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Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org ). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new gland-associated immune niche has implications for respiratory health.

Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1 + profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas., QC 20230710

Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1+ profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas.

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and bears multiple spike mutations2. Here we show that Omicron spike has higher affinity for ACE2 compared to Delta as well as a marked change of antigenicity conferring significant evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralising antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralisation. Importantly, 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 lower airway organoids, lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared to Delta. Replication differences mapped to entry efficiency using spike pseudotyped virus (PV) assays. The defect for Omicron PV to enter specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and knock down of TMPRSS2 impacted Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently utilises the cellular protease TMPRSS2 that promotes cell entry via plasma membrane fusion, with greater dependency on cell entry via the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to utilise TMPRSS2, syncytium formation by the Omicron spike was markedly impaired compared to the Delta spike. Omicron’s less efficient spike cleavage at S1/S2 is associated with shift in cellular tropism away from TMPRSS2 expressing cells, with implications for altered pathogenesis.

The Human Cell Atlas (HCA) consortium aims to establish an atlas of all organs in the healthy human body at single-cell resolution to increase our understanding of basic biological processes that govern development, physiology and anatomy, and to accelerate diagnosis and treatment of disease. The Lung Biological Network of the HCA aims to generate the Human Lung Cell Atlas as a reference for the cellular repertoire, molecular cell states and phenotypes, and cell-cell interactions that characterise normal lung homeostasis in healthy lung tissue. Such a reference atlas of the healthy human lung will facilitate mapping the changes in the cellular landscape in disease. The discovAIR project is one of six pilot actions for the HCA funded by the European Commission in the context of the H2020 framework programme. discovAIR aims to establish the first draft of an integrated Human Lung Cell Atlas, combining single-cell transcriptional and epigenetic profiling with spatially resolving techniques on matched tissue samples, as well as including a number of chronic and infectious diseases of the lung. The integrated Human Lung Cell Atlas will be available as a resource for the wider respiratory community, including basic and translational scientists, clinical medicine, and the private sector, as well as for patients with lung disease and the interested lay public. We anticipate that the Human Lung Cell Atlas will be the founding stone for a more detailed understanding of the pathogenesis of lung diseases, guiding the design of novel diagnostics and preventive or curative interventions.

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2(+)TMPRSS2(+) cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention. An integrated analysis of over 100 single-cell and single-nucleus transcriptomics studies illustrates severe acute respiratory syndrome coronavirus 2 viral entry gene coexpression patterns across different human tissues, and shows association of age, smoking status and sex with viral entry gene expression in respiratory cell populations.

Millions of couples worldwide have difficulties in conceiving a child. These couples, affected by infertility, suffer from symptoms of stress. The causes of infertility are largely unknown and current available treatment, in vitro fertilization has moderate success rates. The in vitro fertilization covers pre-implantation stage of the human embryo development. Better understanding of the molecular mechanisms in the early development might help to improve the in vitro fertilization methods. Much of the knowledge on early development has been gained from model organisms: nematode, fruit fly, zebrafish and mouse. Mouse is a commonly used model organism for mammalian pre-implantation development. Global gene expression studies have been performed on mouse and human. Overall, gene expression changes seem to be similar in principle between the organisms. However, the expression of the first transcribed genes in the early development may not be as conserved between species as other genes expressed later in development. Therefore, human pre-implantation development should also be studied on human material for better translation of the results. Genes with a dynamic expression profile in human pre-implantation development were identified in Paper I. Various criteria such as conservation, expression profile in mouse, relevance in cancer and novelty were applied to choose seventy genes of potential importance in human early development. Expression of those genes was studied in mouse and compared with human orthologues. The results showed differences in the expression profiles between human and mouse. Paper II found novel regulatory elements and potentially important transcription factors from human early development by single-cell RNA sequencing. About 350 of oocytes and blastomeres from early embryos were studied, the promoters of activated genes were analyzed, and many PRD-like homeodomain genes with first-time evidence of expression were cloned. These genes were suggested to

