Publications

Below is the list of papers that are part of the Human Cell Atlas.

These include papers that have been approved by the HCA Publication Committee (composed of HCA Organising Committee members). The HCA Publication Committee reviews submitted publications to check if they fit within HCA technical scope. This review by the HCA Publication Committee does not serve as peer review.

We encourage you to submit your paper for inclusion within the Human Cell Atlas publications, which can help your paper gain more visibility. Please see here for the Publication Review process.

This list also includes papers containing datasets that have been integrated into HCA Atlases available at the HCA Data Portal.

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Pre-print
Networks
Liver
Topics
Human Subjects, Healthy Donors

Spatial transcriptomics of healthy and fibrotic human liver at single-cell resolution

Brianna Watson; Biplab Paul; Liat Amir-Zilberstein; Asa Segerstolpe; Raza Rahman; Angela Shih; Jacques Deguine; Ramnik Xavier; Jeffrey R Moffitt; Alan C Mullen

bioRxiv 2024;2024.02.02.578633

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Musculoskeletal
Topics
Human Subjects, Healthy Donors, Open Access Data

Single nucleus and spatial transcriptomic profiling of healthy human hamstring tendon.

Mimpen JY; Ramos-Mucci L; Paul C; Kurjan A; Hulley PA; Ikwuanusi CT; Cohen CJ; Gwilym SE; Baldwin MJ; Cribbs AP et al

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2024;38;10;e23629

The molecular and cellular basis of health in human tendons remains poorly understood. Among human tendons, hamstring tendon has markedly low pathology and can provide a prototypic healthy tendon reference. The aim of this study was to determine the transcriptomes and location of all cell types in healthy hamstring tendon. Using single nucleus RNA sequencing, we profiled the transcriptomes of 10 533 nuclei from four healthy donors and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and identified cell types previously unreported in tendons, including different skeletal muscle cell types, satellite cells, adipocytes, and undefined nervous system cells. The location of these cell types within tendon was defined using spatial transcriptomics and imaging, and potential transcriptional networks and cell-cell interactions were analyzed. We demonstrate that fibroblasts have the highest number of potential cell-cell interactions in our dataset, are present throughout the tendon, and play an important role in the production and organization of extracellular matrix, thus confirming their role as key regulators of hamstring tendon homeostasis. Overall, our findings underscore the complexity of the cellular networks that underpin healthy human tendon function and the central role of fibroblasts as key regulators of hamstring tendon tissue homeostasis.

Peer reviewed
Networks
Development, Immune, Reproduction
Topics
Human Subjects, Healthy Donors, Disease Donors, Open Access Data, Experimental Methods

Acute response to pathogens in the early human placenta at single-cell resolution.

Hoo R; Ruiz-Morales ER; Kelava I; Rawat M; Mazzeo CI; Tuck E; Sancho-Serra C; Chelaghma S; Predeus AV; Murray S et al

Cell systems 2024

The placenta is a selective maternal-fetal barrier that provides nourishment and protection from infections. However, certain pathogens can attach to and even cross the placenta, causing pregnancy complications with potential lifelong impacts on the child's health. Here, we profiled at the single-cell level the placental responses to three pathogens associated with intrauterine complications-Plasmodium falciparum, Listeria monocytogenes, and Toxoplasma gondii. We found that upon exposure to the pathogens, all placental lineages trigger inflammatory responses that may compromise placental function. Additionally, we characterized the responses of fetal macrophages known as Hofbauer cells (HBCs) to each pathogen and propose that they are the probable niche for T. gondii. Finally, we revealed how P. falciparum adapts to the placental microenvironment by modulating protein export into the host erythrocyte and nutrient uptake pathways. Altogether, we have defined the cellular networks and signaling pathways mediating acute placental inflammatory responses that could contribute to pregnancy complications.

Press release

Peer reviewed
Networks
Breast
Topics
Human Subjects, Healthy Donors, Disease Donors

A single-cell atlas enables mapping of homeostatic cellular shifts in the adult human breast.

Reed AD; Pensa S; Steif A; Stenning J; Kunz DJ; Porter LJ; Hua K; He P; Twigger AJ; Siu AJQ et al

Nature Genetics 2024

Here we use single-cell RNA sequencing to compile a human breast cell atlas assembled from 55 donors that had undergone reduction mammoplasties or risk reduction mastectomies. From more than 800,000 cells we identified 41 cell subclusters across the epithelial, immune and stromal compartments. The contribution of these different clusters varied according to the natural history of the tissue. Age, parity and germline mutations, known to modulate the risk of developing breast cancer, affected the homeostatic cellular state of the breast in different ways. We found that immune cells from BRCA1 or BRCA2 carriers had a distinct gene expression signature indicative of potential immune exhaustion, which was validated by immunohistochemistry. This suggests that immune-escape mechanisms could manifest in non-cancerous tissues very early during tumor initiation. This atlas is a rich resource that can be used to inform novel approaches for early detection and prevention of breast cancer.

Peer reviewed
Networks
Eye
Topics
Human Subjects, Healthy Donors

Single-cell analyses reveal transient retinal progenitor cells in the ciliary margin of developing human retina.

Dorgau B; Collin J; Rozanska A; Zerti D; Unsworth A; Crosier M; Hussain R; Coxhead J; Dhanaseelan T; Patel A et al

Nature Communications 2024;15;1;3567

The emergence of retinal progenitor cells and differentiation to various retinal cell types represent fundamental processes during retinal development. Herein, we provide a comprehensive single cell characterisation of transcriptional and chromatin accessibility changes that underline retinal progenitor cell specification and differentiation over the course of human retinal development up to midgestation. Our lineage trajectory data demonstrate the presence of early retinal progenitors, which transit to late, and further to transient neurogenic progenitors, that give rise to all the retinal neurons. Combining single cell RNA-Seq with spatial transcriptomics of early eye samples, we demonstrate the transient presence of early retinal progenitors in the ciliary margin zone with decreasing occurrence from 8 post-conception week of human development. In retinal progenitor cells, we identified a significant enrichment for transcriptional enhanced associate domain transcription factor binding motifs, which when inhibited led to loss of cycling progenitors and retinal identity in pluripotent stem cell derived organoids.

Peer reviewed
Networks
Immune
Topics
Human Subjects, Healthy Donors, COVID-19

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

Lindeboom RGH; Worlock KB; Dratva LM; Yoshida M; Scobie D; Wagstaffe HR; Richardson L; Wilbrey-Clark A; Barnes JL; Kretschmer L et al

Nature 2024

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.

Editorial Press release

Peer reviewed
Networks
Development, Musculoskeletal
Topics
Human Subjects, Healthy Donors

Human skeletal muscle aging atlas.

Kedlian VR; Wang Y; Liu T; Chen X; Bolt L; Tudor C; Shen Z; Fasouli ES; Prigmore E; Kleshchevnikov V et al

Nature Aging 2024

Skeletal muscle aging is a key contributor to age-related frailty and sarcopenia with substantial implications for global health. Here we profiled 90,902 single cells and 92,259 single nuclei from 17 donors to map the aging process in the adult human intercostal muscle, identifying cellular changes in each muscle compartment. We found that distinct subsets of muscle stem cells exhibit decreased ribosome biogenesis genes and increased CCL2 expression, causing different aging phenotypes. Our atlas also highlights an expansion of nuclei associated with the neuromuscular junction, which may reflect re-innervation, and outlines how the loss of fast-twitch myofibers is mitigated through regeneration and upregulation of fast-type markers in slow-twitch myofibers with age. Furthermore, we document the function of aging muscle microenvironment in immune cell attraction. Overall, we present a comprehensive human skeletal muscle aging resource ( https://www.muscleageingcellatlas.org/ ) together with an in-house mouse muscle atlas to study common features of muscle aging across species.

Press release

Peer reviewed
Networks
Immune
Topics
Computational Methods

Label-aware distance mitigates temporal and spatial variability for clustering and visualization of single-cell gene expression data.

Liang S; Dou J; Iqbal R; Chen K

Communications biology 2024;7;1;326

Clustering and visualization are essential parts of single-cell gene expression data analysis. The Euclidean distance used in most distance-based methods is not optimal. The batch effect, i.e., the variability among samples gathered from different times, tissues, and patients, introduces large between-group distance and obscures the true identities of cells. To solve this problem, we introduce Label-Aware Distance (LAD), a metric using temporal/spatial locality of the batch effect to control for such factors. We validate LAD on simulated data as well as apply it to a mouse retina development dataset and a lung dataset. We also found the utility of our approach in understanding the progression of the Coronavirus Disease 2019 (COVID-19). LAD provides better cell embedding than state-of-the-art batch correction methods on longitudinal datasets. It can be used in distance-based clustering and visualization methods to combine the power of multiple samples to help make biological findings.

Peer reviewed
Networks
Oral & Craniofacial
Topics
Human Subjects, Healthy Donors, Open Access Data, Computational Methods

Single-cell and spatially resolved interactomics of tooth-associated keratinocytes in periodontitis.

Easter QT; Fernandes Matuck B; Beldorati Stark G; Worth CL; Predeus AV; Fremin B; Huynh K; Ranganathan V; Ren Z; Pereira D et al

Nature Communications 2024;15;1;5016

Periodontitis affects billions of people worldwide. To address relationships of periodontal niche cell types and microbes in periodontitis, we generated an integrated single-cell RNA sequencing (scRNAseq) atlas of human periodontium (34-sample, 105918-cell), including sulcular and junctional keratinocytes (SK/JKs). SK/JKs displayed altered differentiation states and were enriched for effector cytokines in periodontitis. Single-cell metagenomics revealed 37 bacterial species with cell-specific tropism. Fluorescence in situ hybridization detected intracellular 16 S and mRNA signals of multiple species and correlated with SK/JK proinflammatory phenotypes in situ. Cell-cell communication analysis predicted keratinocyte-specific innate and adaptive immune interactions. Highly multiplexed immunofluorescence (33-antibody) revealed peri-epithelial immune foci, with innate cells often spatially constrained around JKs. Spatial phenotyping revealed immunosuppressed JK-microniches and SK-localized tertiary lymphoid structures in periodontitis. Here, we demonstrate impacts on and predicted interactomics of SK and JK cells in health and periodontitis, which requires further investigation to support precision periodontal interventions in states of chronic inflammation.

Peer reviewed
Topics
Human Subjects, Disease Donors

Systematic dissection of tumor-normal single-cell ecosystems across a thousand tumors of 30 cancer types.

Kang J; Lee JH; Cha H; An J; Kwon J; Lee S; Kim S; Baykan MY; Kim SY; An D et al

Nature Communications 2024;15;1;4067

The complexity of the tumor microenvironment poses significant challenges in cancer therapy. Here, to comprehensively investigate the tumor-normal ecosystems, we perform an integrative analysis of 4.9 million single-cell transcriptomes from 1070 tumor and 493 normal samples in combination with pan-cancer 137 spatial transcriptomics, 8887 TCGA, and 1261 checkpoint inhibitor-treated bulk tumors. We define a myriad of cell states constituting the tumor-normal ecosystems and also identify hallmark gene signatures across different cell types and organs. Our atlas characterizes distinctions between inflammatory fibroblasts marked by AKR1C1 or WNT5A in terms of cellular interactions and spatial co-localization patterns. Co-occurrence analysis reveals interferon-enriched community states including tertiary lymphoid structure (TLS) components, which exhibit differential rewiring between tumor, adjacent normal, and healthy normal tissues. The favorable response of interferon-enriched community states to immunotherapy is validated using immunotherapy-treated cancers (n = 1261) including our lung cancer cohort (n = 497). Deconvolution of spatial transcriptomes discriminates TLS-enriched from non-enriched cell types among immunotherapy-favorable components. Our systematic dissection of tumor-normal ecosystems provides a deeper understanding of inter- and intra-tumoral heterogeneity.

