Automatic cell-type harmonization and integration across Human Cell Atlas datasets.

Harmonizing cell types across the single-cell community and assembling them into a common framework is central to building a standardized Human Cell Atlas. Here, we present CellHint, a predictive clustering tree-based tool to resolve cell-type differences in annotation resolution and technical biases across datasets. CellHint accurately quantifies cell-cell transcriptomic similarities and places cell types into a relationship graph that hierarchically defines shared and unique cell subtypes. Application to multiple immune datasets recapitulates expert-curated annotations. CellHint also reveals underexplored relationships between healthy and diseased lung cell states in eight diseases. Furthermore, we present a workflow for fast cross-dataset integration guided by harmonized cell types and cell hierarchy, which uncovers underappreciated cell types in adult human hippocampus. Finally, we apply CellHint to 12 tissues from 38 datasets, providing a deeply curated cross-tissue database with ∼3.7 million cells and various machine learning models for automatic cell annotation across human tissues.

A human fetal lung cell atlas uncovers proximal-distal gradients of differentiation and key regulators of epithelial fates.

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.

Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation.

T helper type 2 (Th2) cells are important regulators of mammalian adaptive immunity and have relevance for infection, autoimmunity, and tumor immunology. Using a newly developed, genome-wide retroviral CRISPR knockout (KO) library, combined with RNA-seq, ATAC-seq, and ChIP-seq, we have dissected the regulatory circuitry governing activation and differentiation of these cells. Our experiments distinguish cell activation versus differentiation in a quantitative framework. We demonstrate that these two processes are tightly coupled and are jointly controlled by many transcription factors, metabolic genes, and cytokine/receptor pairs. There are only a small number of genes regulating differentiation without any role in activation. By combining biochemical and genetic data, we provide an atlas for Th2 differentiation, validating known regulators and identifying factors, such as Pparg and Bhlhe40, as part of the core regulatory network governing Th2 helper cell fates.

Distinct microbial and immune niches of the human colon.

Gastrointestinal microbiota and immune cells interact closely and display regional specificity; however, little is known about how these communities differ with location. Here, we simultaneously assess microbiota and single immune cells across the healthy, adult human colon, with paired characterization of immune cells in the mesenteric lymph nodes, to delineate colonic immune niches at steady state. We describe distinct helper T cell activation and migration profiles along the colon and characterize the transcriptional adaptation trajectory of regulatory T cells between lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal expansion and mutational frequency from the cecum to the sigmoid colon and link this to the increasing number of reactive bacterial species.

Human SARS-CoV-2 challenge uncovers local and systemic response dynamics.

The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the dynamics of early cellular responses to this disease remains limited1. Here in our SARS-CoV-2 human challenge study, we used single-cell multi-omics profiling of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in seronegative individuals challenged with pre-Alpha SARS-CoV-2. Our analyses revealed rapid changes in cell-type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific time points and infection status. We observed that the interferon response in blood preceded the nasopharyngeal response. Moreover, nasopharyngeal immune infiltration occurred early in samples from individuals with only transient infection and later in samples from individuals with sustained infection. High expression of HLA-DQA2 before inoculation was associated with preventing sustained infection. Ciliated cells showed multiple immune responses and were most permissive for viral replication, whereas nasopharyngeal T cells and macrophages were infected non-productively. We resolved 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our new computational pipeline Cell2TCR identifies activated antigen-responding T cells based on a gene expression signature and clusters these into clonotype groups and motifs. Overall, our detailed time series data can serve as a Rosetta stone for epithelial and immune cell responses and reveals early dynamic responses associated with protection against infection.

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity.

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.

Molecular connectomics: Placing cells into morphological tissue context.

Here we propose "molecular connectomics" to link molecular and morphological cell features in three dimensions across scales, using machine learning and artificial intelligence to reveal emergent properties of complex biological systems.

A roadmap for the Human Developmental Cell Atlas.

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.

Cells of the human intestinal tract mapped across space and time.

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.

Blood and immune development in human fetal bone marrow and Down syndrome.

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 conception1,2, 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).

SOX9 maintains human foetal lung tip progenitor state by enhancing WNT and RTK signalling.

The balance between self-renewal and differentiation in human foetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self-renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self-renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID, we place SOX9 at the centre of a transcriptional network, which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof-of-principle CRISPRi screen and Targeted DamID tools establish a new workflow for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease.

Decoding human fetal liver haematopoiesis.

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.

Single-cell reconstruction of the early maternal-fetal interface in humans.

