Search

Genome sequencing of cancers often reveals mosaics of different subclones present in the same tumour1–3. Although these are believed to arise according to the principles of somatic evolution, the exact spatial growth patterns and underlying mechanisms remain elusive4,5. Here, to address this need, we developed a workflow that generates detailed quantitative maps of genetic subclone composition across whole-tumour sections. These provide the basis for studying clonal growth patterns, and the histological characteristics, microanatomy and microenvironmental composition of each clone. The approach rests on whole-genome sequencing, followed by highly multiplexed base-specific in situ sequencing, single-cell resolved transcriptomics and dedicated algorithms to link these layers. Applying the base-specific in situ sequencing workflow to eight tissue sections from two multifocal primary breast cancers revealed intricate subclonal growth patterns that were validated by microdissection. In a case of ductal carcinoma in situ, polyclonal neoplastic expansions occurred at the macroscopic scale but segregated within microanatomical structures. Across the stages of ductal carcinoma in situ, invasive cancer and lymph node metastasis, subclone territories are shown to exhibit distinct transcriptional and histological features and cellular microenvironments. These results provide examples of the benefits afforded by spatial genomics for deciphering the mechanisms underlying cancer evolution and microenvironmental ecology.

Methods for analyzing single-cell data perform a core set of computational tasks. These tasks include dimensionality reduction, cell clustering, cell-state annotation, removal of unwanted variation, analysis of differential expression, identification of spatial patterns of gene expression, and joint analysis of multi-modal omics data. Many of these methods rely on likelihood-based models to represent variation in the data; we refer to these as ‘probabilistic models’. Probabilistic models provide principled ways to capture uncertainty in biological systems and are convenient for decomposing the many sources of variation that give rise to omics data.

The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated dense single-cell and spatial reference maps of the human uterus and three-dimensional endometrial organoid cultures. We dissect the signaling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids reveals the pathways and cell states regulating differentiation of the secretory and ciliated lineages both in vivo and in vitro. In vitro downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. We utilize our cellular maps to deconvolute bulk data from endometrial cancers and endometriotic lesions, illuminating the cell types dominating in each of these disorders. These mechanistic insights provide a platform for future development of treatments for common conditions including endometriosis and endometrial carcinoma. Single-cell and spatial transcriptomic profiling of the human endometrium highlights pathways governing the proliferative and secretory phases of the menstrual cycle. Analyses of endometrial organoids show that WNT and NOTCH signaling modulate differentiation into the secretory and ciliated epithelial lineages, respectively. Correction in: Nature Genetics, 55, page 165 (2023)DOI: 10.1038/s41588-022-01287-6

Skeletal muscle ageing increases the incidence of age-associated frailty and sarcopenia in the elderly worldwide, leading to increased morbidity and mortality. However, our understanding of the cellular and molecular mechanisms of muscle ageing is still far from complete. Here, we generate a single-cell and single-nucleus transcriptomic atlas of skeletal muscle ageing from 15 donors across the adult human lifespan, accompanied by myofiber typing using imaging. Our atlas reveals ageing mechanisms acting across different compartments of the muscle, including muscle stem cells (MuSCs), myofibers and the muscle microenvironment. Firstly, we uncover two mechanisms driving MuSC ageing, namely a decrease in ribosome biogenesis and an increase in inflammation. Secondly, we identify a set of nuclei populations explaining the preferential degeneration of the fast-twitch myofibers and suggest two mechanisms acting to compensate for their loss. Importantly, we identify a neuromuscular junction accessory population, which helps myofiber to compensate for aged-related denervation. Thirdly, we reveal multiple microenvironment cell types contributing to the inflammatory milieu of ageing muscle by producing cytokines and chemokines to attract immune cells. Finally, we provide a comparable mouse muscle ageing atlas and further investigate conserved and specific ageing hallmarks across species. In summary, we present a comprehensive human skeletal muscle ageing resource by combining different data modalities, which significantly expands our understanding of muscle biology and ageing.