Peer reviewed
Topics
Computational Methods

GRouNdGAN: GRN-guided simulation of single-cell RNA-seq data using causal generative adversarial networks.

Zinati Y; Takiddeen A; Emad A

Nature Communications 2024;15;1;4055

We introduce GRouNdGAN, a gene regulatory network (GRN)-guided reference-based causal implicit generative model for simulating single-cell RNA-seq data, in silico perturbation experiments, and benchmarking GRN inference methods. Through the imposition of a user-defined GRN in its architecture, GRouNdGAN simulates steady-state and transient-state single-cell datasets where genes are causally expressed under the control of their regulating transcription factors (TFs). Training on six experimental reference datasets, we show that our model captures non-linear TF-gene dependencies and preserves gene identities, cell trajectories, pseudo-time ordering, and technical and biological noise, with no user manipulation and only implicit parameterization. GRouNdGAN can synthesize cells under new conditions to perform in silico TF knockout experiments. Benchmarking various GRN inference algorithms reveals that GRouNdGAN effectively bridges the existing gap between simulated and biological data benchmarks of GRN inference algorithms, providing gold standard ground truth GRNs and realistic cells corresponding to the biological system of interest.

Peer reviewed
Networks
Immune
Topics
Human Subjects, Disease Donors, Experimental Methods, Computational Methods

An atlas of cells in the human tonsil.

Massoni-Badosa R; Aguilar-Fernández S; Nieto JC; Soler-Vila P; Elosua-Bayes M; Marchese D; Kulis M; Vilas-Zornoza A; Bühler MM; Rashmi S et al

Immunity 2024;57;2;379-399.e18

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.

Pre-print
Networks
Reproduction
Topics
Human Subjects, Healthy Donors

Cellular heterogeneity and dynamics of the human uterus in healthy premenopausal women.

Ulrich ND; Vargo A; Ma Q; Shen YC; Hannum DF; Gurczynski SJ; Moore BB; Schon S; Lieberman R; Shikanov A et al

bioRxiv 2024

The human uterus is a complex and dynamic organ whose lining grows, remodels, and regenerates in every menstrual cycle or upon tissue damage. Here we applied single-cell RNA sequencing to profile more the 50,000 uterine cells from both the endometrium and myometrium of 5 healthy premenopausal individuals, and jointly analyzed the data with a previously published dataset from 15 subjects. The resulting normal uterus cell atlas contains more than 167K cells representing the lymphatic endothelium, blood endothelium, stromal, ciliated epithelium, unciliated epithelium, and immune cell populations. Focused analyses within each major cell type and comparisons with subtype labels from prior studies allowed us to document supporting evidence, resolve naming conflicts, and to propose a consensus annotation system of 39 subtypes. We release their gene expression centroids, differentially expressed genes, and mRNA patterns of literature-based markers as a shared community resource. We find many subtypes show dynamic changes over different phases of the cycle and identify multiple potential progenitor cells: compartment-wide progenitors for each major cell type, transitional cells that are upstream of other subtypes, and potential cross-lineage multipotent stromal progenitors that may be capable of replenishing the epithelial, stromal, and endothelial compartments. When compared to the healthy premenopausal samples, a postpartum and a postmenopausal uterus sample revealed substantially altered tissue composition, involving the rise or fall of stromal, endothelial, and immune cells. The cell taxonomy and molecular markers we report here are expected to inform studies of both basic biology of uterine function and its disorders.

Peer reviewed
Networks
Development, Gut, Heart, Kidney, Nervous system
Topics
Human Subjects, Healthy Donors, Disease Donors, Open Access Data

Single-cell atlas of the human brain vasculature across development, adulthood and disease.

Wälchli T; Ghobrial M; Schwab M; Takada S; Zhong H; Suntharalingham S; Vetiska S; Gonzalez DR; Wu R; Rehrauer H et al

Nature 2024

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

Peer reviewed
Networks
Reproduction
Topics
Healthy Donors, Open Access Data, Computational Methods

Cellular atlas of the human ovary using morphologically guided spatial transcriptomics and single-cell sequencing.

Jones ASK; Hannum DF; Machlin JH; Tan A; Ma Q; Ulrich ND; Shen YC; Ciarelli M; Padmanabhan V; Marsh EE et al

Science advances 2024;10;14;eadm7506

The reproductive and endocrine functions of the ovary involve spatially defined interactions among specialized cell populations. Despite the ovary's importance in fertility and endocrine health, functional attributes of ovarian cells are largely uncharacterized. Here, we profiled >18,000 genes in 257 regions from the ovaries of two premenopausal donors to examine the functional units in the ovary. We also generated single-cell RNA sequencing data for 21,198 cells from three additional donors and identified four major cell types and four immune cell subtypes. Custom selection of sampling areas revealed distinct gene activities for oocytes, theca, and granulosa cells. These data contributed panels of oocyte-, theca-, and granulosa-specific genes, thus expanding the knowledge of molecular programs driving follicle development. Serial samples around oocytes and across the cortex and medulla uncovered previously unappreciated variation of hormone and extracellular matrix remodeling activities. This combined spatial and single-cell atlas serves as a resource for future studies of rare cells and pathological states in the ovary.

Peer reviewed
Networks
Gut, Immune
Topics
Model Organism Samples

Exploring the Immune Landscape of Disulfidptosis in Ulcerative Colitis and the Role of Modified Gegen Qinlian Decoction in Mediating Disulfidptosis to Alleviate Colitis in Mice.

Huang J; Zhang J; Wang F; Tang X

Journal of ethnopharmacology 2024;118527

Ulcerative colitis (UC), a recurrent inflammatory bowel disease, continues to challenge effective pharmacologic management. Disulfidptosis, a recently identified form of cell death, appears implicated in the progression of various diseases. Scientific studies have demonstrated that Modified Gegen Qinlian decoction (MGQD) alleviates UC symptoms. However, the underlying mechanisms remain inadequately elucidated.

Peer reviewed
Networks
Nervous system
Topics
Human Subjects, Healthy Donors, Open Access Data

Cell-type-specific effects of age and sex on human cortical neurons.

Chien JF; Liu H; Wang BA; Luo C; Bartlett A; Castanon R; Johnson ND; Nery JR; Osteen J; Li J et al

Neuron 2024

Altered transcriptional and epigenetic regulation of brain cell types may contribute to cognitive changes with advanced age. Using single-nucleus multi-omic DNA methylation and transcriptome sequencing (snmCT-seq) in frontal cortex from young adult and aged donors, we found widespread age- and sex-related variation in specific neuron types. The proportion of inhibitory SST- and VIP-expressing neurons was reduced in aged donors. Excitatory neurons had more profound age-related changes in their gene expression and DNA methylation than inhibitory cells. Hundreds of genes involved in synaptic activity, including EGR1, were less expressed in aged adults. Genes located in subtelomeric regions increased their expression with age and correlated with reduced telomere length. We further mapped cell-type-specific sex differences in gene expression and X-inactivation escape genes. Multi-omic single-nucleus epigenomes and transcriptomes provide new insight into the effects of age and sex on human neurons.

Peer reviewed
Networks
Development, Gut, Kidney, Lung, Organoid
Topics
Human Subjects, Healthy Donors, Disease Donors

Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids.

Gerli MFM; Calà G; Beesley MA; Sina B; Tullie L; Sun KY; Panariello F; Michielin F; Davidson JR; Russo FM et al

Nature Medicine 2024

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

Peer reviewed

Pan-cancer profiling of tumor-infiltrating natural killer cells through transcriptional reference mapping.

Netskar H; Pfefferle A; Goodridge JP; Sohlberg E; Dufva O; Teichmann SA; Brownlie D; Michaëlsson J; Marquardt N; Clancy T et al

Nature Immunology 2024

The functional diversity of natural killer (NK) cell repertoires stems from differentiation, homeostatic, receptor-ligand interactions and adaptive-like responses to viral infections. In the present study, we generated a single-cell transcriptional reference map of healthy human blood- and tissue-derived NK cells, with temporal resolution and fate-specific expression of gene-regulatory networks defining NK cell differentiation. Transfer learning facilitated incorporation of tumor-infiltrating NK cell transcriptomes (39 datasets, 7 solid tumors, 427 patients) into the reference map to analyze tumor microenvironment (TME)-induced perturbations. Of the six functionally distinct NK cell states identified, a dysfunctional stressed CD56bright state susceptible to TME-induced immunosuppression and a cytotoxic TME-resistant effector CD56dim state were commonly enriched across tumor types, the ratio of which was predictive of patient outcome in malignant melanoma and osteosarcoma. This resource may inform the design of new NK cell therapies and can be extended through transfer learning to interrogate new datasets from experimental perturbations or disease conditions.

Pre-print
Networks
Lung
Topics
Human Subjects, Open Access Data

High-parametric protein maps reveal the spatial organization in early-developing human lung

Sanem Sariyar; Alexandros Sountoulidis; Jan Niklas Hansen; Sergio Marco Salas; Mariya Mardamshina; Anna Martinez Casals; Frederic Ballllosera Navarro; Zaneta Andrusivova; Xiaofei Li; Paulo Czarnewski et al

bioRxiv 2024;2024.01.25.577163

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Topics
Human Subjects, Open Access Data, Experimental Methods, Computational Methods

Harnessing Agent-Based Modeling in CellAgentChat to Unravel Cell-Cell Interactions from Single-Cell Data

Vishvak Raghavan; Yue Li; Jun Ding

bioRxiv 2024;2023.08.23.554489

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Networks
Nervous system
Topics
Human Subjects, Healthy Donors, Disease Donors

Integrative Transcriptomics Reveals Layer 1 Astrocytes Altered in Schizophrenia

Julio Leon; Satoshi Yoshinaga; Mizuki Hino; Atsuko Nagaoka; Yoshinari Ando; Jonathan Moody; Miki Kojima; Ayako Kitazawa; Kanehiro Hayashi; Kazunori Nakajima et al

bioRxiv 2024;2024.06.27.601103

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Development, Musculoskeletal, Skin
Topics
Human Subjects, Healthy Donors, Open Access Data

A human embryonic limb cell atlas resolved in space and time

Zhang, Bao; He, Peng; Lawrence, John E. G.; Wang, Shuaiyu; Tuck, Elizabeth; Williams, Brian A.; Roberts, Kenny; Kleshchevnikov, Vitalii; Mamanova, Lira; Bolt, Liam et al

Nature 2023;1-11

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species. Using single-cell and spatial transcriptomics, human embryonic limb development across space and time and the diversification and cross-species conservation of cells are demonstrated.

