Raquel Bartolomé-Casado

Single-cell RNA-sequencing (scRNA-seq) studies have transformed our understanding of cellular diversity in the human intestine, but differences in anatomical sampling, experimental processing and cell-type nomenclature limit comparison across datasets. Here, we present the Human Gut Cell Atlas (HGCA) V1, a community-curated reference of 944,390 healthy and disease-adjacent intestinal cells from 502 samples, 265 donors and 24 landmark studies, linked to detailed anatomical, clinical, and technical metadata. As this metadata is of critical importance to human biology, we developed MetaManager, an open source data management framework for standardized metadata collection and tracking. A five-level consensus taxonomy resolves 94 homeostatic cell identities and enables transferrable, high-resolution annotation of new gut datasets. By assembling cell identity, sampling metadata, and independent spatial validation, intestinal segment and radial tissue depth were revealed to systematically shape cellular composition and transcriptional state, enabling inference of the presence of sampling-dependent tissue niches, including lymphoid follicles. Reference mapping accurately and rapidly recovered cohort-level Crohn’s disease and treatment signatures without de novo annotation, while mapping human intestinal organoids quantified cell-type-specific identity, regional specification, maturation, and transcriptional fidelity to native tissue. The HGCA V1 provides a standardized cellular, anatomical coordinate, and metadata system for reproducible, high-quality analysis of intestinal biology across healthy tissue, disease-associated remodelling, and experimental models, providing a continuously evolving community reference for cumulative discovery in human intestinal biology.

The gastrointestinal tract is a multi-organ system crucial for efficient nutrient uptake and barrier immunity. Advances in genomics and a surge in gastrointestinal diseases^1,2 has fuelled efforts to catalogue cells constituting gastrointestinal tissues in health and disease³. Here we present systematic integration of 25 single-cell RNA sequencing datasets spanning the entire healthy gastrointestinal tract in development and in adulthood. We uniformly processed 385 samples from 189 healthy controls using a newly developed automated quality control approach (scAutoQC), leading to a healthy reference atlas with approximately 1.1 million cells and 136 fine-grained cell states. We anchor 12 gastrointestinal disease datasets spanning gastrointestinal cancers, coeliac disease, ulcerative colitis and Crohn’s disease to this reference. Utilizing this 1.6 million cell resource (gutcellatlas.org), we discover epithelial cell metaplasia originating from stem cells in intestinal inflammatory diseases with transcriptional similarity to cells found in pyloric and Brunner’s glands. Although previously linked to mucosal healing⁴, we now implicate pyloric gland metaplastic cells in inflammation through recruitment of immune cells including T cells and neutrophils. Overall, we describe inflammation-induced changes in stem cells that alter mucosal tissue architecture and promote further inflammation, a concept applicable to other tissues and diseases.

The gastrointestinal (GI) tract consists of connected organs, from the oral cavity to rectum, which function to ensure efficient nutrient uptake and barrier immunity. Diseases of the GI tract affect millions worldwide and as such there are now over 25 published single cell RNA-sequencing (scRNAseq) datasets surveying the GI tract, profiling specific anatomical regions, cell lineages, ages and diseases. To consolidate these efforts, we harmonised and integrated scRNAseq datasets across the whole GI tract from developing and adult human tissues, as well as newly generated data from preterm gut. We uniformly processed 385 samples from 189 healthy controls using a newly developed automated QC approach (scAutoQC). In total, our healthy reference contains ~1.1 million cells which we annotated to a total of 137 fine-grained cell states. We anchor 13 published and 1 unpublished GI disease datasets covering gastric and colorectal (CRC) cancers, celiac disease, ulcerative colitis (UC) and Crohn's disease (CD) to this reference, taking our atlas to a total of 1.6 million cells. We provide our atlas as a valuable resource to the community (available at gutcellatlas.org). Using this resource, we discover epithelial cell metaplasia arising from stem cells across intestinal inflammatory diseases (celiac, UC and CD) and CRC with transcriptional similarity to cells of the gastric and Brunner's glands. Whilst previously linked to mucosal healing, we now implicate these cells in inflammation through recruitment of immune cells including T cells and neutrophils, and through direct interactions with T cells. Overall, we discover a shift in paradigm whereby changes in stem cells during inflammation lead to altered mucosal tissue architecture, which in turn contributes to ongoing inflammation. These findings highlight that in addition to barrier function, epithelial cells actively contribute to progression of inflammation which may be a function applicable to other tissues and diseases.