To (re)define tissue architecture of the lung and airways at the cellular and molecular level, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and Visium Spatial Transcriptomics. Using computational data integration and analysis, we extend beyond the suspension cell paradigm of lung atlases to date, to define and discover macro and micro-anatomical tissue compartments. We describe novel cell types and states in vascular, stromal and nerve bundle microenvironments. From our spatial transcriptomics, we discover and validate a novel survival niche for IgA plasma cells in the airway submucosal glands (SMG). In this niche we define a supporting role for SMG epithelial cells in mucosal immunity through recruitment and maintenance of IgA plasma, B and CD4 T cells locally at the airway SMG. We identify an immune-supporting role for SMG duct and serous cells with distinct signalling circuits to recruit B cells and IgA plasma cells, promoting longevity and antibody secretion through expression of CCL28, APRIL and IL6. We find high expression of MHC-II in SMG duct and serous cells, which are localised closely with memory CD4 T cells, suggesting local modulation of antigen specific immune responses locally at the glands. This new tissue microenvironment, which we term the “gland-associated immune niche” (GAIN) has major implications for respiratory immunity and infection response. Our single cell and spatial data is available for download and query at lungcellatlas.org.

Human limbs emerge during the fourth post-conception week as mesenchymal buds which develop into fully-formed limbs over the subsequent months. Limb development is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common. Decades of work with model organisms has outlined the fundamental processes underlying vertebrate limb development, but an in-depth characterisation of this process in humans has yet to be performed. Here we detail the development of the human embryonic limb across space and time, using both single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells, progressing from a restricted number of multipotent progenitors to myriad mature cell states, and identify several novel cell populations, including neural fibroblasts and multiple distinct mesenchymal states. We uncover two waves of human muscle development, each characterised by different cell states regulated by separate gene expression programmes. We identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity and validate this by performing MSC knock down in human embryonic myoblasts, which results in significant upregulation of late myogenic genes. Through integration of multiple anatomically continuous spatial transcriptomic samples, we spatially map single-cell clusters across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of mesenchyme in the autopod. Finally, we perform scRNA-seq on murine embryonic limbs to facilitate cross-species developmental comparison at single-cell resolution, finding substantial homology between the two species.

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 SummaryBy 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.

SummaryMultiple distinct cell types of the human lung and airways have been defined by single cell RNA sequencing (scRNAseq). Here we present a multi-omics spatial lung atlas to define novel cell types which we map back into the macro- and micro-anatomical tissue context to define functional tissue microenvironments. Firstly, we have generated single cell and nuclei RNA sequencing, VDJ-sequencing and Visium Spatial Transcriptomics data sets from 5 different locations of the human lung and airways. Secondly, we define additional cell types/states, as well as spatially map novel and known human airway cell types, such as adult lung chondrocytes, submucosal gland (SMG) duct cells, distinct pericyte and smooth muscle subtypes, immune-recruiting fibroblasts, peribronchial and perichondrial fibroblasts, peripheral nerve associated fibroblasts and Schwann cells. Finally, we define a survival niche for IgA-secreting plasma cells at the SMG, comprising the newly defined epithelial SMG-Duct cells, and B and T lineage immune cells. Using our transcriptomic data for cell-cell interaction analysis, we propose a signalling circuit that establishes and supports this niche. Overall, we provide a transcriptional and spatial lung atlas with multiple novel cell types that allows for the study of specific tissue microenvironments such as the newly defined gland-associated lymphoid niche (GALN).

Funder: Medical Research Council, 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.

Linear motifs have an integral role in dynamic cell functions, including cell signaling. However, due to their small size, low complexity, and frequent mutations, identifying novel functional motifs poses a challenge. Viruses rely extensively on the molecular mimicry of cellular linear motifs. In this study, we apply systematic motif prediction combined with functional filters to identify human linear motifs convergently evolved also in viral proteins. We observe an increase in the sensitivity of motif prediction and improved enrichment in known instances. We identify >7,300 non-redundant motif instances at various confidence levels, 99 of which are supported by all functional and structural filters. Overall, we provide a pipeline to improve the identification of functional linear motifs from interactomics datasets and a comprehensive catalog of putative human motifs that can contribute to our understanding of the human domain-linear motif code and the associated mechanisms of viral interference.

