high-resolution molecular resource of intra- and interpatient lineage states across distinct gastric cancer subtypes.SignificanceWe profiled gastric malignancies at single-cell resolution and identified increased plasma cell proportions as a novel feature of diffuse-type tumors. We also uncovered distinct cancer-associated fibroblast subtypes with INHBA-FAP-high cell populations as predictors of poor clinical prognosis. Our findings highlight potential origins of deregulated cell states in the gastric tumor ecosystem. This article is highlighted in the In This Issue feature, p. 587.

Single-Cell Atlas of Lineage States, Tumor Microenvironment, and Subtype-Specific Expression Programs in Gastric Cancer

The project was performed to characterize all kinds of human gastric cells and clarify the physiology of normal gastric mucosa and the development of intestinal metaplasia. Single-cell RNA sequence and downstream analyses were applied to elucidate and to build up a hypothesis. Here, we constructed a largest human gastric atlas comprised of 137,610 cells from our university and other two public databases. We identified new possible stem cell marker genes and activated transcription factor in the stem cells. Moreover, we clarified a spatial distribution of the fibroblasts in the gastric mucosa by RNA in situ hybridization and the results suggested the difference of fibroblasts between intestinal metaplasia and genuine colonic mucosa. Cell-cell communication analysis also indicated that a potential interaction in the stem cell niche and intestinal metaplasia-specific interaction between epithelial cells and fibroblasts.

Single-Cell Transcriptome and Downstream Analyses of Human Stomach Reveal the Cell Diversity and Developmental Features of Gastric and Metaplastic Mucosae

Crohn's disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Here, we profile, via multiple single-cell technologies, T cells purified from the intestinal epithelium and lamina propria (LP) from terminal ileum resections of adult severe CD cases. We find that intraepithelial lymphocytes (IEL) contain several unique T cell subsets, including NKp30+γδT cells expressing RORγt and producing IL-26 upon NKp30 engagement. Further analyses comparing tissues from non-inflamed and inflamed regions of patients with CD versus healthy controls show increased activated TH17 but decreased CD8+T, γδT, TFH and Treg cells in inflamed tissues. Similar analyses of LP find increased CD8+, as well as reduced CD4+T cells with an elevated TH17 over Treg/TFH ratio. Our analyses of CD tissues thus suggest a potential link, pending additional validations, between transmural inflammation, reduced IEL γδT cells and altered spatial distribution of IEL and LP T cell subsets.

Single-cell analyses of Crohn's disease tissues reveal intestinal intraepithelial T cells heterogeneity and altered subset distributions

Clinical benefits of cytokine blockade in ileal Crohn’s disease (iCD) are limited to a subset of patients. Here we applied single cell technologies to iCD lesions to address whether cellular heterogeneity contributes to treatment resistance. We found that a subset of patients expressed a unique cellular module in inflamed tissues that consisted of IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells and stromal cells, which we named the GIMATS module. Analysis of ligand-receptor interaction pairs identified a distinct connectivity network that likely drives the GIMATS module. Strikingly, the GIMATS module was also present in a subset of patients in 4 independent iCD cohorts (n=441), and its presence at diagnosis correlated with failure to achieve corticosteroid-free durable remission upon anti-TNF therapy. These results emphasize the limitations of current diagnostic assays and the potential for single cell mapping tools to identify novel biomarkers of treatment response and tailored therapeutic opportunities

Single-cell analysis of Crohn’s disease lesions identifies a pathogenic cellular module associated with resistance to anti-TNF therapy

Analysis of genomic and transcriptomic profiling has proposed new insights for diagnosis and prognosis in gastric cancer (GC). Human GCs are a unified model composed of diverse cells, including premalignant, immune and cancer. In this study, we present a human cell population of GC and premalignant states at single cell resolution using the 10X Chromium platform. We profiled 112,041 gastric cell landscape across 24 individuals with adjacent normal and GC lesions. Overall design: For a total of 24 patients with 9 intestinal gastric cancers (IGC) and 15 diffuse gastric cancers (DGC), 13,022 cells consisted of 47 biopsies both 24 GC lesions and 23 adjacent normals were selected after a quality control.

