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Neuronal vulnerability and multilineage diversity in multiple sclerosis

Multiple sclerosis (MS) is a neuroinflammatory disease with a relapsing–remitting disease course at early stages, distinct lesion characteristics in cortical grey versus subcortical white matter and neurodegeneration at chronic stages. Here we used single-nucleus RNA sequencing to assess changes in expression in multiple cell lineages in MS lesions and validated the results using multiplex in situ hybridization. We found selective vulnerability and loss of excitatory CUX2-expressing projection neurons in upper-cortical layers underlying meningeal inflammation; such MS neuron populations exhibited upregulation of stress pathway genes and long non-coding RNAs. Signatures of stressed oligodendrocytes, reactive astrocytes and activated microglia mapped most strongly to the rim of MS plaques. Notably, single-nucleus RNA sequencing identified phagocytosing microglia and/or macrophages by their ingestion and perinuclear import of myelin transcripts, confirmed by functional mouse and human culture assays. Our findings indicate lineage- and region-specific transcriptomic changes associated with selective cortical neuron damage and glial activation contributing to progression of MS lesions.

Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq

Tumors harbor multiple cell types that are thought to play a role in the development of resistance to drug treatments. Tirosh et al. used single-cell sequencing to investigate the distribution of these differing genetic profiles within melanomas. Many cells harbored heterogeneous genetic programs that reflected two different states of genetic expression, one of which was linked to resistance development. Following drug treatment, the resistance-linked expression state was found at a much higher level. Furthermore, the environment of the melanoma cells affected their gene expression programs.

IFNγ-dependent tissue immune homeostasis is co-opted in the tumor microenvironment

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.

A Cancer Cell Program Promotes T Cell Exclusion and Resistance to Checkpoint Blockade

Immune checkpoint inhibitors (ICIs) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA sequencing (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients. CDK4/6-inhibition represses this program in individual malignant cells, induces senescence, and reduces melanoma tumor outgrowth in mouse models in vivo when given in combination with immunotherapy. Our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and suggests new therapeutic strategies to overcome immunotherapy resistance.

A single-nucleus RNA-sequencing pipeline to decipher the molecular anatomy and pathophysiology of human kidneys

Defining cellular and molecular identities within the kidney is necessary to understand its organization and function in health and disease. Here we demonstrate a reproducible method with minimal artifacts for single-nucleus Droplet-based RNA sequencing (snDrop-Seq) that we use to resolve thirty distinct cell populations in human adult kidney. We define molecular transition states along more than ten nephron segments spanning two major kidney regions. We further delineate cell type-specific expression of genes associated with chronic kidney disease, diabetes and hypertension, providing insight into possible targeted therapies. This includes expression of a hypertension-associated mechano-sensory ion channel in mesangial cells, and identification of proximal tubule cell populations defined by pathogenic expression signatures. Our fully optimized, quality-controlled transcriptomic profiling pipeline constitutes a tool for the generation of healthy and diseased molecular atlases applicable to clinical samples.

Single-Cell RNA-Seq Reveals AML Hierarchies Relevant to Disease Progression and Immunity. (Bone Marrow)

Acute myeloid leukemia (AML) is a heterogeneous disease that resides within a complex microenvironment, complicating efforts to understand how different cell types contribute to disease progression. We combined single-cell RNA sequencing and genotyping to profile 38,410 cells from 40 bone marrow aspirates, including 16 AML patients and five healthy donors. We then applied a machine learning classifier to distinguish a spectrum of malignant cell types whose abundances varied between patients and between subclones in the same tumor. Cell type compositions correlated with prototypic genetic lesions, including an association of FLT3-ITD with abundant progenitor-like cells. Primitive AML cells exhibited dysregulated transcriptional programs with co-expression of stemness and myeloid priming genes and had prognostic significance. Differentiated monocyte-like AML cells expressed diverse immunomodulatory genes and suppressed T cell activity in vitro. In conclusion, we provide single-cell technologies and an atlas of AML cell states, regulators, and markers with implications for precision medicine and immune therapies.

Single-Cell Transcriptomics of the Human Endocrine Pancreas

Human pancreatic islets consist of multiple endocrine cell types. To facilitate the detection of rare cellular states and uncover population heterogeneity, we performed single-cell RNA sequencing (RNA-seq) on islets from multiple deceased organ donors, including children, healthy adults, and individuals with type 1 or type 2 diabetes. We developed a robust computational biology framework for cell type annotation. Using this framework, we show that α- and β-cells from children exhibit less well-defined gene signatures than those in adults. Remarkably, α- and β-cells from donors with type 2 diabetes have expression profiles with features seen in children, indicating a partial dedifferentiation process. We also examined a naturally proliferating α-cell from a healthy adult, for which pathway analysis indicated activation of the cell cycle and repression of checkpoint control pathways. Importantly, this replicating α-cell exhibited activated Sonic hedgehog signaling, a pathway not previously known to contribute to human α-cell proliferation. Our study highlights the power of single-cell RNA-seq and provides a stepping stone for future explorations of cellular heterogeneity in pancreatic endocrine cells.

