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Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries 1 . Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal–mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15 + and TREM2 + fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy, which can guide in vitro gonadogenesis.

Introduction Otitis media is an umbrella term for middle ear inflammation; ranging from acute infection to chronic mucosal disease. It is a leading cause of antimicrobial therapy prescriptions and surgery in children. Despite this, treatments have changed little in over 50 years. Research has been limited by the lack of physiological models of middle ear epithelium. Methods We develop a novel human middle ear epithelial culture using an air‐liquid interface (ALI) system; akin to the healthy ventilated middle ear in vivo. We validate this using immunohistochemistry, immunofluorescence, scanning and transmission electron microscopy, and membrane conductance studies. We also utilize this model to perform a pilot challenge of middle ear epithelial cells with SARS‐CoV‐2. Results We demonstrate that human middle ear epithelial cells cultured at an ALI undergo mucociliary differentiation to produce diverse epithelial subtypes including basal (p63+), goblet (MUC5AC+, MUC5B+), and ciliated (FOXJ1+) cells. Mature ciliagenesis is visualized and tight junction formation is shown with electron microscopy, and confirmed by membrane conductance. Together, these demonstrate this model reflects the complex epithelial cell types which exist in vivo. Following SARS‐CoV‐2 challenge, human middle ear epithelium shows positive viral uptake, as measured by polymerase chain reaction and immunohistochemistry. Conclusion We describe a novel physiological system to study the human middle ear. This can be utilized for translational research into middle ear diseases. We also demonstrate, for the first time under controlled conditions, that human middle ear epithelium is susceptible to SARS‐CoV‐2 infection, which has important clinical implications for safe otological surgery. Level of Evidence NA.

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. Cells from embryonic, fetal, paediatric and adult human intestinal tissue are analysed at different locations along the intestinal tract to construct a single-cell atlas of the developing and adult human intestinal tract, encompassing all cell lineages.

Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11–12 weeks after conception 1 , 2 , yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6–7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21). A single-cell atlas of human fetal bone marrow in healthy fetuses and fetuses with Down syndrome provides insight into developmental haematopoiesis in humans and the transcription and functional differences that occur in Down syndrome.

KMT2A- rearranged infant ALL is an aggressive childhood leukemia with poor prognosis. Here, we investigated the developmental state of KMT2A -rearranged infant B-cell acute lymphoblastic leukemia (B-ALL) using bulk messenger RNA (mRNA) meta-analysis and examination of single lymphoblast transcriptomes against a developing bone marrow reference. KMT2A -rearranged infant B-ALL was uniquely dominated by an early lymphocyte precursor (ELP) state, whereas less adverse NUTM1 -rearranged infant ALL demonstrated signals of later developing B cells, in line with most other childhood B-ALLs. We compared infant lymphoblasts with ELP cells and revealed that the cancer harbored hybrid myeloid–lymphoid features, including nonphysiological antigen combinations potentially targetable to achieve cancer specificity. We validated surface coexpression of exemplar combinations by flow cytometry. Through analysis of shared mutations in separate leukemias from a child with infant KMT2A -rearranged B-ALL relapsing as AML, we established that KMT2A rearrangement occurred in very early development, before hematopoietic specification, emphasizing that cell of origin cannot be inferred from the transcriptional state. Single-cell transcriptomic and phylogenetic analyses reveal new insights into the developmental origin and potential therapeutic targets for a particularly aggressive form of B-cell acute lymphoblastic leukemia in infants.

T cells develop from circulating precursor cells, which enter the thymus and migrate through specialized subcompartments that support their maturation and selection 1 . In humans, this process starts in early fetal development and is highly active until thymic involution in adolescence. To map the microanatomical underpinnings of this process in pre- and early postnatal stages, we established a quantitative morphological framework for the thymus—the Cortico-Medullary Axis—and used it to perform a spatially resolved analysis. Here, by applying this framework to a curated multimodal single-cell atlas, spatial transcriptomics and high-resolution multiplex imaging data, we demonstrate establishment of the lobular cytokine network, canonical thymocyte trajectories and thymic epithelial cell distributions by the beginning of the the second trimester of fetal development. We pinpoint tissue niches of thymic epithelial cell progenitors and distinct subtypes associated with Hassall’s corpuscles and identify divergence in the timing of medullary entry between CD4 and CD8 T cell lineages. These findings provide a basis for a detailed understanding of T lymphocyte development and are complemented with a holistic toolkit for cross-platform imaging data analysis, annotation and OrganAxis construction (TissueTag), which can be applied to any tissue. A quantitative morphological framework for the human thymus reveals the establishment of the lobular cytokine network, canonical thymocyte trajectories and thymic epithelial cell distributions in fetal and paediatric thymic development.

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 disease 3 . 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 healing 4 , 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 study provides a comprehensive transcriptomic atlas of the human gastrointestinal tract across the lifespan, highlighting inflammation-induced changes in epithelial stem cells that alter mucosal architecture and promote further inflammation.

Cutaneous T cell lymphoma (CTCL) is a potentially fatal clonal malignancy of T cells primarily affecting the skin. The most common form of CTCL, mycosis fungoides, can be difficult to diagnose, resulting in treatment delay. We performed single-cell and spatial transcriptomics analysis of skin from patients with mycosis fungoides-type CTCL and an integrated comparative analysis with human skin cell atlas datasets from healthy and inflamed skin. We revealed the co-optation of T helper 2 (T H 2) cell-immune gene programs by malignant CTCL cells and modeling of the tumor microenvironment to support their survival. We identified MHC-II + fibroblasts and dendritic cells that can maintain T H 2 cell-like tumor cells. CTCL tumor cells are spatially associated with B cells, forming tertiary lymphoid structure-like aggregates. Finally, we validated the enrichment of B cells in CTCL and its association with disease progression across three independent patient cohorts. Our findings provide diagnostic aids, potential biomarkers for disease staging and therapeutic strategies for CTCL. Haniffa and colleagues provide diagnostic aids, potential biomarkers for disease staging and therapeutic strategies for cutaneous T cell lymphoma.

Cutaneous T cell lymphoma (CTCL) is a potentially fatal clonal malignancy of T cells primarily affecting the skin. The most common form of CTCL, mycosis fungoides, can be difficult to diagnose, resulting in treatment delay. We performed single-cell and spatial transcriptomics analysis of skin from patients with mycosis fungoides-type CTCL and an integrated comparative analysis with human skin cell atlas datasets from healthy and inflamed skin. We revealed the co-optation of T helper 2 (T 2) cell-immune gene programs by malignant CTCL cells and modeling of the tumor microenvironment to support their survival. We identified MHC-II fibroblasts and dendritic cells that can maintain T 2 cell-like tumor cells. CTCL tumor cells are spatially associated with B cells, forming tertiary lymphoid structure-like aggregates. Finally, we validated the enrichment of B cells in CTCL and its association with disease progression across three independent patient cohorts. Our findings provide diagnostic aids, potential biomarkers for disease staging and therapeutic strategies for CTCL.