Single-cell analysis of gastric pre-cancerous and cancer lesions reveals cell lineage diversity and intratumoral heterogeneity

Immunotherapies have been explored in treating solid tumors, albeit with disparate clinical effects in distinct cancer types. Systematic interrogation of immune cells in the tumor microenvironment (TME) is vital to the prediction of immunotherapy response and the development of innovative immunotherapeutics. To comprehensively characterize the immune microenvironment in advanced biliary tract cancer (BTC), we utilized single-cell RNA sequencing in unselected viable cells from 16 matched samples. We identified nineteen cell subsets from a total of 45,851 cells, in which exhausted CD8+ T cells, macrophages, and dendritic cells (DCs) in BTC were shown to augment and communicate within the TME. Transcriptional profiles coupled with T cell receptor (TCR) sequences revealed that exhausted CD8+ T cells retained clonal expansion and high proliferation in the TME, and some of them highly expressed the endoplasmic reticulum stress (ER) sensor XBP1, indicating its key role in remodeling TME. Functional assays demonstrated that XBP1 and common immune checkpoints (PD1, TIGIT) were significantly upregulated in CD8+ T cells cocultured within the TME of BTC cell lines (GBC-SD, HCCC-9810). When treating the coculture groups with the specific inhibitor of IRE1α-XBP1 (4μ8C), the downregulation of TIGIT was observed in the treatment group. Collectively, comprehensive transcriptome profiling provides deeper insights into the immune atlas in advanced BTC, which might be instrumental in exploring novel immunotherapy strategies. Overall design: Five patients diagnosed with cholangiocarcinoma in the Eastern Hepatobiliary Surgery Hospital were incorporated into the current study, including two intrahepatic cholangiocarcinoma (iCCA), two gallbladder carcinoma (GBC) and one dCCA patients. Of note, none of the patients have undergone any preoperative treatments (chemotherapy, radiation or anti-tumor medicines) prior to tumor resection. Tumor primary locus and metastasis as well as lymph node were consistently harvested for each participant, if available. Meanwhile, peripheral blood samples were collected prior to surgical procedures using anticoagulant tubes.

Single-cell atlas of diverse immune populations in the advanced biliary tract cancer microenvironment

The functions of innate lymphoid cells (ILCs) in immune system are increasingly appreciated, whereas the early development of ILCs in human remains elusive. In this study, we sorted humanhematopoietic stem progenitor cells, lymphoid progenitors, presumed ILC progenitor/precursors and mature ILCs in the fetal hematopoietic, lymphoid and non-lymphoid tissues, from 8 to 12 PCW,for single-cell RNA-sequencing, followed by computational analysis and functional validation. We delineated the early phase of ILC development, from hematopoietic stem progenitor cells to multipotent lymphoid progenitors and to ILC progenitors, which mainly occurred in fetal liver and intestine. We further unveiled interleukin-3 receptor alpha (IL-3RA) as the surface marker for the lymphoid progenitors with T cell, B cell and ILC potentials. Notably, we determined the heterogeneity and tissue distribution of each ILC subpopulation, revealing the shared proliferating characteristics of the precursors of each ILC subtype. Additionally, a novel unconventional ILC2 subpopulation (CRTH2-CCR9+ ILC2) was identified in fetal thymus. Taken together, our study illuminates the precise cellular and molecular features underlying the stepwise formation of human fetal ILC hierarchy with remarkable spatiotemporal heterogeneity. Overall design: Here, we provided an unprecedented single-cell transcriptome atlas of stepwise ILC ontogeny in early human fetus using cells from fetal hematopoietic, lymphoid and non-lymphoid tissues spanning 8 to 12 post-conception weeks (PCWs).