During early human pregnancy the uterine mucosa transforms into the decidua, into which the fetal placenta implants and where placental trophoblast cells intermingle and communicate with maternal cells. Trophoblast-decidual interactions underlie common diseases of pregnancy, including pre-eclampsia and stillbirth. Here we profile the transcriptomes of about 70,000 single cells from first-trimester placentas with matched maternal blood and decidual cells. The cellular composition of human decidua reveals subsets of perivascular and stromal cells that are located in distinct decidual layers. There are three major subsets of decidual natural killer cells that have distinctive immunomodulatory and chemokine profiles. We develop a repository of ligand-receptor complexes and a statistical tool to predict the cell-type specificity of cell-cell communication via these molecular interactions. Our data identify many regulatory interactions that prevent harmful innate or adaptive immune responses in this environment. Our single-cell atlas of the maternal-fetal interface reveals the cellular organization of the decidua and placenta, and the interactions that are critical for placentation and reproductive success.

Obesity Is Associated with Attenuated Tissue Immunity in COVID-19.

Rationale: Obesity affects 40% of U.S. adults, is associated with a proinflammatory state, and presents a significant risk factor for the development of severe coronavirus disease (COVID-19). To date, there is limited information on how obesity might affect immune cell responses in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Objectives: To determine the impact of obesity on respiratory tract immunity in COVID-19 across the human lifespan. Methods: We analyzed single-cell transcriptomes from BAL in three ventilated adult cohorts with (n = 24) or without (n = 9) COVID-19 from nasal immune cells in children with (n = 14) or without (n = 19) COVID-19, and from peripheral blood mononuclear cells in an independent adult COVID-19 cohort (n = 42), comparing obese and nonobese subjects. Measurements and Main Results: Surprisingly, we found that obese adult subjects had attenuated lung immune or inflammatory responses in SARS-CoV-2 infection, with decreased expression of IFN-α, IFN-γ, and TNF-α (tumor necrosis factor α) response gene signatures in almost all lung epithelial and immune cell subsets, and lower expression of IFNG and TNF in specific lung immune cells. Peripheral blood immune cells in an independent adult cohort showed a similar but less marked reduction in type-I IFN and IFNγ response genes, as well as decreased serum IFNα, in obese patients with SARS-CoV-2. Nasal immune cells from obese children with COVID-19 also showed reduced enrichment of IFN-α and IFN-γ response genes. Conclusions: These findings show blunted tissue immune responses in obese patients with COVID-19, with implications for treatment stratification, supporting the specific application of inhaled recombinant type-I IFNs in this vulnerable subset.

Expression Atlas update: gene and protein expression in multiple species.

The EMBL-EBI Expression Atlas is an added value knowledge base that enables researchers to answer the question of where (tissue, organism part, developmental stage, cell type) and under which conditions (disease, treatment, gender, etc) a gene or protein of interest is expressed. Expression Atlas brings together data from >4500 expression studies from >65 different species, across different conditions and tissues. It makes these data freely available in an easy to visualise form, after expert curation to accurately represent the intended experimental design, re-analysed via standardised pipelines that rely on open-source community developed tools. Each study's metadata are annotated using ontologies. The data are re-analyzed with the aim of reproducing the original conclusions of the underlying experiments. Expression Atlas is currently divided into Bulk Expression Atlas and Single Cell Expression Atlas. Expression Atlas contains data from differential studies (microarray and bulk RNA-Seq) and baseline studies (bulk RNA-Seq and proteomics), whereas Single Cell Expression Atlas is currently dedicated to Single Cell RNA-Sequencing (scRNA-Seq) studies. The resource has been in continuous development since 2009 and it is available at https://www.ebi.ac.uk/gxa.

FastCAR: fast correction for ambient RNA to facilitate differential gene expression analysis in single-cell RNA-sequencing datasets.