A Single-Cell Transcriptome Atlas of the Human Pancreas.

To understand organ function, it is important to have an inventory of its cell types and of their corresponding marker genes. This is a particularly challenging task for human tissues like the pancreas, because reliable markers are limited. Hence, transcriptome-wide studies are typically done on pooled islets of Langerhans, obscuring contributions from rare cell types and of potential subpopulations. To overcome this challenge, we developed an automated platform that uses FACS, robotics, and the CEL-Seq2 protocol to obtain the transcriptomes of thousands of single pancreatic cells from deceased organ donors, allowing in silico purification of all main pancreatic cell types. We identify cell type-specific transcription factors and a subpopulation of REG3A-positive acinar cells. We also show that CD24 and TM4SF4 expression can be used to sort live alpha and beta cells with high purity. This resource will be useful for developing a deeper understanding of pancreatic biology and pathophysiology of diabetes mellitus.

Intra-and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis (Fibroblast cells)

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 (Epithelial cells)

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 (Immunes cells)

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.

A human liver cell atlas reveals heterogeneity and epithelial progenitors

The human liver is an essential multifunctional organ. The incidence of liver diseases is rising and there are limited treatment options. However, the cellular composition of the liver remains poorly understood. Here we performed single-cell RNA sequencing of about 10,000 cells from normal liver tissue from nine human donors to construct a human liver cell atlas. Our analysis identified previously unknown subtypes of endothelial cells, Kupffer cells, and hepatocytes, with transcriptome-wide zonation of some of these populations. We show that the EPCAM+ population is heterogeneous, comprising hepatocyte-biased and cholangiocyte populations as well as a TROP2int progenitor population with strong potential to form bipotent liver organoids. As a proof-of-principle, we used our atlas to unravel the phenotypic changes that occur in hepatocellular carcinoma cells and in human hepatocytes and liver endothelial cells engrafted into a mouse liver. Our human liver cell atlas provides a powerful resource to enable the discovery of previously unknown cell types in normal and diseased livers. Single-cell RNA sequencing of cells from healthy human liver, hepatocellular carcinoma and chimaeric mouse liver identifies subtypes of liver cells, epithelial progenitors and differences between healthy and diseased cells.

Single-cell multiomics sequencing and analyses of human colorectal cancer

To better design treatments for cancer, it is important to understand the heterogeneity in tumors and how this contributes to metastasis. To examine this process, Bian et al. used a single-cell triple omics sequencing (scTrio-seq) technique to examine the mutations, transcriptome, and methylome within colorectal cancer tumors and metastases from 10 individual patients. The analysis provided insights into tumor evolution, linked DNA methylation to genetic lineages, and showed that DNA methylation levels are consistent within lineages but can differ substantially among clones.

A Cellular Anatomy of the Normal Adult Human Prostate and Prostatic Urethra

Using single-cell RNA sequencing, immunofluorescence, and flow cytometry, Henry et al. create a cellular anatomy of the normal human prostate and provide the tools to identify, isolate, and localize every cell type. They identify two additional epithelial cell types enriched in the prostatic urethra and proximal prostatic ducts.

Lipid-Associated Macrophages Control Metabolic Homeostasis in a Trem2-Dependent Manner (Human data)

Immune cells residing in white adipose tissue have been highlighted as important factors contributing to the pathogenesis of metabolic diseases, but the molecular regulators that drive adipose tissue immune cell remodeling during obesity remain largely unknown. Using index and transcriptional single-cell sorting, we comprehensively map all adipose tissue immune populations in both mice and humans during obesity. We describe a novel and conserved Trem2+ lipid-associated macrophage (LAM) subset and identify markers, spatial localization, origin, and functional pathways associated with these cells. Genetic ablation of Trem2 in mice globally inhibits the downstream molecular LAM program, leading to adipocyte hypertrophy as well as systemic hypercholesterolemia, body fat accumulation, and glucose intolerance. These findings identify Trem2 signaling as a major pathway by which macrophages respond to loss of tissue-level lipid homeostasis, highlighting Trem2 as a key sensor of metabolic pathologies across multiple tissues and a potential therapeutic target in metabolic diseases