Single-cell deciphering of human fetal innate lymphoid cell development

Background: As core units of organ tissues, cells of various types play their harmonious rhythms to maintain the homeostasis of the human body. It is essential to identify the characteristics of cells in human organs and their regulatory networks for understanding the biological mechanisms related to health and disease. However, a systematic and comprehensive single-cell transcriptional profile across multiple organs of a normal human adult is missing. Results: We perform single-cell transcriptomes of 84,363 cells derived from 15 tissue organs of one adult donor and generate an adult human cell atlas. The adult human cell atlas depicts 252 subtypes of cells, including major cell types such as T, B, myeloid, epithelial, and stromal cells, as well as novel COCH+ fibroblasts and FibSmo cells, each of which is distinguished by multiple marker genes and transcriptional profiles. These collectively contribute to the heterogeneity of major human organs. Moreover, T cell and B cell receptor repertoire comparisons and trajectory analyses reveal direct clonal sharing of T and B cells with various developmental states among different tissues. Furthermore, novel cell markers, transcription factors and ligand-receptor pairs are identified with potential functional regulations in maintaining the homeostasis of human cells among tissues. Conclusions: The adult human cell atlas reveals the inter- and intra-organ heterogeneity of cell characteristics and provides a useful resource in uncovering key events during the development of human diseases in the context of the heterogeneity of cells and organs. Overall design: We performed single-cell transcriptomes of 84,363 cells derived from 15 tissue organs of one adult donor and generated an adult human cell atlas (AHCA). Keywords: Human cell atlas, Single-cell RNA sequencing, TCR, BCR, Transcriptome

Single-cell transcriptome profiling of an adult human cell atlas of 15 major organs

Neuroblastoma is a pediatric tumor of the developing sympathetic nervous system. However, the cellular origin of neuroblastoma has yet to be defined. Here we studied the single-cell transcriptomes of neuroblastomas and normal human developing adrenal glands at various stages of embryonic and fetal development. We defined normal differentiation trajectories from Schwann cell precursors over intermediate states to neuroblasts or chromaffin cells and showed that neuroblastomas transcriptionally resemble normal fetal adrenal neuroblasts. Importantly, neuroblastomas with varying clinical phenotypes matched different temporal states along normal neuroblast differentiation trajectories, with the degree of differentiation corresponding to clinical prognosis. Our work highlights the roles of oncogenic MYCN and loss of TFAP2B in blocking differentiation and may provide the basis for designing therapeutic interventions to overcome differentiation blocks.

Single-cell transcriptomic analyses provide insights into the developmental origins of neuroblastoma.

Understanding gene function and regulation in homeostasis and disease requires knowledge of the cellular and tissue contexts in which genes are expressed. Here, we applied four single-nucleus RNA sequencing methods to eight diverse, archived, frozen tissue types from 16 donors and 25 samples, generating a cross-tissue atlas of 209,126 nuclei profiles, which we integrated across tissues, donors, and laboratory methods with a conditional variational autoencoder. Using the resulting cross-tissue atlas, we highlight shared and tissue-specific features of tissue-resident cell populations; identify cell types that might contribute to neuromuscular, metabolic, and immune components of monogenic diseases and the biological processes involved in their pathology; and determine cell types and gene modules that might underlie disease mechanisms for complex traits analyzed by genome-wide association studies.

Single-nucleus cross-tissue molecular reference maps toward understanding disease gene function.

Fibroblasts synthesize the extracellular matrix of connective tissue and play an essential role in maintaining tissue integrity. We have previously shown that mouse skin connective tissue, the dermis, is comprised of functionally distinct fibroblast lineages. However, the extent of fibroblast heterogeneity in human skin is unknown. Here, using a combination of spatial transcriptional profiling of human and mouse dermis and single cell transcriptional profiling of human dermal fibroblasts, we show that there are at least four distinct fibroblast populations in adult human skin. We define markers permitting prospective isolation of these cells and show that although marker expression is rapidly lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signalling, T cell communication and the ability to support human epidermal reconstitution in organotypic culture. Furthermore, while some fibroblast subpopulations are spatially segregated, others are not. These findings have profound implications for normal wound healing and diseases characterized by excessive fibrosis, and suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications.

Spatial and single-cell transcriptional profiling identifies functionally distinct human dermal fibroblast subpopulations

The invasive potential of gastric cancers (GCs) defines the hallmarks of malignancies; however, the cellular components and their molecular features associated with tumor invasiveness in GC microenvironments (TME) are poorly understood. In this study, we constructed a comprehensive atlas of 23,060 single cells obtained from surgically dissected superficial and deep layers of five diffuse-type GC along with matched normal. As a result, seven major cell types were identified. Fibroblasts, endothelial cells, and myeloid cells were categorized as being enriched in the deep layers. Cell type-specific clustering further revealed that the superficial-to-deep layer transition is associated with enrichment in inflammatory endothelial cells and fibroblasts with upregulated CCL2 transcripts. This study reveals the spatial reprogramming of the TME that may underlie invasive tumor potential in diffuse-type GC. This TME profiling across tumor layers suggests new targets, such as CCL2, that can modify the TME to inhibit tumor progression in diffuse-type GC. Overall design: A scRNAseq analysis of 30,365 cells from diffuse-type gastric cancer using 10X Genomics.

