We have not yet reached a point at which routine sequencing of large numbers of whole eukaryotic genomes is feasible, and so it is often necessary to select genomic regions of interest and to enrich ...these regions before sequencing. There are several enrichment approaches, each with unique advantages and disadvantages. Here we describe our experiences with the leading target-enrichment technologies, the optimizations that we have performed and typical results that can be obtained using each. We also provide detailed protocols for each technology so that end users can find the best compromise between sensitivity, specificity and uniformity for their particular project.
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. Here, to ...comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue 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. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.
Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, ...using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.
Messenger RNA encodes cellular function and phenotype. In the context of human cancer, it defines the identities of malignant cells and the diversity of tumor tissue. We studied 72,501 single-cell ...transcriptomes of human renal tumors and normal tissue from fetal, pediatric, and adult kidneys. We matched childhood Wilms tumor with specific fetal cell types, thus providing evidence for the hypothesis that Wilms tumor cells are aberrant fetal cells. In adult renal cell carcinoma, we identified a canonical cancer transcriptome that matched a little-known subtype of proximal convoluted tubular cell. Analyses of the tumor composition defined cancer-associated normal cells and delineated a complex vascular endothelial growth factor (VEGF) signaling circuit. Our findings reveal the precise cellular identities and compositions of human kidney tumors.
Spatiotemporal immune zonation of the human kidney Stewart, Benjamin J; Ferdinand, John R; Young, Matthew D ...
Science (American Association for the Advancement of Science),
09/2019, Letnik:
365, Številka:
6460
Journal Article
Recenzirano
Odprti dostop
Tissue-resident immune cells are important for organ homeostasis and defense. The epithelium may contribute to these functions directly or by cross-talk with immune cells. We used single-cell RNA ...sequencing to resolve the spatiotemporal immune topology of the human kidney. We reveal anatomically defined expression patterns of immune genes within the epithelial compartment, with antimicrobial peptide transcripts evident in pelvic epithelium in the mature, but not fetal, kidney. A network of tissue-resident myeloid and lymphoid immune cells was evident in both fetal and mature kidney, with postnatal acquisition of transcriptional programs that promote infection-defense capabilities. Epithelial-immune cross-talk orchestrated localization of antibacterial macrophages and neutrophils to the regions of the kidney most susceptible to infection. Overall, our study provides a global overview of how the immune landscape of the human kidney is zonated to counter the dominant immunological challenge.
We present a multiomic cell atlas of human lung development that combines single-cell RNA and ATAC sequencing, high-throughput spatial transcriptomics, and single-cell imaging. Coupling single-cell ...methods with spatial analysis has allowed a comprehensive cellular survey of the epithelial, mesenchymal, endothelial, and erythrocyte/leukocyte compartments from 5–22 post-conception weeks. We identify previously uncharacterized cell states in all compartments. These include developmental-specific secretory progenitors and a subtype of neuroendocrine cell related to human small cell lung cancer. Our datasets are available through our web interface (https://lungcellatlas.org). To illustrate its general utility, we use our cell atlas to generate predictions about cell-cell signaling and transcription factor hierarchies which we rigorously test using organoid models.
Display omitted
•Spatiotemporal atlas of human lung development identifies 144 cell types/states•Tracking the developmental origins of multiple cell types, including new progenitors•Functional diversity of fibroblasts in distinct anatomical signaling niches•Experimental validation of TFs controlling neuroendocrine cell heterogeneity
Multiomic analysis of human fetal lungs from 5–22 post-conception weeks unveils cell-lineage trajectories across different cell types during development and will provide fresh insights into lung disease progression in adults.
Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and ...airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org ). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new 'gland-associated immune niche' has implications for respiratory health.
Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we ...studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8+ T cell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.
Display omitted
•Single-cell and spatial sequencing reveals key features of renal cell carcinoma (RCC)•Intra-tumoral heterogeneity of CD8+ clonotypes dwarfs that from somatic mutations•RCC cells showing epithelial-mesenchymal transition (EMT) enrich at tumor edges•Macrophages expressing IL1B correlate with EMT and could have therapeutic importance
Li et al. use single-cell and spatial sequencing to examine the cellular features of kidney tumors and classify cancer cells according to function. They find a more invasive phenotype at the interface separating tumor with normal kidney, which appears to be driven by IL-1β signaling in macrophages.
Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, displays impaired terminal B-cell differentiation and defective antibody responses. Incomplete ...genetic penetrance and ample phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. Here, we generate a single-cell epigenomics and transcriptomics census of naïve-to-memory B cell differentiation in a CVID-discordant MZ twin pair. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B-cells mirroring defective cell-cell communication upon activation. These findings are validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naïve-to-memory B-cell transition in CVID and indicate links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, gives insight into future diagnosis and treatments of CVID patients.
PDF