Press release

Pre-print
Topics
Human Subjects, Open Access Data, Experimental Methods, Computational Methods

scSemiProfiler: Advancing Large-scale Single-cell Studies through Semi-profiling with Deep Generative Models and Active Learning

Jingtao Wang; Gregory Joseph Fonseca; Jun Ding

bioRxiv 2023;2023.11.20.567929

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print

scSemiProfiler: Advancing Large-scale Single-cell Studies through Semi-profiling with Deep Generative Models and Active Learning

Jingtao Wang; Gregory Joseph Fonseca; Jun Ding

bioRxiv 2023;2023.11.20.567929

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print

Spatially resolved single-cell atlas of the lung in fatal Covid19 in an African population reveals a distinct cellular signature and an interferon gamma dominated response

James Nyirenda; Olympia Hardy; João Da Silva Filho; Vanessa Herder; Charalampos Attipa; Charles Ndovi; Memory Siwombo; Takondwa Namalima; Leticia Suwedi; Watipenge Nyasulu et al

bioRxiv 2023;2023.11.16.566964

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Networks
Genetic diversity
Topics
Human Subjects, Healthy Donors, Open Access Data, Computational Methods

A single-cell atlas of transcribed cis-regulatory elements in the human genome

Jonathan Moody; Tsukasa Kouno; Miki Kojima; Ikuko Koya; Julio Leon; Akari Suzuki; Akira Hasegawa; Taishin Akiyama; Nobuko Akiyama; Masayuki Amagai et al

bioRxiv 2023;2023.11.13.566791

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print

Integrated multi-omics single cell atlas of the human retina

Jin Li; Jun Wang; Ignacio L Ibarra; Xuesen Cheng; Malte D Luecken; Jiaxiong Lu; Aboozar Monavarfeshani; Wenjun Yan; Yiqiao Zheng; Zhen Zuo et al

bioRxiv 2023;2023.11.07.566105

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print

Oncogenic RAS-Pathway Activation Drives Oncofetal Reprogramming and Creates Therapeutic Vulnerabilities in Juvenile Myelomonocytic Leukemia

Mark Hartmann; Maximilian Schönung; Jovana Rajak; Valentin Maurer; Ling Hai; Katharina Bauer; Mariam Hakobyan; Sina Staeble; Jens Langstein; Laura Jardine et al

bioRxiv 2023;2023.10.27.563754

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print

Early human fetal lung atlas reveals the temporal dynamics of epithelial cell plasticity

Henry Quach; Spencer Farrell; Kayshani Kanagarajah; Michael Wu; Xiaoqiao Xu; Prajkta Kallurkar; Andrei Turinsky; Christine E. Bear; Felix Ratjen; Sidhartha Goyal et al

bioRxiv 2023;2023.10.27.564403

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print

Iterative spatial protein and RNA analysis on the same tissue section using MICS technology

Emily Neil; Dongju Park; Rebecca C. Hennessey; Eric C. DiBiasio; Michael DiBuono; Hanna Lafayette; Erica Lloyd; Hsinyi Lo; Julia Femel; Alex Makrigiorgos et al

bioRxiv 2023;2023.10.27.564191

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Networks
Genetic diversity, Immune
Topics
Human Subjects, Healthy Donors, Open Access Data, Computational Methods

Quantification of the escape from X chromosome inactivation with the million cell-scale human single-cell omics datasets reveals heterogeneity of escape across cell types and tissues

Yoshihiko Tomofuji; Ryuya Edahiro; Yuya Shirai; Kian Hong Kock; Kyuto Sonehara; Qingbo S. Wang; Shinichi Namba; Jonathan Moody; Yoshinari Ando; Akari Suzuki et al

bioRxiv 2023;2023.10.14.561800

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Musculoskeletal
Topics
Healthy Donors, Disease Donors, Open Access Data, Experimental Methods, Computational Methods, Benchmarking

A roadmap for delivering a human musculoskeletal cell atlas

Baldwin, Mathew; Buckley, Christopher D.; Guilak, Farshid; Hulley, Philippa; Cribbs, Adam P.; Snelling, Sarah

Nature Reviews Rheumatology 2023;19;11;738-752

Advances in single-cell technologies have transformed the ability to identify the individual cell types present within tissues and organs. The musculoskeletal bionetwork, part of the wider Human Cell Atlas project, aims to create a detailed map of the healthy musculoskeletal system at a single-cell resolution throughout tissue development and across the human lifespan, with complementary generation of data from diseased tissues. Given the prevalence of musculoskeletal disorders, this detailed reference dataset will be critical to understanding normal musculoskeletal function in growth, homeostasis and ageing. The endeavour will also help to identify the cellular basis for disease and lay the foundations for novel therapeutic approaches to treating diseases of the joints, soft tissues and bone. Here, we present a Roadmap delineating the critical steps required to construct the first draft of a human musculoskeletal cell atlas. We describe the key challenges involved in mapping the extracellular matrix-rich, but cell-poor, tissues of the musculoskeletal system, outline early milestones that have been achieved and describe the vision and directions for a comprehensive musculoskeletal cell atlas. By embracing cutting-edge technologies, integrating diverse datasets and fostering international collaborations, this endeavour has the potential to drive transformative changes in musculoskeletal medicine. A single-cell atlas of the human musculoskeletal system could help to improve the understanding of musculoskeletal function in growth, homeostasis, ageing and disease. This Roadmap delineates the steps required and challenges involved in creating such an atlas.

Pre-print

Human subcutaneous and visceral adipocyte atlases uncover classical and specialized adipocytes and depot-specific patterns

Or Lazarescu; Maya Ziv-Agam; Yulia Haim; Idan Hekselman; Juman Jubran; Ariel Shneyour; Danny Kitsberg; Liron Levin; Idit F Liberty; Uri Yoel et al

bioRxiv 2023;2023.09.04.555678

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Nervous system
Topics
Computational Methods

Comparative single-cell transcriptomic analysis of primate brains highlights human-specific regulatory evolution

Suresh, Hamsini; Crow, Megan; Jorstad, Nikolas; Hodge, Rebecca; Lein, Ed; Dobin, Alexander; Bakken, Trygve; Gillis, Jesse

Nature Ecology & Evolution 2023;1-14

Enhanced cognitive function in humans is hypothesized to result from cortical expansion and increased cellular diversity. However, the mechanisms that drive these phenotypic innovations remain poorly understood, in part because of the lack of high-quality cellular resolution data in human and non-human primates. Here, we take advantage of single-cell expression data from the middle temporal gyrus of five primates (human, chimp, gorilla, macaque and marmoset) to identify 57 homologous cell types and generate cell type-specific gene co-expression networks for comparative analysis. Although orthologue expression patterns are generally well conserved, we find 24% of genes with extensive differences between human and non-human primates (3,383 out of 14,131), which are also associated with multiple brain disorders. To assess the functional significance of gene expression differences in an evolutionary context, we evaluate changes in network connectivity across meta-analytic co-expression networks from 19 animals. We find that a subset of these genes has deeply conserved co-expression across all non-human animals, and strongly divergent co-expression relationships in humans (139 out of 3,383, <1% of primate orthologues). Genes with human-specific cellular expression and co-expression profiles (such as NHEJ1, GTF2H2, C2 and BBS5) typically evolve under relaxed selective constraints and may drive rapid evolutionary change in brain function. Analysis of single-cell expression data from the middle temporal gyrus of five primates together with analysis of network connectivity across 19 animals identifies genes with human-specific cellular expression and co-expression profiles that might contribute to evolutionary change in brain function.

Pre-print
Networks
Immune
Topics
Human Subjects, Open Access Data, Experimental Methods, Computational Methods

Scalable querying of human cell atlases via a foundational model reveals commonalities across fibrosis-associated macrophages

Graham Heimberg; Tony CY Kuo; Daryle DePianto; Tobias Heigl; Nathaniel Diamant; Omar Salem; Gabriele Scalia; Tommaso Biancalani; Jason Rock; Shannon Turley et al

bioRxiv 2023;2023.07.18.549537

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Networks
Lung
Topics
Human Subjects, Computational Methods

Scalable querying of human cell atlases via a foundational model reveals commonalities across fibrosis-associated macrophages

Graham Heimberg; Tony CY Kuo; Daryle DePianto; Tobias Heigl; Nathaniel Diamant; Omar Salem; Gabriele Scalia; Tommaso Biancalani; Jason Rock; Shannon Turley et al

bioRxiv 2023;2023.07.18.549537

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Networks
Oral & Craniofacial
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Disease Donors, Open Access Data, Experimental Methods, Computational Methods

Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

Quinn T. Easter; Bruno Fernandes Matuck; Germán Beldorati Stark; Catherine L. Worth; Alexander V. Predeus; Brayon Fremin; Khoa Huynh; Vaishnavi Ranganathan; Diana Pereira; Theresa Weaver et al

bioRxiv 2023;2023.08.23.554343

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Topics
Human Subjects, Open Access Data, Experimental Methods, Computational Methods

multiDGD: A versatile deep generative model for multi-omics data

Viktoria Schuster; Emma Dann; Anders Krogh; Sarah A. Teichmann

bioRxiv 2023;2023.08.23.554420

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print

multiDGD: A versatile deep generative model for multi-omics data

Viktoria Schuster; Emma Dann; Anders Krogh; Sarah A. Teichmann

bioRxiv 2023;2023.08.23.554420

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print

Consensus prediction of cell type labels with popV

Can Ergen; Galen Xing; Chenling Xu; Michael Jayasuriya; Erin McGeever; Angela Oliveira Pisco; Aaron Streets; Nir Yosef

bioRxiv 2023;2023.08.18.553912

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Breast, Eye
Topics
Human Subjects, Healthy Donors, Computational Methods

Single-nucleotide variant calling in single-cell sequencing data with Monopogen

Dou, Jinzhuang; Tan, Yukun; Kock, Kian Hong; Wang, Jun; Cheng, Xuesen; Tan, Le Min; Han, Kyung Yeon; Hon, Chung-Chau; Park, Woong-Yang; Shin, Jay W. et al

Nature Biotechnology 2023;1-10

Single-cell omics technologies enable molecular characterization of diverse cell types and states, but how the resulting transcriptional and epigenetic profiles depend on the cell’s genetic background remains understudied. We describe Monopogen, a computational tool to detect single-nucleotide variants (SNVs) from single-cell sequencing data. Monopogen leverages linkage disequilibrium from external reference panels to identify germline SNVs and detects putative somatic SNVs using allele cosegregating patterns at the cell population level. It can identify 100 K to 3 M germline SNVs achieving a genotyping accuracy of 95%, together with hundreds of putative somatic SNVs. Monopogen-derived genotypes enable global and local ancestry inference and identification of admixed samples. It identifies variants associated with cardiomyocyte metabolic levels and epigenomic programs. It also improves putative somatic SNV detection that enables clonal lineage tracing in primary human clonal hematopoiesis. Monopogen brings together population genetics, cell lineage tracing and single-cell omics to uncover genetic determinants of cellular processes. Monopogen identifies single-nucleotide variants in single-cell sequencing data.