Spatially distinct reprogramming of the tumor microenvironment based on tumor invasion in diffuse-type gastric cancers

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.

Spatially organized multicellular immune hubs in human colorectal cancer

Development of the human intestine is not well understood. Here we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including novel epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe new functions for these including as vascular niche cells. We pinpoint the origins of Peyer’s patches and gut-associated lymphoid tissue (GALT) and describe location specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. Overall design: Colon and terminal ileum tissue sections from 8-22 post-conceptual week fetal intestine samples were subject to single cell RNA-seq analysis.

Spatiotemporal Analysis of Human Intestinal Development at Single Cell Resolution - scRNA-Seq

Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.

Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease

We have created a compendium of single cell transcriptome data from the model
organism Mus musculus comprising more than 100,000 cells from 20 organs and
tissues. These data represent a new resource for cell biology, revealing gene
expression in poorly characterized cell populations and allowing for direct and
controlled comparison of gene expression in cell types shared between tissues, such
as T-lymphocytes and endothelial cells from distinct anatomical locations. Two
distinct technical approaches were used for most tissues: one approach, microfluidic
droplet-based 3’-end counting, enabled the survey of thousands of cells at relatively
low coverage, while the other, FACS-based full length transcript analysis, enabled
characterization of cell types with high sensitivity and coverage. The cumulative
data provide the foundation for an atlas of transcriptomic cell biology.

Tabula Muris: Transcriptomic characterization of 20 organs and tissues from Mus musculus at single cell resolution

The authors develop two methods to profile the Enteric Nervous System of adult mice and humans at single-cell resolution: RAISIN RNA-seq for profiling intact nuclei with ribosome-bound mRNA and MIRACL-seq for label-free enrichment of rare cell types by droplet-based profiling.

The Human and Mouse Enteric Nervous System at Single-Cell Resolution.

We profiled the transcriptomes of about 14,000 CD45+ single cells from 3 normal and 3 URPL deciduas within 8-10 gestational weeks. The cellular composition revealed six major subsets of immune cells including dNK, T cell, macrophage, dendritic cell (DC), mast cell, and B cell in the decidua.We identified previously unknown variations of immune cells including the decreased proportion of CSF1+CD59+ KIRs-expressing dNK subset and the increase of activated DC subset in URPL deciduas. Overall design: Profiling the transcriptomes of about 14,000 CD45+ single cells from 3 normal and 3 URPL deciduas using single-cell RNA Seq.

The Immune Atlas of Human Deciduas With Unexplained Recurrent Pregnancy Loss

In recent years there has been tremendous progress towards deep molecular characterization of cell types using single cell transcriptome sequencing. Here we report a single cell transcriptomic atlas comprising nearly 500,000 cells from 24 different human tissues and organs. In several instances multiple organs were analyzed from the same donor. Analyzing organs from the same individual controls for genetic background, age, environment, and epigenetic effects, and enables a detailed comparison of cell types that are shared between tissues. This resource provides a rich molecular characterization of more than 400 cell types, their distribution across tissues, and detailed information about tissue specific variation in gene expression. We have used the fact that multiple tissues came from the same donor to study the clonal distribution of T cells between tissues, to understand the tissue specific mutation rate in B cells, and to analyze the cell cycle state and proliferative potential of shared cell types across tissues. Finally, we have also used this data to characterize cell type specific RNA splicing and how such splicing varies across tissues within an individual.

