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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).

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues, in contrast to those circulating in the blood, remains limited. Here, we surveyed the immune compartment of lymphoid and non-lymphoid tissues of six adult donors by single-cell RNA sequencing, including alpha beta T-cell receptor (αβ TCR), gamma delta (γδ) TCR and B-cell receptor (BCR) variable regions. To aid systematic cell type identification we developed CellTypist, a tool for automated and accurate cell type annotation. Using this approach combined with manual curation, we determined the tissue distribution of finely phenotyped immune cell types and cell states. This revealed tissue-specific features within cell subsets, such as a subtype of activated dendritic cells in the airways expressing CSF2RA, GPR157, CRLF2, ITGAD-expressing γδ T cells in spleen and liver, and ITGAX+ splenic memory B cells. Single cell paired chain TCR analysis revealed cell type-specific biases in VDJ usage, and BCR analysis revealed characteristic patterns of somatic hypermutation and isotype usage in plasma and memory B cell subsets. 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.

SummaryHuman gut development requires the orchestrated interaction of various differentiating cell types. Here we generate an in-depth single-cell map of the developing human intestine at 6–10 weeks post-conception, a period marked by crypt-villus formation. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells, which are distinct from LGR5-expressing cells. We use computational analyses to show that these cells contribute to differentiated cell subsets directly and indirectly via the generation of LGR5-expressing stem cells and receive signals from the surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes ofex vivotissues andin vitrofetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNAseq profiles from paediatric Crohn’s disease epithelium alongside matched healthy controls to reveal disease associated changes in epithelial composition. Contrasting these with the fetal profiles reveals re-activation of fetal transcription factors in Crohn’s disease epithelium. Our study provides a unique resource, available atwww.gutcellatlas.org, and underscores the importance of unravelling fetal development in understanding disease.

Summary Human gut development requires the orchestrated interaction of differentiating cell types. Here, we generate an in-depth single-cell map of the developing human intestine at 6–10 weeks post-conception. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells; distinct from LGR5-expressing cells. We propose that these cells may contribute to differentiated cell subsets via the generation of LGR5-expressing stem cells and receive signals from surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes of ex vivo tissues and in vitro fetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNA-seq profiles from pediatric Crohn’s disease epithelium alongside matched healthy controls to reveal disease-associated changes in the epithelial composition. Contrasting these with the fetal profiles reveals the re-activation of fetal transcription factors in Crohn’s disease. Our study provides a resource available at www.gutcellatlas.org, and underscores the importance of unraveling fetal development in understanding disease., Graphical Abstract, Highlights • Single-cell RNA-seq map of the developing and pediatric human intestine • Cycling BEX5+ epithelial precursors are distinct from adult LGR5+ stem cells • Human fetal intestinal organoids mature in culture • Fetal transcription factors are reactivated in the Crohn’s disease epithelium, Elmentaite and Ross et al. generated a detailed single-cell RNA-seq map of the developing human intestine. Data analyses revealed the presence of an early intestinal epithelial precursor cell and the re-activation of fetal transcription factors in the intestinal epithelium of children diagnosed with Crohn’s disease. The study provides a unique resource accessible to all researchers at www.gutcellatlas.org.