Pre-print

SuperCellCyto: enabling efficient analysis of large scale cytometry datasets

Givanna H. Putri; George Howitt; Felix Marsh-Wakefield; Thomas Ashhurst; Belinda Phipson

bioRxiv 2023;2023.08.14.553168

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print

Identification of novel myeloid-derived cell states with implication in cancer outcome

Gabriela Rapozo Guimarães; Giovanna Resk Maklouf; Cristiane Esteves Teixeira; Leandro de Oliveira Santos; Nayara Gusmão Tessarollo; Marco Antônio Pretti; Nayara Evelin Toledo; Jéssica Gonçalves Vieira da Cruz; Marcelo Falchetti; Mylla M. Dimas et al

bioRxiv 2023;2023.01.04.522727

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Networks
Eye, Organoid
Topics
Model Organism Samples, Open Access Data

Deciphering the spatio-temporal transcriptional and chromatin accessibility of human retinal organoid development at the single cell level

Birthe Dorgau; Joseph Collin; Agata Rozanska; Veronika Boczonadi; Marina Moya-Molina; Rafiqul Hussain; Jonathan Coxhead; Tamil Dhanaseelan; Lyle Armstrong; Rachel Queen et al

bioRxiv 2023;2023.07.19.549507

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Kidney

An atlas of healthy and injured cell states and niches in the human kidney

Lake, Blue B.; Menon, Rajasree; Winfree, Seth; Hu, Qiwen; Ferreira, Ricardo Melo; Kalhor, Kian; Barwinska, Daria; Otto, Edgar A.; Ferkowicz, Michael; Diep, Dinh et al

Nature 2023;619;7970;585-594

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations. A high-resolution kidney cellular atlas of 51 main cell types, including rare and previously undescribed cell populations, represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

Peer reviewed
Networks
Heart
Topics
Human Subjects, Healthy Donors, Open Access Data, Computational Methods

Spatially resolved multiomics of human cardiac niches

Kanemaru, Kazumasa; Cranley, James; Muraro, Daniele; Miranda, Antonio M. A.; Ho, Siew Yen; Wilbrey-Clark, Anna; Patrick Pett, Jan; Polanski, Krzysztof; Richardson, Laura; Litvinukova, Monika et al

Nature 2023;1-10

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system1. The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug–target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG+ and IgA+ plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs. Single-cell and spatial transcriptomic analysis of eight human heart tissues reveals the cellular profiles and tissue architecture of niches including the cardiac conduction system, and a new tool, drug2cell, identifies drug target expression.

Peer reviewed
Networks
Breast
Topics
Human Subjects, Healthy Donors, Open Access Data

A spatially resolved single-cell genomic atlas of the adult human breast

Kumar, Tapsi; Nee, Kevin; Wei, Runmin; He, Siyuan; Nguyen, Quy H.; Bai, Shanshan; Blake, Kerrigan; Pein, Maren; Gong, Yanwen; Sei, Emi et al

Nature 2023;1-11

The adult human breast is comprised of an intricate network of epithelial ducts and lobules that are embedded in connective and adipose tissue1–3. Although most previous studies have focused on the breast epithelial system4–6, many of the non-epithelial cell types remain understudied. Here we constructed the comprehensive Human Breast Cell Atlas (HBCA) at single-cell and spatial resolution. Our single-cell transcriptomics study profiled 714,331 cells from 126 women, and 117,346 nuclei from 20 women, identifying 12 major cell types and 58 biological cell states. These data reveal abundant perivascular, endothelial and immune cell populations, and highly diverse luminal epithelial cell states. Spatial mapping using four different technologies revealed an unexpectedly rich ecosystem of tissue-resident immune cells, as well as distinct molecular differences between ductal and lobular regions. Collectively, these data provide a reference of the adult normal breast tissue for studying mammary biology and diseases such as breast cancer. The Human Breast Cell Atlas identifies 12 major breast cell types and 58 biological cell states, revealing abundant pericyte, endothelial and immune cell populations, and highly diverse luminal epithelial cell states.

Pre-print

TooManyCellsInteractive: a visualization tool for dynamic exploration of single-cell data

Conor Klamann; Christie Lau; Gregory W. Schwartz

bioRxiv 2023;2023.06.16.544954

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Networks
Eye
Topics
Human Subjects, Open Access Data

Multi-omics analysis in human retina uncovers ultraconserved cis-regulatory elements at rare eye disease loci

Victor Lopez Soriano; Alfredo Dueñas Rey; Rajarshi Mukherjee; Genomics England Research Consortium; Frauke Coppieters; Miriam Bauwens; Andy Willaert; Elfride De Baere

medRxiv 2023;2023.06.08.23291052

medRxiv - The Preprint Server for Health Sciences

Peer reviewed
Networks
Lung
Topics
Human Subjects, Healthy Donors, Disease Donors, Open Access Data, Computational Methods

An integrated cell atlas of the lung in health and disease

Sikkema, Lisa; Ramírez-Suástegui, Ciro; Strobl, Daniel C.; Gillett, Tessa E.; Zappia, Luke; Madissoon, Elo; Markov, Nikolay S.; Zaragosi, Laure-Emmanuelle; Ji, Yuge; Ansari, Meshal et al

Nature Medicine 2023;1-15

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. A single-cell atlas of the human lungs, integrating data from 2.4 million cells from 486 individuals and including samples from healthy and diseased lungs, provides a roadmap for the generation of organ-scale cell atlases.

Press release Press release Press release Press release

Peer reviewed
Networks
Gut
Topics
Human Subjects, Healthy Donors, Disease Donors, Benchmarking

A Roadmap for the Human Gut Cell Atlas

Zilbauer, Matthias; James, Kylie R.; Kaur, Mandeep; Pott, Sebastian; Li, Zhixin; Burger, Albert; Thiagarajah, Jay R.; Burclaff, Joseph; Jahnsen, Frode L.; Perrone, Francesca et al

Nature Reviews Gastroenterology & Hepatology 2023;20;9;597-614

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. Single-cell profiling studies of the human gastrointestinal tract are increasing, offering an excellent opportunity to generate the first Human Gut Cell Atlas. This Roadmap presents a structured direction towards this goal and provides a detailed overview of the major challenges.

Pre-print

WebAtlas pipeline for integrated single cell and spatial transcriptomic data

Tong Li; David Horsfall; Daniela Basurto-Lozada; Kenny Roberts; Martin Prete; John E G Lawrence; Peng He; Elisabeth Tuck; Josh Moore; Shila Ghazanfar et al

bioRxiv 2023;2023.05.19.541329

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Immune
Topics
Healthy Donors, Experimental Methods, Computational Methods

Dandelion uses the single-cell adaptive immune receptor repertoire to explore lymphocyte developmental origins

Suo, Chenqu; Polanski, Krzysztof; Dann, Emma; Lindeboom, Rik G. H.; Vilarrasa-Blasi, Roser; Vento-Tormo, Roser; Haniffa, Muzlifah; Meyer, Kerstin B.; Dratva, Lisa M.; Tuong, Zewen Kelvin et al

Nature Biotechnology 2023;1-12

Assessment of single-cell gene expression (single-cell RNA sequencing) and adaptive immune receptor (AIR) sequencing (scVDJ-seq) has been invaluable in studying lymphocyte biology. Here we introduce Dandelion, a computational pipeline for scVDJ-seq analysis. It enables the application of standard V(D)J analysis workflows to single-cell datasets, delivering improved V(D)J contig annotation and the identification of nonproductive and partially spliced contigs. We devised a strategy to create an AIR feature space that can be used for both differential V(D)J usage analysis and pseudotime trajectory inference. The application of Dandelion improved the alignment of human thymic development trajectories of double-positive T cells to mature single-positive CD4/CD8 T cells, generating predictions of factors regulating lineage commitment. Dandelion analysis of other cell compartments provided insights into the origins of human B1 cells and ILC/NK cell development, illustrating the power of our approach. Dandelion is available at https://www.github.com/zktuong/dandelion . A computational pipeline enables differential V(D)J usage analysis and pseudotime trajectory inference from single-cell AIR sequencing.

Pre-print
Networks
Nervous system
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Integrated platform for multi-scale molecular imaging and phenotyping of the human brain

Juhyuk Park; Ji Wang; Webster Guan; Lars A. Gjesteby; Dylan Pollack; Lee Kamentsky; Nicholas B. Evans; Jeff Stirman; Xinyi Gu; Chuanxi Zhao et al

bioRxiv 2023;2022.03.13.484171

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Development, Organoid, Reproduction
Topics
Human Subjects, Healthy Donors, Open Access Data, Computational Methods, Benchmarking

Spatial multiomics map of trophoblast development in early pregnancy

Arutyunyan, Anna; Roberts, Kenny; Troulé, Kevin; Wong, Frederick C. K.; Sheridan, Megan A.; Kats, Ilia; Garcia-Alonso, Luz; Velten, Britta; Hoo, Regina; Ruiz-Morales, Elias R. et al

Nature 2023;1-9

The relationship between the human placenta—the extraembryonic organ made by the fetus, and the decidua—the mucosal layer of the uterus, is essential to nurture and protect the fetus during pregnancy. Extravillous trophoblast cells (EVTs) derived from placental villi infiltrate the decidua, transforming the maternal arteries into high-conductance vessels1. Defects in trophoblast invasion and arterial transformation established during early pregnancy underlie common pregnancy disorders such as pre-eclampsia2. Here we have generated a spatially resolved multiomics single-cell atlas of the entire human maternal–fetal interface including the myometrium, which enables us to resolve the full trajectory of trophoblast differentiation. We have used this cellular map to infer the possible transcription factors mediating EVT invasion and show that they are preserved in in vitro models of EVT differentiation from primary trophoblast organoids3,4 and trophoblast stem cells5. We define the transcriptomes of the final cell states of trophoblast invasion: placental bed giant cells (fused multinucleated EVTs) and endovascular EVTs (which form plugs inside the maternal arteries). We predict the cell–cell communication events contributing to trophoblast invasion and placental bed giant cell formation, and model the dual role of interstitial EVTs and endovascular EVTs in mediating arterial transformation during early pregnancy. Together, our data provide a comprehensive analysis of postimplantation trophoblast differentiation that can be used to inform the design of experimental models of the human placenta in early pregnancy. A multiomics single-cell atlas of the human maternal–fetal interface including the myometrium, combining spatial transcriptomics data with chromatin accessibility, provides a comprehensive analysis of cell states as placental cells infiltrate the uterus during early pregnancy.

Press release

Pre-print
Networks
Lung
Topics
Human Subjects, Healthy Donors, Disease Donors

Airway epithelial response to RSV is impaired in multiciliated and goblet cells in asthma

Aurore C. A. Gay; Martin Banchero; Orestes A. Carpaij; Tessa Kole; Leonie Apperloo; Djoke van Gosliga; Putri Ayu Fajar; Gerard H. Koppelman; Louis Bont; Rudi W. Hendriks et al

bioRxiv 2023;2023.03.16.532356

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Networks
Development, Immune, Organoid
Topics
Open Access Data, Computational Methods

Gene-level alignment of single cell trajectories informs the progression of in vitro T cell differentiation

Dinithi Sumanaweera; Chenqu Suo; Daniele Muraro; Emma Dann; Krzysztof Polanski; Alexander Steemers; Jong-Eun Park; Bianca Dumitrascu; Sarah Teichmann

bioRxiv 2023;2023.03.08.531713

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Topics
Computational Methods

TACCO unifies annotation transfer and decomposition of cell identities for single-cell and spatial omics

Mages, Simon; Moriel, Noa; Avraham-Davidi, Inbal; Murray, Evan; Watter, Jan; Chen, Fei; Rozenblatt-Rosen, Orit; Klughammer, Johanna; Regev, Aviv; Nitzan, Mor

Nature Biotechnology 2023;1-9

Transferring annotations of single-cell-, spatial- and multi-omics data is often challenging owing both to technical limitations, such as low spatial resolution or high dropout fraction, and to biological variations, such as continuous spectra of cell states. Based on the concept that these data are often best described as continuous mixtures of cells or molecules, we present a computational framework for the transfer of annotations to cells and their combinations (TACCO), which consists of an optimal transport model extended with different wrappers to annotate a wide variety of data. We apply TACCO to identify cell types and states, decipher spatiomolecular tissue structure at the cell and molecular level and resolve differentiation trajectories using synthetic and biological datasets. While matching or exceeding the accuracy of specialized tools for the individual tasks, TACCO reduces the computational requirements by up to an order of magnitude and scales to larger datasets (for example, considering the runtime of annotation transfer for 1 M simulated dropout observations). Annotation transfer from reference to new datasets is improved with a probabilistic approach.