Spatial genomic technologies can map gene expression in tissues, but provide limited potential for transcriptome-wide discovery approaches and application to fixed tissue samples. Here, we introduce the GeoMX Whole Transcriptome Atlas (WTA), a new technology for transcriptome-wide spatial profiling of tissues with cellular resolution. WTA significantly expands the Digital Spatial Profiling approach to enable in situ hybridisation against 18,190 genes at high-throughput using a sequencing readout. We applied WTA to generate the first spatial transcriptomic map of the fetal human neocortex, validating transcriptome-wide spatial profiling on formalin-fixed tissue material and demonstrating the spatial enrichment of autism gene expression in deep cortical layers. To demonstrate the value of WTA for cell atlasing, we integrated single-cell RNA-sequencing (scRNA-seq) and WTA data to spatially map dozens of neural cell types and showed that WTA can be used to directly measure cell type specific transcriptomes in situ. Moreover, we developed computational tools for background correction of WTA data and accurate integration with scRNA-seq. Our results present WTA as a versatile transcriptome-wide discovery tool for cell atlasing and fixed tissue spatial transcriptomics.

The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated single-cell and spatial reference maps of the human uterus and 3D endometrial organoid cultures. We dissect the signalling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids highlights common pathways regulating the differentiation of secretory and ciliated lineage in vivo and in vitro. We show in vitro that downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. These mechanistic insights provide a platform for future development of treatments for a range of common endometrial disorders including endometriosis and carcinoma.

The spatial organization of cell types in tissues fundamentally shapes cellular interactions and function, but the high-throughput spatial mapping of complex tissues remains a challenge. We present сell2location, a principled and versatile Bayesian model that integrates single-cell and spatial transcriptomics to map cell types in situ in a comprehensive manner. We show that сell2location outperforms existing tools in accuracy and comprehensiveness and we demonstrate its utility by mapping two complex tissues. In the mouse brain, we use a new paired single nucleus and spatial RNA-sequencing dataset to map dozens of cell types and identify tissue regions in an automated manner. We discover novel regional astrocyte subtypes including fine subpopulations in the thalamus and hypothalamus. In the human lymph node, we resolve spatially interlaced immune cell states and identify co-located groups of cells underlying tissue organisation. We spatially map a rare pre-germinal centre B-cell population and predict putative cellular interactions relevant to the interferon response. Collectively our results demonstrate how сell2location can serve as a versatile first-line analysis tool to map tissue architectures in a high-throughput manner.

Spatial transcriptomic technologies promise to resolve cellular wiring diagrams of tissues in health and disease, but comprehensive mapping of cell types in situ remains a challenge. Here we present сell2location, a Bayesian model that can resolve fine-grained cell types in spatial transcriptomic data and create comprehensive cellular maps of diverse tissues. Cell2location accounts for technical sources of variation and borrows statistical strength across locations, thereby enabling the integration of single-cell and spatial transcriptomics with higher sensitivity and resolution than existing tools. We assessed cell2location in three different tissues and show improved mapping of fine-grained cell types. In the mouse brain, we discovered fine regional astrocyte subtypes across the thalamus and hypothalamus. In the human lymph node, we spatially mapped a rare pre-germinal center B cell population. In the human gut, we resolved fine immune cell populations in lymphoid follicles. Collectively, our results present сell2location as a versatile analysis tool for mapping tissue architectures in a comprehensive manner.

The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR-Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments.

Although the cerebral cortex is organized into six excitatory neuronal layers, it is unclear whether glial cells show distinct layering. In the present study, we developed a high-content pipeline, the large-area spatial transcriptomic (LaST) map, which can quantify single-cell gene expression in situ. Screening 46 candidate genes for astrocyte diversity across the mouse cortex, we identified superficial, mid and deep astrocyte identities in gradient layer patterns that were distinct from those of neurons. Astrocyte layer features, established in the early postnatal cortex, mostly persisted in adult mouse and human cortex. Single-cell RNA sequencing and spatial reconstruction analysis further confirmed the presence of astrocyte layers in the adult cortex. Satb2 and Reeler mutations that shifted neuronal post-mitotic development were sufficient to alter glial layering, indicating an instructive role for neuronal cues. Finally, astrocyte layer patterns diverged between mouse cortical regions. These findings indicate that excitatory neurons and astrocytes are organized into distinct lineage-associated laminae. ispartof: NATURE NEUROSCIENCE vol:23 issue:4 pages:500-+ ispartof: location:United States status: published