The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans

To delineate the ontogeny of the mammalian endoderm, we generated 112,217 single-cell transcriptomes representing all endoderm populations within the mouse embryo until midgestation. By employing graph-based approaches, we modelled differentiating cells for spatio-temporal characterization of developmental trajectories and defined the transcriptional architecture that accompanies the emergence of the first (primitive or extra-embryonic) endodermal population and its sister pluripotent (embryonic) epiblast lineage. We uncovered a relationship between descendants of these two lineages, whereby epiblast cells differentiate into endoderm at two distinct time-points, before and during gastrulation. Trajectories of endoderm cells were mapped as they acquired embryonic versus extra-embryonic fates, and as they spatially converged within the nascent gut endoderm; revealing them to be globally similar but retaining aspects of their lineage history. We observed the regionalized identity of cells along the anterior-posterior axis of the emergent gut tube, reflecting their embryonic or extra-embryonic origin, and their coordinate patterning into organ-specific territories.

The emergent landscape of the mouse gut endoderm at single-cell resolution

Our current knowledge of the different immune cells in neuroblastoma is based on in vitro and in vivo studies mainly focusing on a single cell type. Importantly, different studies have conveyed conflicting results. Moreover, a comprehensive immune cell overview at the single-cell level is still missing and understanding the complete immune cell composition of neuroblastoma will be crucial for the development of novel immunotherapeutics against the disease. In this study, we performed single-cell RNA-sequencing on nineteen human neuroblastoma samples coupled with multiplex immunohistochemistry and survival analysis using additional datasets to provide a comprehensive cellular and molecular immune cell landscape of human neuroblastoma. Further, we contrasted our data with single-cell RNA-sequencing data from normal fetal adrenal gland to characterize cell-state changes from normal tissue to cancerous neuroblastoma. Our analysis revealed 27 immune cell subtypes including distinct subpopulations of myeloid, NK, B and T cells not identified in neuroblastoma before. Several immune cell subtypes demonstrated a survival benefit such as inflammatory monocytes, tumor associated macrophages, various T cell populations, and Active NK cells. Furthermore, in contrast to adult cancers and previous neuroblastoma studies, we demonstrated an increase in inflammatory monocyte cell-state when contrasting normal and tumor tissue, while we do not observe differences in cytotoxicity and exhaustion score for cytotoxic T cells, nor in Treg activity. Finally, we performed a systemic receptor-ligand interaction analysis between tumor, stroma and immune cells, where we showed the neuroblastoma tumor microenvironment is highly complex and strongly correlated to survival. In addition, we highlighted several interactions that we suggest to be tested in future studies as a therapeutic option in human neuroblastoma. Taken together, our study significantly adds to the in depth understanding of the immune cell landscape, the complexity of the tumor microenvironment and it provides a resource for the development of novel immunotherapeutics for neuroblastoma.

The immune cell atlas of human neuroblastoma

Crohn's disease (CD) is a chronic gastrointestinal disease that is increasing in prevalence worldwide. CD is multifactorial, involving the complex interplay of genetic, immune, and environmental factors, necessitating a system-level understanding of its etiology. To characterize cell-type-specific transcriptional heterogeneity in active CD, we profiled 720,633 cells from the terminal ileum and colon of 71 donors with varying inflammation status. Our integrated datasets revealed organ- and compartment-specific responses to acute and chronic inflammation; most immune changes were in cell composition, whereas transcriptional changes dominated among epithelial and stromal cells. These changes correlated with endoscopic inflammation, but small and large intestines exhibited distinct responses, which were particularly apparent when focusing on IBD risk genes. Finally, we mapped markers of disease-associated myofibroblast activation and identified CHMP1A, TBX3, and RNF168 as regulators of fibrotic complications. Altogether, our results provide a roadmap for understanding cell-type- and organ-specific differences in CD and potential directions for therapeutic development.

The landscape of immune dysregulation in Crohn's disease revealed through single-cell transcriptomic profiling in the ileum and colon