Pre-print
Networks
Lung
Topics
Human Subjects, Healthy Donors, Experimental Methods

The emergence of goblet inflammatory or ITGB6hi nasal progenitor cells determines age-associated SARS-CoV-2 pathogenesis

Maximillian Woodall; Ana-Maria Cujba; Kaylee B. Worlock; Katie-Marie Case; Tereza Masonou; Masahiro Yoshida; Krzysztof Polanski; Ni Huang; Rik G. H. Lindeboom; Lira Mamanova et al

bioRxiv 2023;2023.01.16.524211

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Gut
Topics
Human Subjects, Disease Donors

Stomach encyclopedia: Combined single-cell and spatial transcriptomics reveal cell diversity and homeostatic regulation of human stomach.

Tsubosaka A; Komura D; Kakiuchi M; Katoh H; Onoyama T; Yamamoto A; Abe H; Seto Y; Ushiku T; Ishikawa S

Cell reports 2023;42;10;113236

The stomach is an important digestive organ with various biological functions. However, because of the complexity of its cellular and glandular composition, its precise cellular biology has yet to be elucidated. In this study, we conducted single-cell RNA sequencing (scRNA-seq) and subcellular-level spatial transcriptomics analysis of the human stomach and constructed the largest dataset to date: a stomach encyclopedia. This dataset consists of approximately 380,000 cells from scRNA-seq and the spatial transcriptome, enabling integrated analyses of transcriptional and spatial information of gastric and metaplastic cells. This analysis identified LEFTY1 as an uncharacterized stem cell marker, which was confirmed through lineage tracing analysis. A wide variety of cell-cell interactions between epithelial and stromal cells, including PDGFRA+BMP4+WNT5A+ fibroblasts, was highlighted in the developmental switch of intestinal metaplasia. Our extensive dataset will function as a fundamental resource in investigations of the stomach, including studies of development, aging, and carcinogenesis.

Peer reviewed
Networks
Lung

Guided construction of single cell reference for human and mouse lung.

Guo M; Morley MP; Jiang C; Wu Y; Li G; Du Y; Zhao S; Wagner A; Cakar AC; Kouril M et al

Nature Communications 2023;14;1;4566

Accurate cell type identification is a key and rate-limiting step in single-cell data analysis. Single-cell references with comprehensive cell types, reproducible and functionally validated cell identities, and common nomenclatures are much needed by the research community for automated cell type annotation, data integration, and data sharing. Here, we develop a computational pipeline utilizing the LungMAP CellCards as a dictionary to consolidate single-cell transcriptomic datasets of 104 human lungs and 17 mouse lung samples to construct LungMAP single-cell reference (CellRef) for both normal human and mouse lungs. CellRefs define 48 human and 40 mouse lung cell types catalogued from diverse anatomic locations and developmental time points. We demonstrate the accuracy and stability of LungMAP CellRefs and their utility for automated cell type annotation of both normal and diseased lungs using multiple independent methods and testing data. We develop user-friendly web interfaces for easy access and maximal utilization of the LungMAP CellRefs.

Pre-print

A deep lung cell atlas reveals cytokine-mediated lineage switching of a rare cell progenitor of the human airway epithelium.

Waghray A; Monga I; Lin B; Shah V; Slyper M; Giotti B; Xu J; Waldman J; Dionne D; Nguyen LT et al

bioRxiv 2023

The human airway contains specialized rare epithelial cells whose roles in respiratory disease are not well understood. Ionocytes express the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), while chemosensory tuft cells express asthma-associated alarmins. However, surprisingly, exceedingly few mature tuft cells have been identified in human lung cell atlases despite the ready identification of rare ionocytes and neuroendocrine cells. To identify human rare cell progenitors and define their lineage relationship to mature tuft cells, we generated a deep lung cell atlas containing 311,748 single cell RNA-Seq (scRNA-seq) profiles from discrete anatomic sites along the large and small airways and lung lobes of explanted donor lungs that could not be used for organ transplantation. Of 154,222 airway epithelial cells, we identified 687 ionocytes (0.45%) that are present in similar proportions in both large and small airways, suggesting that they may contribute to both large and small airways pathologies in CF. In stark contrast, we recovered only 3 mature tuft cells (0.002%). Instead, we identified rare bipotent progenitor cells that can give rise to both ionocytes and tuft cells, which we termed tuft-ionocyte progenitor cells (TIP cells). Remarkably, the cycling fraction of these TIP cells was comparable to that of basal stem cells. We used scRNA-seq and scATAC-seq to predict transcription factors that mark this novel rare cell progenitor population and define intermediate states during TIP cell lineage transitions en route to the differentiation of mature ionocytes and tuft cells. The default lineage of TIP cell descendants is skewed towards ionocytes, explaining the paucity of mature tuft cells in the human airway. However, Type 2 and Type 17 cytokines, associated with asthma and CF, diverted the lineage of TIP cell descendants in vitro , resulting in the differentiation of mature tuft cells at the expense of ionocytes. Consistent with this model of mature tuft cell differentiation, we identify mature tuft cells in a patient who died from an asthma flare. Overall, our findings suggest that the immune signaling pathways active in asthma and CF may skew the composition of disease-relevant rare cells and illustrate how deep atlases are required for identifying physiologically-relevant scarce cell populations.

Pre-print

Cell type-specific gene expression dynamics during human brain maturation.

Steyn C; Mishi R; Fillmore S; Verhoog MB; More J; Rohlwink UK; Melvill R; Butler J; Enslin JMN; Jacobs M et al

bioRxiv 2023

The human brain undergoes protracted post-natal maturation, guided by dynamic changes in gene expression. To date, studies exploring these processes have used bulk tissue analyses, which mask cell type-specific gene expression dynamics. Here, using single nucleus (sn)RNA-Sseq on temporal lobe tissue, including samples of African ancestry, we build a joint paediatric and adult atlas of 54 cell subtypes, which we verify with spatial transcriptomics. We explore the differences in cell states between paediatric and adult cell types, revealing the genes and pathways that change during brain maturation. Our results highlight excitatory neuron subtypes, including the LTK and FREM subtypes, that show elevated expression of genes associated with cognition and synaptic plasticity in paediatric tissue. The new resources we present here improve our understanding of the brain during a critical period of its development and contribute to global efforts to build an inclusive cell map of the brain.

Pre-print
Networks
Nervous system

Cell type-specific gene expression dynamics during human brain maturation.

Steyn C; Mishi R; Fillmore S; Verhoog MB; More J; Rohlwink UK; Melvill R; Butler J; Enslin JMN; Jacobs M et al

bioRxiv 2023

The human brain undergoes protracted post-natal maturation, guided by dynamic changes in gene expression. To date, studies exploring these processes have used bulk tissue analyses, which mask cell type-specific gene expression dynamics. Here, using single nucleus (sn)RNA-Sseq on temporal lobe tissue, including samples of African ancestry, we build a joint paediatric and adult atlas of 54 cell subtypes, which we verify with spatial transcriptomics. We explore the differences in cell states between paediatric and adult cell types, revealing the genes and pathways that change during brain maturation. Our results highlight excitatory neuron subtypes, including the LTK and FREM subtypes, that show elevated expression of genes associated with cognition and synaptic plasticity in paediatric tissue. The new resources we present here improve our understanding of the brain during a critical period of its development and contribute to global efforts to build an inclusive cell map of the brain.

Pre-print
Networks
Immune
Topics
Human Subjects, Open Access Data

A spatial human thymus cell atlas mapped to a continuous tissue axis.

Yayon N; Kedlian VR; Boehme L; Suo C; Wachter B; Beuschel RT; Amsalem O; Polanski K; Koplev S; Tuck E et al

bioRxiv 2023

T cells develop from circulating precursors, which enter the thymus and migrate throughout specialised sub-compartments to support maturation and selection. This process starts already in early fetal development and is highly active until the involution of the thymus in adolescence. To map the micro-anatomical underpinnings of this process in pre- vs. post-natal states, we undertook a spatially resolved analysis and established a new quantitative morphological framework for the thymus, the Cortico-Medullary Axis. Using this axis in conjunction with the curation of a multimodal single-cell, spatial transcriptomics and high-resolution multiplex imaging atlas, we show that canonical thymocyte trajectories and thymic epithelial cells are highly organised and fully established by post-conception week 12, pinpoint TEC progenitor states, find that TEC subsets and peripheral tissue genes are associated with Hassall's Corpuscles and uncover divergence in the pace and drivers of medullary entry between CD4 vs. CD8 T cell lineages. These findings are complemented with a holistic toolkit for spatial analysis and annotation, providing a basis for a detailed understanding of T lymphocyte development.

Pre-print
Topics
Human Subjects, Open Access Data, Experimental Methods, Computational Methods

Inference of single cell profiles from histology stains with the Single-Cell omics from Histology Analysis Framework (SCHAF).

Comiter C; Vaishnav ED; Ciampricotti M; Li B; Yang Y; Rodig SJ; Turner M; Pfaff KL; Jané-Valbuena J; Slyper M et al

bioRxiv 2023

Tissue biology involves an intricate balance between cell-intrinsic processes and interactions between cells organized in specific spatial patterns, which can be respectively captured by single-cell profiling methods, such as single-cell RNA-seq (scRNA-seq), and histology imaging data, such as Hematoxylin-and-Eosin (H&E) stains. While single-cell profiles provide rich molecular information, they can be challenging to collect routinely and do not have spatial resolution. Conversely, histological H&E assays have been a cornerstone of tissue pathology for decades, but do not directly report on molecular details, although the observed structure they capture arises from molecules and cells. Here, we leverage adversarial machine learning to develop SCHAF (Single-Cell omics from Histology Analysis Framework), to generate a tissue sample's spatially-resolved single-cell omics dataset from its H&E histology image. We demonstrate SCHAF on two types of human tumors-from lung and metastatic breast cancer-training with matched samples analyzed by both sc/snRNA-seq and by H&E staining. SCHAF generated appropriate single-cell profiles from histology images in test data, related them spatially, and compared well to ground-truth scRNA-Seq, expert pathologist annotations, or direct MERFISH measurements. SCHAF opens the way to next-generation H&E2.0 analyses and an integrated understanding of cell and tissue biology in health and disease.

Pre-print
Networks
Breast, Lung
Topics
Human Subjects, Disease Donors, Computational Methods

Inference of single cell profiles from histology stains with the Single-Cell omics from Histology Analysis Framework (SCHAF).