Cell type-specific differential gene expression analyses based on single-cell transcriptome datasets are sensitive to the presence of cell-free mRNA in the droplets containing single cells. This so-called ambient RNA contamination may differ between samples obtained from patients and healthy controls. Current ambient RNA correction methods were not developed specifically for single-cell differential gene expression (sc-DGE) analyses and might therefore not sufficiently correct for ambient RNA-derived signals. Here, we show that ambient RNA levels are highly sample-specific. We found that without ambient RNA correction, sc-DGE analyses erroneously identify transcripts originating from ambient RNA as cell type-specific disease-associated genes. We therefore developed a computationally lean and intuitive correction method, Fast Correction for Ambient RNA (FastCAR), optimized for sc-DGE analysis of scRNA-Seq datasets generated by droplet-based methods including the 10XGenomics Chromium platform. FastCAR uses the profile of transcripts observed in libraries that likely represent empty droplets to determine the level of ambient RNA in each individual sample, and then corrects for these ambient RNA gene expression values. FastCAR can be applied as part of the data pre-processing and QC in sc-DGE workflows comparing scRNA-Seq data in a health versus disease experimental design. We compared FastCAR with two methods previously developed to remove ambient RNA, SoupX and CellBender. All three methods identified additional genes in sc-DGE analyses that were not identified in the absence of ambient RNA correction. However, we show that FastCAR performs better at correcting gene expression values attributed to ambient RNA, resulting in a lower frequency of false-positive observations. Moreover, the use of FastCAR in a sc-DGE workflow increases the cell-type specificity of sc-DGE analyses across disease conditions.

Th2 high and mast cell gene signatures are associated with corticosteroid sensitivity in COPD.

RATIONALE: Severe asthma and chronic obstructive pulmonary disease (COPD) share common pathophysiological traits such as relative corticosteroid insensitivity. We recently published three transcriptome-associated clusters (TACs) using hierarchical analysis of the sputum transcriptome in asthmatics from the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort comprising one Th2-high inflammatory signature (TAC1) and two Th2-low signatures (TAC2 and TAC3). OBJECTIVE: We examined whether gene expression signatures obtained in asthma can be used to identify the subgroup of patients with COPD with steroid sensitivity. METHODS: Using gene set variation analysis, we examined the distribution and enrichment scores (ES) of the 3 TACs in the transcriptome of bronchial biopsies from 46 patients who participated in the Groningen Leiden Universities Corticosteroids in Obstructive Lung Disease COPD study that received 30 months of treatment with inhaled corticosteroids (ICS) with and without an added long-acting ß-agonist (LABA). The identified signatures were then associated with longitudinal clinical variables after treatment. Differential gene expression and cellular convolution were used to define key regulated genes and cell types. MEASUREMENTS AND MAIN RESULTS: Bronchial biopsies in patients with COPD at baseline showed a wide range of expression of the 3 TAC signatures. After ICS±LABA treatment, the ES of TAC1 was significantly reduced at 30 months, but those of TAC2 and TAC3 were unaffected. A corticosteroid-sensitive TAC1 signature was developed from the TAC1 ICS-responsive genes. This signature consisted of mast cell-specific genes identified by single-cell RNA-sequencing and positively correlated with bronchial biopsy mast cell numbers following ICS±LABA. Baseline levels of gene transcription correlated with the change in RV/TLC %predicted following 30-month ICS±LABA. CONCLUSION: Sputum-derived transcriptomic signatures from an asthma cohort can be recapitulated in bronchial biopsies of patients with COPD and identified a signature of airway mast cells as a predictor of corticosteroid responsiveness.

TreeAndLeaf: an R/Bioconductor package for graphs and trees with focus on the leaves.

MOTIVATION: Dendrogram is a classical diagram for visualizing binary trees. Although efficient to represent hierarchical relations, it provides limited space for displaying information on the leaf elements, especially for large trees. RESULTS: Here, we present TreeAndLeaf, an R/Bioconductor package that implements a hybrid layout strategy to represent tree diagrams with focus on the leaves. The TreeAndLeaf package combines force-directed graph and tree layout algorithms using a single visualization system, allowing projection of multiple layers of information onto a graph-tree diagram. The Supplementary Information provides two case studies that use breast cancer data from epidemiological and experimental studies. AVAILABILITY AND IMPLEMENTATION: TreeAndLeaf is written in the R language, and is available from the Bioconductor project at http://bioconductor.org/packages/TreeAndLeaf/ (version≥1.4.2). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Integrated Single-Cell Atlas of Endothelial Cells of the Human Lung.

BACKGROUND: Cellular diversity of the lung endothelium has not been systematically characterized in humans. We provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. METHODS: We reprocessed human control single-cell RNA sequencing (scRNAseq) data from 6 datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in vitro was performed. The signaling network between different lung cell types was studied. For cross-species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. RESULTS: Six lung scRNAseq datasets were reanalyzed and annotated to identify >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial, and venous ECs, we found previously indistinguishable subpopulations; among venous EC, we identified 2 previously indistinguishable populations: pulmonary-venous ECs (COL15A1neg) localized to the lung parenchyma and systemic-venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1, and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic-venous ECs and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). CONCLUSIONS: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.