Millions of couples worldwide have difficulties in conceiving a child. These couples, affected by infertility, suffer from symptoms of stress. The causes of infertility are largely unknown and current available treatment, in vitro fertilization has moderate success rates. The in vitro fertilization covers pre-implantation stage of the human embryo development. Better understanding of the molecular mechanisms in the early development might help to improve the in vitro fertilization methods. Much of the knowledge on early development has been gained from model organisms: nematode, fruit fly, zebrafish and mouse. Mouse is a commonly used model organism for mammalian pre-implantation development. Global gene expression studies have been performed on mouse and human. Overall, gene expression changes seem to be similar in principle between the organisms. However, the expression of the first transcribed genes in the early development may not be as conserved between species as other genes expressed later in development. Therefore, human pre-implantation development should also be studied on human material for better translation of the results. Genes with a dynamic expression profile in human pre-implantation development were identified in Paper I. Various criteria such as conservation, expression profile in mouse, relevance in cancer and novelty were applied to choose seventy genes of potential importance in human early development. Expression of those genes was studied in mouse and compared with human orthologues. The results showed differences in the expression profiles between human and mouse. Paper II found novel regulatory elements and potentially important transcription factors from human early development by single-cell RNA sequencing. About 350 of oocytes and blastomeres from early embryos were studied, the promoters of activated genes were analyzed, and many PRD-like homeodomain genes with first-time evidence of expression were cloned. These genes were suggested to

Millions of couples worldwide have difficulties in conceiving a child. These couples, affected by infertility, suffer from symptoms of stress. The causes of infertility are largely unknown and current available treatment, in vitro fertilization has moderate success rates. The in vitro fertilization covers pre-implantation stage of the human embryo development. Better understanding of the molecular mechanisms in the early development might help to improve the in vitro fertilization methods. Much of the knowledge on early development has been gained from model organisms: nematode, fruit fly, zebrafish and mouse. Mouse is a commonly used model organism for mammalian pre-implantation development. Global gene expression studies have been performed on mouse and human. Overall, gene expression changes seem to be similar in principle between the organisms. However, the expression of the first transcribed genes in the early development may not be as conserved between species as other genes expressed later in development. Therefore, human pre-implantation development should also be studied on human material for better translation of the results. Genes with a dynamic expression profile in human pre-implantation development were identified in Paper I. Various criteria such as conservation, expression profile in mouse, relevance in cancer and novelty were applied to choose seventy genes of potential importance in human early development. Expression of those genes was studied in mouse and compared with human orthologues. The results showed differences in the expression profiles between human and mouse. Paper II found novel regulatory elements and potentially important transcription factors from human early development by single-cell RNA sequencing. About 350 of oocytes and blastomeres from early embryos were studied, the promoters of activated genes were analyzed, and many PRD-like homeodomain genes with first-time evidence of expression were cloned. These genes were suggested to

Millions of couples worldwide have difficulties in conceiving a child. These couples, affected by infertility, suffer from symptoms of stress. The causes of infertility are largely unknown and current available treatment, in vitro fertilization has moderate success rates. The in vitro fertilization covers pre-implantation stage of the human embryo development. Better understanding of the molecular mechanisms in the early development might help to improve the in vitro fertilization methods. Much of the knowledge on early development has been gained from model organisms: nematode, fruit fly, zebrafish and mouse. Mouse is a commonly used model organism for mammalian pre-implantation development. Global gene expression studies have been performed on mouse and human. Overall, gene expression changes seem to be similar in principle between the organisms. However, the expression of the first transcribed genes in the early development may not be as conserved between species as other genes expressed later in development. Therefore, human pre-implantation development should also be studied on human material for better translation of the results. Genes with a dynamic expression profile in human pre-implantation development were identified in Paper I. Various criteria such as conservation, expression profile in mouse, relevance in cancer and novelty were applied to choose seventy genes of potential importance in human early development. Expression of those genes was studied in mouse and compared with human orthologues. The results showed differences in the expression profiles between human and mouse. Paper II found novel regulatory elements and potentially important transcription factors from human early development by single-cell RNA sequencing. About 350 of oocytes and blastomeres from early embryos were studied, the promoters of activated genes were analyzed, and many PRD-like homeodomain genes with first-time evidence of expression were cloned. These genes were suggested to

Millions of couples worldwide have difficulties in conceiving a child. These couples, affected by infertility, suffer from symptoms of stress. The causes of infertility are largely unknown and current available treatment, in vitro fertilization has moderate success rates. The in vitro fertilization covers pre-implantation stage of the human embryo development. Better understanding of the molecular mechanisms in the early development might help to improve the in vitro fertilization methods. Much of the knowledge on early development has been gained from model organisms: nematode, fruit fly, zebrafish and mouse. Mouse is a commonly used model organism for mammalian pre-implantation development. Global gene expression studies have been performed on mouse and human. Overall, gene expression changes seem to be similar in principle between the organisms. However, the expression of the first transcribed genes in the early development may not be as conserved between species as other genes expressed later in development. Therefore, human pre-implantation development should also be studied on human material for better translation of the results. Genes with a dynamic expression profile in human pre-implantation development were identified in Paper I. Various criteria such as conservation, expression profile in mouse, relevance in cancer and novelty were applied to choose seventy genes of potential importance in human early development. Expression of those genes was studied in mouse and compared with human orthologues. The results showed differences in the expression profiles between human and mouse. Paper II found novel regulatory elements and potentially important transcription factors from human early development by single-cell RNA sequencing. About 350 of oocytes and blastomeres from early embryos were studied, the promoters of activated genes were analyzed, and many PRD-like homeodomain genes with first-time evidence of expression were cloned. These genes were suggested to