Comiter C; Vaishnav ED; Ciampricotti M; Li B; Yang Y; Rodig SJ; Turner M; Pfaff KL; Jané-Valbuena J; Slyper M et al

bioRxiv 2023

Tissue biology involves an intricate balance between cell-intrinsic processes and interactions between cells organized in specific spatial patterns, which can be respectively captured by single-cell profiling methods, such as single-cell RNA-seq (scRNA-seq), and histology imaging data, such as Hematoxylin-and-Eosin (H&E) stains. While single-cell profiles provide rich molecular information, they can be challenging to collect routinely and do not have spatial resolution. Conversely, histological H&E assays have been a cornerstone of tissue pathology for decades, but do not directly report on molecular details, although the observed structure they capture arises from molecules and cells. Here, we leverage adversarial machine learning to develop SCHAF (Single-Cell omics from Histology Analysis Framework), to generate a tissue sample's spatially-resolved single-cell omics dataset from its H&E histology image. We demonstrate SCHAF on two types of human tumors-from lung and metastatic breast cancer-training with matched samples analyzed by both sc/snRNA-seq and by H&E staining. SCHAF generated appropriate single-cell profiles from histology images in test data, related them spatially, and compared well to ground-truth scRNA-Seq, expert pathologist annotations, or direct MERFISH measurements. SCHAF opens the way to next-generation H&E2.0 analyses and an integrated understanding of cell and tissue biology in health and disease.

Pre-print

Inference of single cell profiles from histology stains with the Single-Cell omics from Histology Analysis Framework (SCHAF).

Comiter C; Vaishnav ED; Ciampricotti M; Li B; Yang Y; Rodig SJ; Turner M; Pfaff KL; Jané-Valbuena J; Slyper M et al

bioRxiv 2023

Tissue biology involves an intricate balance between cell-intrinsic processes and interactions between cells organized in specific spatial patterns, which can be respectively captured by single-cell profiling methods, such as single-cell RNA-seq (scRNA-seq), and histology imaging data, such as Hematoxylin-and-Eosin (H&E) stains. While single-cell profiles provide rich molecular information, they can be challenging to collect routinely and do not have spatial resolution. Conversely, histological H&E assays have been a cornerstone of tissue pathology for decades, but do not directly report on molecular details, although the observed structure they capture arises from molecules and cells. Here, we leverage adversarial machine learning to develop SCHAF (Single-Cell omics from Histology Analysis Framework), to generate a tissue sample's spatially-resolved single-cell omics dataset from its H&E histology image. We demonstrate SCHAF on two types of human tumors-from lung and metastatic breast cancer-training with matched samples analyzed by both sc/snRNA-seq and by H&E staining. SCHAF generated appropriate single-cell profiles from histology images in test data, related them spatially, and compared well to ground-truth scRNA-Seq, expert pathologist annotations, or direct MERFISH measurements. SCHAF opens the way to next-generation H&E2.0 analyses and an integrated understanding of cell and tissue biology in health and disease.

Pre-print
Topics
Human Subjects, Open Access Data

Bering: joint cell segmentation and annotation for spatial transcriptomics with transferred graph embeddings.

Jin K; Zhang Z; Zhang K; Viggiani F; Callahan C; Tang J; Aronow BJ; Shu J

bioRxiv 2023

Single-cell spatial transcriptomics such as in-situ hybridization or sequencing technologies can provide subcellular resolution that enables the identification of individual cell identities, locations, and a deep understanding of subcellular mechanisms. However, accurate segmentation and annotation that allows individual cell boundaries to be determined remains a major challenge that limits all the above and downstream insights. Current machine learning methods heavily rely on nuclei or cell body staining, resulting in the significant loss of both transcriptome depth and the limited ability to learn latent representations of spatial colocalization relationships. Here, we propose Bering, a graph deep learning model that leverages transcript colocalization relationships for joint noise-aware cell segmentation and molecular annotation in 2D and 3D spatial transcriptomics data. Graph embeddings for the cell annotation are transferred as a component of multi-modal input for cell segmentation, which is employed to enrich gene relationships throughout the process. To evaluate performance, we benchmarked Bering with state-of-the-art methods and observed significant improvement in cell segmentation accuracies and numbers of detected transcripts across various spatial technologies and tissues. To streamline segmentation processes, we constructed expansive pre-trained models, which yield high segmentation accuracy in new data through transfer learning and self-distillation, demonstrating the generalizability of Bering.

Peer reviewed
Networks
Gut, Organoid

EPIREGULIN creates a developmental niche for spatially organized human intestinal enteroids.

Childs CJ; Holloway EM; Sweet CW; Tsai YH; Wu A; Vallie A; Eiken MK; Capeling MM; Zwick RK; Palikuqi B et al

JCI insight 2023;8;6

Epithelial organoids derived from intestinal tissue, called enteroids, recapitulate many aspects of the organ in vitro and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identified an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells and feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown enteroids, and EREG-grown enteroids showed that EGF enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.

Peer reviewed
Networks
Development

A tridimensional atlas of the developing human head.

Blain R; Couly G; Shotar E; Blévinal J; Toupin M; Favre A; Abjaghou A; Inoue M; Hernández-Garzón E; Clarençon F et al

Cell 2023;186;26;5910-5924.e17

The evolution and development of the head have long captivated researchers due to the crucial role of the head as the gateway for sensory stimuli and the intricate structural complexity of the head. Although significant progress has been made in understanding head development in various vertebrate species, our knowledge of early human head ontogeny remains limited. Here, we used advanced whole-mount immunostaining and 3D imaging techniques to generate a comprehensive 3D cellular atlas of human head embryogenesis. We present detailed developmental series of diverse head tissues and cell types, including muscles, vasculature, cartilage, peripheral nerves, and exocrine glands. These datasets, accessible through a dedicated web interface, provide insights into human embryogenesis. We offer perspectives on the branching morphogenesis of human exocrine glands and unknown features of the development of neurovascular and skeletomuscular structures. These insights into human embryology have important implications for understanding craniofacial defects and neurological disorders and advancing diagnostic and therapeutic strategies.

Press release

Peer reviewed
Networks
Immune, Nervous system
Topics
Computational Methods, Benchmarking

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

Xu C; Prete M; Webb S; Jardine L; Stewart BJ; Hoo R; He P; Meyer KB; Teichmann SA

Cell 2023;186;26;5876-5891.e20

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.

Press release

Peer reviewed
Networks
Immune, Lung

Distinct immune microenvironment of lung adenocarcinoma in never-smokers from smokers.

Luo W; Zeng Z; Jin Y; Yang L; Fan T; Wang Z; Pan Y; Yang Y; Yao M; Li Y et al

Cell reports. Medicine 2023;4;6;101078

Lung cancer in never-smokers (LCINS) presents clinicopathological and molecular features distinct from that in smokers. Tumor microenvironment (TME) plays important roles in cancer progression and therapeutic response. To decipher the difference in TME between never-smoker and smoker lung cancers, we conduct single-cell RNA sequencing on 165,753 cells from 22 treatment-naive lung adenocarcinoma (LUAD) patients. We find that the dysfunction of alveolar cells induced by cigarette smoking contributes more to the aggressiveness of smoker LUADs, while the immunosuppressive microenvironment exerts more effects on never-smoker LUADs' aggressiveness. Moreover, the SPP1hi pro macrophage is identified to be another independent source of monocyte-derived macrophage. Importantly, higher expression of immune checkpoint CD47 and lower expression of major histocompatibility complex (MHC)-I in cancer cells of never-smoker LUADs imply that CD47 may be a better immunotherapy target for LCINS. Therefore, this study reveals the difference of tumorigenesis between never-smoker and smoker LUADs and provides a potential immunotherapy strategy for LCINS.

Peer reviewed
Networks
Development, Gut, Immune, Kidney, Liver, Organoid, Reproduction, Skin
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Experimental Methods, Computational Methods, Benchmarking

Yolk sac cell atlas reveals multiorgan functions during human early development.

Goh I; Botting RA; Rose A; Webb S; Engelbert J; Gitton Y; Stephenson E; Quiroga Londoño M; Mather M; Mende N et al

Science 2023;381;6659;eadd7564

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

Press release

Peer reviewed
Networks
Development, Immune, Lung
Topics
Human Subjects, Healthy Donors

Early human lung immune cell development and its role in epithelial cell fate.

Barnes JL; Yoshida M; He P; Worlock KB; Lindeboom RGH; Suo C; Pett JP; Wilbrey-Clark A; Dann E; Mamanova L et al

Science immunology 2023;8;90;eadf9988

Studies of human lung development have focused on epithelial and mesenchymal cell types and function, but much less is known about the developing lung immune cells, even though the airways are a major site of mucosal immunity after birth. An unanswered question is whether tissue-resident immune cells play a role in shaping the tissue as it develops in utero. Here, we profiled human embryonic and fetal lung immune cells using scRNA-seq, smFISH, and immunohistochemistry. At the embryonic stage, we observed an early wave of innate immune cells, including innate lymphoid cells, natural killer cells, myeloid cells, and lineage progenitors. By the canalicular stage, we detected naive T lymphocytes expressing high levels of cytotoxicity genes and the presence of mature B lymphocytes, including B-1 cells. Our analysis suggests that fetal lungs provide a niche for full B cell maturation. Given the presence and diversity of immune cells during development, we also investigated their possible effect on epithelial maturation. We found that IL-1β drives epithelial progenitor exit from self-renewal and differentiation to basal cells in vitro. In vivo, IL-1β-producing myeloid cells were found throughout the lung and adjacent to epithelial tips, suggesting that immune cells may direct human lung epithelial development.

Press release

Peer reviewed
Networks
Gut, Immune

The landscape of immune dysregulation in Crohn's disease revealed through single-cell transcriptomic profiling in the ileum and colon.

Kong L; Pokatayev V; Lefkovith A; Carter GT; Creasey EA; Krishna C; Subramanian S; Kochar B; Ashenberg O; Lau H et al

Immunity 2023;56;2;444-458.e5

Crohn's disease (CD) is a chronic gastrointestinal disease that is increasing in prevalence worldwide. CD is multifactorial, involving the complex interplay of genetic, immune, and environmental factors, necessitating a system-level understanding of its etiology. To characterize cell-type-specific transcriptional heterogeneity in active CD, we profiled 720,633 cells from the terminal ileum and colon of 71 donors with varying inflammation status. Our integrated datasets revealed organ- and compartment-specific responses to acute and chronic inflammation; most immune changes were in cell composition, whereas transcriptional changes dominated among epithelial and stromal cells. These changes correlated with endoscopic inflammation, but small and large intestines exhibited distinct responses, which were particularly apparent when focusing on IBD risk genes. Finally, we mapped markers of disease-associated myofibroblast activation and identified CHMP1A, TBX3, and RNF168 as regulators of fibrotic complications. Altogether, our results provide a roadmap for understanding cell-type- and organ-specific differences in CD and potential directions for therapeutic development.

Peer reviewed
Networks
Immune, Lung

Comparative immunological landscape between pre- and early-stage LUAD manifested as ground-glass nodules revealed by scRNA and scTCR integrated analysis.

Wang Z; Yang L; Wang W; Zhou H; Chen J; Ma Z; Wang X; Zhang Q; Liu H; Zhou C et al

Cell communication and signaling : CCS 2023;21;1;325

Mechanism underlying the malignant progression of precancer to early-stage lung adenocarcinoma (LUAD) as well as their indolence nature remains elusive.

Peer reviewed
Collection

BICCN-Brain - Science

Networks
Nervous system
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Interindividual variation in human cortical cell type abundance and expression.

Johansen N; Somasundaram S; Travaglini KJ; Yanny AM; Shumyatcher M; Casper T; Cobbs C; Dee N; Ellenbogen R; Ferreira M et al

Science 2023;382;6667;eadf2359

Single-cell transcriptomic studies have identified a conserved set of neocortical cell types from small postmortem cohorts. We extended these efforts by assessing cell type variation across 75 adult individuals undergoing epilepsy and tumor surgeries. Nearly all nuclei map to one of 125 robust cell types identified in the middle temporal gyrus. However, we found interindividual variance in abundances and gene expression signatures, particularly in deep-layer glutamatergic neurons and microglia. A minority of donor variance is explainable by age, sex, ancestry, disease state, and cell state. Genomic variation was associated with expression of 150 to 250 genes for most cell types. This characterization of cellular variation provides a baseline for cell typing in health and disease.