Rhabdomyosarcoma (RMS) is the most soft tissue sarcoma of childhood and histologically mimics developing skeletal muscle. To better understand RMS, we performed single-cell RNA-seq of one embryonal subtype RMS (ERMS) and one alveolar subtype RMS (ARMS). We expanded our observations by performing single-nucleus RNA-seq of 18 patient tumors (12 ERMS, 6 ARMS). All 18 of these tumors were used to generate orthotopic patient-derived xenografts, and we performed single-cell RNA-seq of all 18 O-PDXs to compare heterogeneity between the original tumor and the patient-derived model. 15 of the O-PDXs underwent lentiviral barcoding, and lineage could be tracked using a separate dial-out PCR step during the single-cell RNA-seq workflow. We performed single-cell RNA-seq of an ex vivo organoid, showing that we can preserve heterogeneity in this model. We expanded our study to the epigenetic level by using single-cell ATAC-sequencing of 7 O-PDXs. Finally, we longitudinally tracked shifts in heterogeneity in an O-PDX model treated with chemotherapy. Overall design: We performed single-cell RNA-seq from 2 patient RMS tumors, 18 O-PDXs, and 1 organoid. Additionally, we performed single-nucleus RNA-seq from 18 patient RMS tumors and single-cell ATAC-seq from 7 O-PDXs. We also provide dialout PCR data from 15 RMS O-PDX that were used for lentiviral barcode experiments to track clonal lineages within O-PDXs.

The myogenesis program drives clonal selection and drug resistance in rhabdomyosarcoma

B cells, which are critical for intestinal homeostasis, remain understudied in ulcerative colitis (UC). In this study, we recruited three cohorts of patients with UC (primary cohort, n = 145; validation cohort 1, n = 664; and validation cohort 2, n = 143) to comprehensively define the landscape of B cells during UC-associated intestinal inflammation. Using single-cell RNA sequencing, single-cell IgH gene sequencing and protein-level validation, we mapped the compositional, transcriptional and clonotypic landscape of mucosal and circulating B cells. We found major perturbations within the mucosal B cell compartment, including an expansion of naive B cells and IgG+ plasma cells with curtailed diversity and maturation. Furthermore, we isolated an auto-reactive plasma cell clone targeting integrin αvβ6 from inflamed UC intestines. We also identified a subset of intestinal CXCL13-expressing TFH-like T peripheral helper cells that were associated with the pathogenic B cell response. Finally, across all three cohorts, we confirmed that changes in intestinal humoral immunity are reflected in circulation by the expansion of gut-homing plasmablasts that correlates with disease activity and predicts disease complications. Our data demonstrate a highly dysregulated B cell response in UC and highlight a potential role of B cells in disease pathogenesis.

Ulcerative colitis is characterized by a plasmablast-skewed humoral response associated with disease activity

Bowel function requires coordinated activity of many enteric neuron subtypes. Clear definition of subtype-specific gene expression may facilitate molecular diagnoses for bowel motility disorders. Using adult mouse colon RNAseq data from 635 myenteric neurons and 707 E17.5 neurons, we defined seven adult myenteric neuron subtypes, eight E17.5 neuron subtypes and hundreds of differentially-expressed genes. Manually dissected human colon myenteric plexus yielded data from 48 neurons, 3798 glia, 5568 smooth muscle, 377 interstitial cells, and 2153 macrophages. Immunohistochemistry demonstrated differential protein abundance for BNC2, PBX3, RBFOX1, TBX2, and TBX3 in enteric neuron subtypes. Conditional Tbx3 loss reduced NOS1-expressing myenteric neurons. Differential Gfra1 and Gfra2 expression coupled with calcium imaging revealed that GDNF and neurturin acutely and differentially regulate activity of ~50% of myenteric neurons with distinct effects on smooth muscle contractions. This insight into enteric nervous system biology provides a foundation for future studies of bowel motility disorders. Overall design: 20 scRNAseq datasets were analyzed: (1) 3 single-nucleus seq runs from mouse distal colon, (2) 16 single nucleus seq samples from human, (3) 1 single cell seq from E17.5 mouse whole bowel

scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3

Assessment of the effect of cold ischaemic time on single cell RNAseq data from human tissues using 10x Genomics 3' single cell RNA sequencing. This project contains data for spleen, oesophagus epithelium and lung parenchyma, three tissues that had previously been reported to have differential sensitivity to ischaemia. Samples were collected into Hypothermasol FRS hypothermic preservation media and dissociated fresh (as soon as possible) or at 12h, 24h, 72h post onset of cold ischaemia in the donor. Single cell and bulk RNA sequencing data was generated at each time point and whole genome sequencing was carried out for each donor.

Gut Network

Network Description

Gut Network atlases are still under development.

Network Coordinators