Summary: Visium HD by 10X Genomics is the first commercially available platform capable of capturing full scale transcriptomic data paired with a reference morphology image from archived FFPE blocks at sub-cellular resolution. However, aggregation of capture regions to single cells poses challenges. Bin2cell reconstructs cells from the highest resolution data (2 μm bins) by leveraging morphology image segmentation and gene expression information. It is compatible with established Python single cell and spatial transcriptomics software, and operates efficiently in a matter of minutes without requiring a GPU. We demonstrate improvements in downstream analysis when using the reconstructed cells over default 8 μm bins on mouse brain and human colorectal cancer data. Availability and implementation: Bin2cell is available at https://github.com/Teichlab/bin2cell, along with documentation and usage examples, and can be installed from pip. Probe design functionality is available at https://github.com/Teichlab/gene2probe.

Summary: Visium HD by 10X Genomics is the first commercially available platform capable of capturing full scale transcriptomic data paired with a reference morphology image from archived FFPE blocks at sub-cellular resolution. However, aggregation of capture regions to single cells poses challenges. Bin2cell reconstructs cells from the highest resolution data (2 μ m bins) by leveraging morphology image segmentation and gene expression information. It is compatible with established Python single cell and spatial transcriptomics software, and operates efficiently in a matter of minutes without requiring a GPU. We demonstrate improvements in downstream analysis when using the reconstructed cells over default 8 μ m bins on mouse brain and human colorectal cancer data. Availability and Implementation: Bin2cell is available at https://github.com/Teichlab/bin2cell , along with documentation and usage examples, and can be installed from pip. Probe design functionality is available at https://github.com/Teichlab/gene2probe Contact: ny1@sanger.ac.uk , st9@sanger.ac.uk Supplementary information: Supplementary data are available online.

The intestinal immune system is highly adapted to maintaining tolerance to the commensal microbiota and self-antigens while defending against invading pathogens^1,2. Recognizing how the diverse network of local cells establish homeostasis and maintains it in the complex immune environment of the gut is critical to understanding how tolerance can be re-established following dysfunction, such as in inflammatory disorders. Although cell and molecular interactions that control T regulatory (T_reg) cell development and function have been identified^3,4, less is known about the cellular neighbourhoods and spatial compartmentalization that shapes microorganism-reactive T_reg cell function. Here we used in vivo live imaging, photo-activation-guided single-cell RNA sequencing^5–7 and spatial transcriptomics to follow the natural history of T cells that are reactive towards Helicobacter hepaticus through space and time in the settings of tolerance and inflammation. Although antigen stimulation can occur anywhere in the tissue, the lamina propria—but not embedded lymphoid aggregates—is the key microniche that supports effector T_reg (eT_reg) cell function. eT_reg cells are stable once their niche is established; however, unleashing inflammation breaks down compartmentalization, leading to dominance of CD103⁺SIRPα⁺ dendritic cells in the lamina propria. We identify and validate the putative tolerogenic interaction between CD206⁺ macrophages and eT_reg cells in the lamina propria and identify receptor–ligand pairs that are likely to govern the interaction. Our results reveal a spatial mechanism of tolerance in the lamina propria and demonstrate how knowledge of local interactions may contribute to the next generation of tolerance-inducing therapies.

The number of studies investigating the human gastrointestinal tract using various single-cell profiling methods has increased substantially in the past few years. Although this increase provides a unique opportunity for the generation of the first comprehensive Human Gut Cell Atlas (HGCA), there remains a range of major challenges ahead. Above all, the ultimate success will largely depend on a structured and coordinated approach that aligns global efforts undertaken by a large number of research groups. In this Roadmap, we discuss a comprehensive forward-thinking direction for the generation of the HGCA on behalf of the Gut Biological Network of the Human Cell Atlas. Based on the consensus opinion of experts from across the globe, we outline the main requirements for the first complete HGCA by summarizing existing data sets and highlighting anatomical regions and/or tissues with limited coverage. We provide recommendations for future studies and discuss key methodologies and the importance of integrating the healthy gut atlas with related diseases and gut organoids. Importantly, we critically overview the computational tools available and provide recommendations to overcome key challenges.

Studies in mice and humans have shown that CD8⁺T cell immunosurveillance in non-lymphoid tissues is dominated by resident populations. Whether CD4⁺T cells use the same strategies to survey peripheral tissues is less clear. Here, examining the turnover of CD4⁺T cells in transplanted duodenum in humans, we demonstrate that the majority of CD4⁺T cells were still donor-derived one year after transplantation. In contrast to memory CD4⁺T cells in peripheral blood, intestinal CD4⁺T_RMcells expressed CD69 and CD161, but only a minor fraction expressed CD103. Functionally, intestinal CD4⁺T_RMcells were very potent cytokine producers; the vast majority being polyfunctional T_H1 cells, whereas a minor fraction produced IL-17. Interestingly, a fraction of intestinal CD4⁺T cells produced granzyme-B and perforin after activation. Together, we show that the intestinal CD4⁺T-cell compartment is dominated by resident populations that survive for more than 1 year. This finding is of high relevance for the development of oral vaccines and therapies for diseases in the gut.