Editorial Editorial Editorial

Peer reviewed
Collection

BICCN-Brain - Science

Networks
Nervous system
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Transcriptomic cytoarchitecture reveals principles of human neocortex organization.

Jorstad NL; Close J; Johansen N; Yanny AM; Barkan ER; Travaglini KJ; Bertagnolli D; Campos J; Casper T; Crichton K et al

Science 2023;382;6667;eadf6812

Variation in cytoarchitecture is the basis for the histological definition of cortical areas. We used single cell transcriptomics and performed cellular characterization of the human cortex to better understand cortical areal specialization. Single-nucleus RNA-sequencing of 8 areas spanning cortical structural variation showed a highly consistent cellular makeup for 24 cell subclasses. However, proportions of excitatory neuron subclasses varied substantially, likely reflecting differences in connectivity across primary sensorimotor and association cortices. Laminar organization of astrocytes and oligodendrocytes also differed across areas. Primary visual cortex showed characteristic organization with major changes in the excitatory to inhibitory neuron ratio, expansion of layer 4 excitatory neurons, and specialized inhibitory neurons. These results lay the groundwork for a refined cellular and molecular characterization of human cortical cytoarchitecture and areal specialization.

Editorial Editorial Editorial

Peer reviewed
Collection

BICCN-Brain - Science

Networks
Nervous system
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Single-cell analysis of prenatal and postnatal human cortical development.

Velmeshev D; Perez Y; Yan Z; Valencia JE; Castaneda-Castellanos DR; Wang L; Schirmer L; Mayer S; Wick B; Wang S et al

Science 2023;382;6667;eadf0834

We analyzed >700,000 single-nucleus RNA sequencing profiles from 106 donors during prenatal and postnatal developmental stages and identified lineage-specific programs that underlie the development of specific subtypes of excitatory cortical neurons, interneurons, glial cell types, and brain vasculature. By leveraging single-nucleus chromatin accessibility data, we delineated enhancer gene regulatory networks and transcription factors that control commitment of specific cortical lineages. By intersecting our results with genetic risk factors for human brain diseases, we identified the cortical cell types and lineages most vulnerable to genetic insults of different brain disorders, especially autism. We find that lineage-specific gene expression programs up-regulated in female cells are especially enriched for the genetic risk factors of autism. Our study captures the molecular progression of cortical lineages across human development.

Editorial Editorial Editorial

Peer reviewed
Collection

BICCN-Brain - Science

Networks
Nervous system
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Transcriptomic diversity of cell types across the adult human brain.

Siletti K; Hodge R; Mossi Albiach A; Lee KW; Ding SL; Hu L; Lönnerberg P; Bakken T; Casper T; Clark M et al

Science 2023;382;6667;eadd7046

The human brain directs complex behaviors, ranging from fine motor skills to abstract intelligence, but the diversity of cell types that support these skills has not been fully described. In this work, we used single-nucleus RNA sequencing to systematically survey cells across the entire adult human brain. We sampled more than three million nuclei from approximately 100 dissections across the forebrain, midbrain, and hindbrain in three postmortem donors. Our analysis identified 461 clusters and 3313 subclusters organized largely according to developmental origins and revealing high diversity in midbrain and hindbrain neurons. Astrocytes and oligodendrocyte-lineage cells also exhibited regional diversity at multiple scales. The transcriptomic census of the entire human brain presented in this work provides a resource for understanding the molecular diversity of the human brain in health and disease.

Editorial Editorial Editorial

Peer reviewed
Collection

BICCN-Brain - Science

Networks
Nervous system
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Comprehensive cell atlas of the first-trimester developing human brain.

Braun E; Danan-Gotthold M; Borm LE; Lee KW; Vinsland E; Lönnerberg P; Hu L; Li X; He X; Andrusivová Ž et al

Science 2023;382;6667;eadf1226

The adult human brain comprises more than a thousand distinct neuronal and glial cell types, a diversity that emerges during early brain development. To reveal the precise sequence of events during early brain development, we used single-cell RNA sequencing and spatial transcriptomics and uncovered cell states and trajectories in human brains at 5 to 14 postconceptional weeks (pcw). We identified 12 major classes that are organized as ~600 distinct cell states, which map to precise spatial anatomical domains at 5 pcw. We described detailed differentiation trajectories of the human forebrain and midbrain and found a large number of region-specific glioblasts that mature into distinct pre-astrocytes and pre-oligodendrocyte precursor cells. Our findings reveal the establishment of cell types during the first trimester of human brain development.

Editorial Editorial Editorial

Peer reviewed
Collection

BICCN-Brain - Science

Networks
Nervous system
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Signature morphoelectric properties of diverse GABAergic interneurons in the human neocortex.

Lee BR; Dalley R; Miller JA; Chartrand T; Close J; Mann R; Mukora A; Ng L; Alfiler L; Baker K et al

Science 2023;382;6667;eadf6484

Human cortex transcriptomic studies have revealed a hierarchical organization of γ-aminobutyric acid-producing (GABAergic) neurons from subclasses to a high diversity of more granular types. Rapid GABAergic neuron viral genetic labeling plus Patch-seq (patch-clamp electrophysiology plus single-cell RNA sequencing) sampling in human brain slices was used to reliably target and analyze GABAergic neuron subclasses and individual transcriptomic types. This characterization elucidated transitions between PVALB and SST subclasses, revealed morphological heterogeneity within an abundant transcriptomic type, identified multiple spatially distinct types of the primate-specialized double bouquet cells (DBCs), and shed light on cellular differences between homologous mouse and human neocortical GABAergic neuron types. These results highlight the importance of multimodal phenotypic characterization for refinement of emerging transcriptomic cell type taxonomies and for understanding conserved and specialized cellular properties of human brain cell types.

Editorial Editorial Editorial

Peer reviewed
Collection

BICCN-Brain - Science

Networks
Nervous system
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Single-cell DNA methylation and 3D genome architecture in the human brain.

Tian W; Zhou J; Bartlett A; Zeng Q; Liu H; Castanon RG; Kenworthy M; Altshul J; Valadon C; Aldridge A et al

Science 2023;382;6667;eadf5357

Delineating the gene-regulatory programs underlying complex cell types is fundamental for understanding brain function in health and disease. Here, we comprehensively examined human brain cell epigenomes by probing DNA methylation and chromatin conformation at single-cell resolution in 517 thousand cells (399 thousand neurons and 118 thousand non-neurons) from 46 regions of three adult male brains. We identified 188 cell types and characterized their molecular signatures. Integrative analyses revealed concordant changes in DNA methylation, chromatin accessibility, chromatin organization, and gene expression across cell types, cortical areas, and basal ganglia structures. We further developed single-cell methylation barcodes that reliably predict brain cell types using the methylation status of select genomic sites. This multimodal epigenomic brain cell atlas provides new insights into the complexity of cell-type-specific gene regulation in adult human brains.

Editorial Editorial Editorial

Peer reviewed
Collection

BICCN-Brain - Science

Networks
Nervous system
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Morphoelectric and transcriptomic divergence of the layer 1 interneuron repertoire in human versus mouse neocortex.

Chartrand T; Dalley R; Close J; Goriounova NA; Lee BR; Mann R; Miller JA; Molnar G; Mukora A; Alfiler L et al

Science 2023;382;6667;eadf0805

Neocortical layer 1 (L1) is a site of convergence between pyramidal-neuron dendrites and feedback axons where local inhibitory signaling can profoundly shape cortical processing. Evolutionary expansion of human neocortex is marked by distinctive pyramidal neurons with extensive L1 branching, but whether L1 interneurons are similarly diverse is underexplored. Using Patch-seq recordings from human neurosurgical tissue, we identified four transcriptomic subclasses with mouse L1 homologs, along with distinct subtypes and types unmatched in mouse L1. Subclass and subtype comparisons showed stronger transcriptomic differences in human L1 and were correlated with strong morphoelectric variability along dimensions distinct from mouse L1 variability. Accompanied by greater layer thickness and other cytoarchitecture changes, these findings suggest that L1 has diverged in evolution, reflecting the demands of regulating the expanded human neocortical circuit.

Editorial Editorial Editorial

Peer reviewed
Collection

BICCN-Brain - Science

Networks
Nervous system
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Comparative transcriptomics reveals human-specific cortical features.

Jorstad NL; Song JHT; Exposito-Alonso D; Suresh H; Castro-Pacheco N; Krienen FM; Yanny AM; Close J; Gelfand E; Long B et al

Science 2023;382;6667;eade9516

The cognitive abilities of humans are distinctive among primates, but their molecular and cellular substrates are poorly understood. We used comparative single-nucleus transcriptomics to analyze samples of the middle temporal gyrus (MTG) from adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets to understand human-specific features of the neocortex. Human, chimpanzee, and gorilla MTG showed highly similar cell-type composition and laminar organization as well as a large shift in proportions of deep-layer intratelencephalic-projecting neurons compared with macaque and marmoset MTG. Microglia, astrocytes, and oligodendrocytes had more-divergent expression across species compared with neurons or oligodendrocyte precursor cells, and neuronal expression diverged more rapidly on the human lineage. Only a few hundred genes showed human-specific patterning, suggesting that relatively few cellular and molecular changes distinctively define adult human cortical structure.

Editorial Editorial Editorial

Peer reviewed
Collection

BICCN-Brain - Science

Networks
Nervous system
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Molecular programs of regional specification and neural stem cell fate progression in macaque telencephalon.

Micali N; Ma S; Li M; Kim SK; Mato-Blanco X; Sindhu SK; Arellano JI; Gao T; Shibata M; Gobeske KT et al

Science 2023;382;6667;eadf3786

During early telencephalic development, intricate processes of regional patterning and neural stem cell (NSC) fate specification take place. However, our understanding of these processes in primates, including both conserved and species-specific features, remains limited. Here, we profiled 761,529 single-cell transcriptomes from multiple regions of the prenatal macaque telencephalon. We deciphered the molecular programs of the early organizing centers and their cross-talk with NSCs, revealing primate-biased galanin-like peptide (GALP) signaling in the anteroventral telencephalon. Regional transcriptomic variations were observed along the frontotemporal axis during early stages of neocortical NSC progression and in neurons and astrocytes. Additionally, we found that genes associated with neuropsychiatric disorders and brain cancer risk might play critical roles in the early telencephalic organizers and during NSC progression.

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BICCN-Brain - Science

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Nervous system
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Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Spatiotemporal molecular dynamics of the developing human thalamus.

Kim CN; Shin D; Wang A; Nowakowski TJ

Science 2023;382;6667;eadf9941

The thalamus plays a central coordinating role in the brain. Thalamic neurons are organized into spatially distinct nuclei, but the molecular architecture of thalamic development is poorly understood, especially in humans. To begin to delineate the molecular trajectories of cell fate specification and organization in the developing human thalamus, we used single-cell and multiplexed spatial transcriptomics. We show that molecularly defined thalamic neurons differentiate in the second trimester of human development and that these neurons organize into spatially and molecularly distinct nuclei. We identified major subtypes of glutamatergic neuron subtypes that are differentially enriched in anatomically distinct nuclei and six subtypes of γ-aminobutyric acid-mediated (GABAergic) neurons that are shared and distinct across thalamic nuclei.