Millions of couples worldwide have difficulties in conceiving a child. These couples, affected by infertility, suffer from symptoms of stress. The causes of infertility are largely unknown and current available treatment, in vitro fertilization has moderate success rates. The in vitro fertilization covers pre-implantation stage of the human embryo development. Better understanding of the molecular mechanisms in the early development might help to improve the in vitro fertilization methods. Much of the knowledge on early development has been gained from model organisms: nematode, fruit fly, zebrafish and mouse. Mouse is a commonly used model organism for mammalian pre-implantation development. Global gene expression studies have been performed on mouse and human. Overall, gene expression changes seem to be similar in principle between the organisms. However, the expression of the first transcribed genes in the early development may not be as conserved between species as other genes expressed later in development. Therefore, human pre-implantation development should also be studied on human material for better translation of the results. Genes with a dynamic expression profile in human pre-implantation development were identified in Paper I. Various criteria such as conservation, expression profile in mouse, relevance in cancer and novelty were applied to choose seventy genes of potential importance in human early development. Expression of those genes was studied in mouse and compared with human orthologues. The results showed differences in the expression profiles between human and mouse. Paper II found novel regulatory elements and potentially important transcription factors from human early development by single-cell RNA sequencing. About 350 of oocytes and blastomeres from early embryos were studied, the promoters of activated genes were analyzed, and many PRD-like homeodomain genes with first-time evidence of expression were cloned. These genes were suggested to

Millions of couples worldwide have difficulties in conceiving a child. These couples, affected by infertility, suffer from symptoms of stress. The causes of infertility are largely unknown and current available treatment, in vitro fertilization has moderate success rates. The in vitro fertilization covers pre-implantation stage of the human embryo development. Better understanding of the molecular mechanisms in the early development might help to improve the in vitro fertilization methods. Much of the knowledge on early development has been gained from model organisms: nematode, fruit fly, zebrafish and mouse. Mouse is a commonly used model organism for mammalian pre-implantation development. Global gene expression studies have been performed on mouse and human. Overall, gene expression changes seem to be similar in principle between the organisms. However, the expression of the first transcribed genes in the early development may not be as conserved between species as other genes expressed later in development. Therefore, human pre-implantation development should also be studied on human material for better translation of the results. Genes with a dynamic expression profile in human pre-implantation development were identified in Paper I. Various criteria such as conservation, expression profile in mouse, relevance in cancer and novelty were applied to choose seventy genes of potential importance in human early development. Expression of those genes was studied in mouse and compared with human orthologues. The results showed differences in the expression profiles between human and mouse. Paper II found novel regulatory elements and potentially important transcription factors from human early development by single-cell RNA sequencing. About 350 of oocytes and blastomeres from early embryos were studied, the promoters of activated genes were analyzed, and many PRD-like homeodomain genes with first-time evidence of expression were cloned. These genes were suggested to

Millions of couples worldwide have difficulties in conceiving a child. These couples, affected by infertility, suffer from symptoms of stress. The causes of infertility are largely unknown and current available treatment, in vitro fertilization has moderate success rates. The in vitro fertilization covers pre-implantation stage of the human embryo development. Better understanding of the molecular mechanisms in the early development might help to improve the in vitro fertilization methods. Much of the knowledge on early development has been gained from model organisms: nematode, fruit fly, zebrafish and mouse. Mouse is a commonly used model organism for mammalian pre-implantation development. Global gene expression studies have been performed on mouse and human. Overall, gene expression changes seem to be similar in principle between the organisms. However, the expression of the first transcribed genes in the early development may not be as conserved between species as other genes expressed later in development. Therefore, human pre-implantation development should also be studied on human material for better translation of the results. Genes with a dynamic expression profile in human pre-implantation development were identified in Paper I. Various criteria such as conservation, expression profile in mouse, relevance in cancer and novelty were applied to choose seventy genes of potential importance in human early development. Expression of those genes was studied in mouse and compared with human orthologues. The results showed differences in the expression profiles between human and mouse. Paper II found novel regulatory elements and potentially important transcription factors from human early development by single-cell RNA sequencing. About 350 of oocytes and blastomeres from early embryos were studied, the promoters of activated genes were analyzed, and many PRD-like homeodomain genes with first-time evidence of expression were cloned. These genes were suggested to