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BICCN-Brain - Science

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Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

A comparative atlas of single-cell chromatin accessibility in the human brain.

Li YE; Preissl S; Miller M; Johnson ND; Wang Z; Jiao H; Zhu C; Wang Z; Xie Y; Poirion O et al

Science 2023;382;6667;eadf7044

Recent advances in single-cell transcriptomics have illuminated the diverse neuronal and glial cell types within the human brain. However, the regulatory programs governing cell identity and function remain unclear. Using a single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq), we explored open chromatin landscapes across 1.1 million cells in 42 brain regions from three adults. Integrating this data unveiled 107 distinct cell types and their specific utilization of 544,735 candidate cis-regulatory DNA elements (cCREs) in the human genome. Nearly a third of the cCREs demonstrated conservation and chromatin accessibility in the mouse brain cells. We reveal strong links between specific brain cell types and neuropsychiatric disorders including schizophrenia, bipolar disorder, Alzheimer's disease (AD), and major depression, and have developed deep learning models to predict the regulatory roles of noncoding risk variants in these disorders.

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BICCN-Brain - Science

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Nervous system
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Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

A marmoset brain cell census reveals regional specialization of cellular identities.

Krienen FM; Levandowski KM; Zaniewski H; Del Rosario RCH; Schroeder ME; Goldman M; Wienisch M; Lutservitz A; Beja-Glasser VF; Chen C et al

Science advances 2023;9;41;eadk3986

The mammalian brain is composed of many brain structures, each with its own ontogenetic and developmental history. We used single-nucleus RNA sequencing to sample over 2.4 million brain cells across 18 locations in the common marmoset, a New World monkey primed for genetic engineering, and examined gene expression patterns of cell types within and across brain structures. The adult transcriptomic identity of most neuronal types is shaped more by developmental origin than by neurotransmitter signaling repertoire. Quantitative mapping of GABAergic types with single-molecule FISH (smFISH) reveals that interneurons in the striatum and neocortex follow distinct spatial principles, and that lateral prefrontal and other higher-order cortical association areas are distinguished by high proportions of VIP+ neurons. We use cell type-specific enhancers to drive AAV-GFP and reconstruct the morphologies of molecularly resolved interneuron types in neocortex and striatum. Our analyses highlight how lineage, local context, and functional class contribute to the transcriptional identity and biodistribution of primate brain cell types.

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BICCN-Brain - Science

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Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Multi-omic profiling of the developing human cerebral cortex at the single-cell level.

Zhu K; Bendl J; Rahman S; Vicari JM; Coleman C; Clarence T; Latouche O; Tsankova NM; Li A; Brennand KJ et al

Science advances 2023;9;41;eadg3754

The cellular complexity of the human brain is established via dynamic changes in gene expression throughout development that is mediated, in part, by the spatiotemporal activity of cis-regulatory elements (CREs). We simultaneously profiled gene expression and chromatin accessibility in 45,549 cortical nuclei across six broad developmental time points from fetus to adult. We identified cell type-specific domains in which chromatin accessibility is highly correlated with gene expression. Differentiation pseudotime trajectory analysis indicates that chromatin accessibility at CREs precedes transcription and that dynamic changes in chromatin structure play a critical role in neuronal lineage commitment. In addition, we mapped cell type-specific and temporally specific genetic loci implicated in neuropsychiatric traits, including schizophrenia and bipolar disorder. Together, our results describe the complex regulation of cell composition at critical stages in lineage determination and shed light on the impact of spatiotemporal alterations in gene expression on neuropsychiatric disease.

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BICCN-Brain - Science

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Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

A single-cell multi-omic atlas spanning the adult rhesus macaque brain.

Chiou KL; Huang X; Bohlen MO; Tremblay S; DeCasien AR; O'Day DR; Spurrell CH; Gogate AA; Zintel TM; et al

Science advances 2023;9;41;eadh1914

Cataloging the diverse cellular architecture of the primate brain is crucial for understanding cognition, behavior, and disease in humans. Here, we generated a brain-wide single-cell multimodal molecular atlas of the rhesus macaque brain. Together, we profiled 2.58 M transcriptomes and 1.59 M epigenomes from single nuclei sampled from 30 regions across the adult brain. Cell composition differed extensively across the brain, revealing cellular signatures of region-specific functions. We also identified 1.19 M candidate regulatory elements, many previously unidentified, allowing us to explore the landscape of cis-regulatory grammar and neurological disease risk in a cell type-specific manner. Altogether, this multi-omic atlas provides an open resource for investigating the evolution of the human brain and identifying novel targets for disease interventions.

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BICCN-Brain - Science

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Nervous system
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Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

A cellular resolution atlas of Broca's area.

Costantini I; Morgan L; Yang J; Balbastre Y; Varadarajan D; Pesce L; Scardigli M; Mazzamuto G; Gavryusev V; Castelli FM et al

Science advances 2023;9;41;eadg3844

Brain cells are arranged in laminar, nuclear, or columnar structures, spanning a range of scales. Here, we construct a reliable cell census in the frontal lobe of human cerebral cortex at micrometer resolution in a magnetic resonance imaging (MRI)-referenced system using innovative imaging and analysis methodologies. MRI establishes a macroscopic reference coordinate system of laminar and cytoarchitectural boundaries. Cell counting is obtained with a digital stereological approach on the 3D reconstruction at cellular resolution from a custom-made inverted confocal light-sheet fluorescence microscope (LSFM). Mesoscale optical coherence tomography enables the registration of the distorted histological cell typing obtained with LSFM to the MRI-based atlas coordinate system. The outcome is an integrated high-resolution cellular census of Broca's area in a human postmortem specimen, within a whole-brain reference space atlas.

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BICCN-Brain - Science

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Nervous system
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Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Structural and functional specializations of human fast-spiking neurons support fast cortical signaling.

Wilbers R; Galakhova AA; Driessens SLW; Heistek TS; Metodieva VD; Hagemann J; Heyer DB; Mertens EJ; Deng S; Idema S et al

Science advances 2023;9;41;eadf0708

Fast-spiking interneurons (FSINs) provide fast inhibition that synchronizes neuronal activity and is critical for cognitive function. Fast synchronization frequencies are evolutionary conserved in the expanded human neocortex despite larger neuron-to-neuron distances that challenge fast input-output transfer functions of FSINs. Here, we test in human neurons from neurosurgery tissue, which mechanistic specializations of human FSINs explain their fast-signaling properties in human cortex. With morphological reconstructions, multipatch recordings, and biophysical modeling, we find that despite threefold longer dendritic path, human FSINs maintain fast inhibition between connected pyramidal neurons through several mechanisms: stronger synapse strength of excitatory inputs, larger dendrite diameter with reduced complexity, faster AP initiation, and faster and larger inhibitory output, while Na+ current activation/inactivation properties are similar. These adaptations underlie short input-output delays in fast inhibition of human pyramidal neurons through FSINs, explaining how cortical synchronization frequencies are conserved despite expanded and sparse network topology of human cortex.

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BICCN-Brain - Science

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Nervous system
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Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Whole human-brain mapping of single cortical neurons for profiling morphological diversity and stereotypy.

Han X; Guo S; Ji N; Li T; Liu J; Ye X; Wang Y; Yun Z; Xiong F; Rong J et al

Science advances 2023;9;41;eadf3771

Quantifying neuron morphology and distribution at the whole-brain scale is essential to understand the structure and diversity of cell types. It is exceedingly challenging to reuse recent technologies of single-cell labeling and whole-brain imaging to study human brains. We propose adaptive cell tomography (ACTomography), a low-cost, high-throughput, and high-efficacy tomography approach, based on adaptive targeting of individual cells. We established a platform to inject dyes into cortical neurons in surgical tissues of 18 patients with brain tumors or other conditions and one donated fresh postmortem brain. We collected three-dimensional images of 1746 cortical neurons, of which 852 neurons were reconstructed to quantify local dendritic morphology, and mapped to standard atlases. In our data, human neurons are more diverse across brain regions than by subject age or gender. The strong stereotypy within cohorts of brain regions allows generating a statistical tensor field of neuron morphology to characterize anatomical modularity of a human brain.

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BICCN-Brain - Science

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Nervous system
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Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Human voltage-gated Na+ and K+ channel properties underlie sustained fast AP signaling.

Wilbers R; Metodieva VD; Duverdin S; Heyer DB; Galakhova AA; Mertens EJ; Versluis TD; Baayen JC; Idema S; Noske DP et al

Science advances 2023;9;41;eade3300

Human cortical pyramidal neurons are large, have extensive dendritic trees, and yet have unexpectedly fast input-output properties: Rapid subthreshold synaptic membrane potential changes are reliably encoded in timing of action potentials (APs). Here, we tested whether biophysical properties of voltage-gated sodium (Na+) and potassium (K+) currents in human pyramidal neurons can explain their fast input-output properties. Human Na+ and K+ currents exhibited more depolarized voltage dependence, slower inactivation, and faster recovery from inactivation compared with their mouse counterparts. Computational modeling showed that despite lower Na+ channel densities in human neurons, the biophysical properties of Na+ channels resulted in higher channel availability and contributed to fast AP kinetics stability. Last, human Na+ channel properties also resulted in a larger dynamic range for encoding of subthreshold membrane potential changes. Thus, biophysical adaptations of voltage-gated Na+ and K+ channels enable fast input-output properties of large human pyramidal neurons.

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BICCN-Brain - Science

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Nervous system
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Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Temporal disparity of action potentials triggered in axon initial segments and distal axons in the neocortex.

Rózsa M; Tóth M; Oláh G; Baka J; Lákovics R; Barzó P; Tamás G

Science advances 2023;9;41;eade4511

Neural population activity determines the timing of synaptic inputs, which arrive to dendrites, cell bodies, and axon initial segments (AISs) of cortical neurons. Action potential initiation in the AIS (AIS-APs) is driven by input integration, and the phase preference of AIS-APs during network oscillations is characteristic to cell classes. Distal regions of cortical axons do not receive synaptic inputs, yet experimental induction protocols can trigger retroaxonal action potentials (RA-APs) in axons distal from the soma. We report spontaneously occurring RA-APs in human and rodent cortical interneurons that appear uncorrelated to inputs and population activity. Network-linked triggering of AIS-APs versus input-independent timing of RA-APs of the same interneurons results in disparate temporal contribution of a single cell to in vivo network operation through perisomatic and distal axonal firing.

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BICCN-Brain - Science

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Nervous system
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Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

A single-cell genomic atlas for maturation of the human cerebellum during early childhood.

Ament SA; Cortes-Gutierrez M; Herb BR; Mocci E; Colantuoni C; McCarthy MM

Science translational medicine 2023;eade1283

Inflammation early in life is a clinically established risk factor for autism spectrum disorders and schizophrenia, yet the impact of inflammation on human brain development is poorly understood. The cerebellum undergoes protracted postnatal maturation, making it especially susceptible to perturbations contributing to the risk of developing neurodevelopmental disorders. Here, using single-cell genomics of postmortem cerebellar brain samples, we characterized the postnatal development of cerebellar neurons and glia in 1 to 5 year-old children, comparing individuals who had died while experiencing inflammation to those who had died as a result of an accident. Our analyses revealed that inflammation and postnatal cerebellar maturation are associated with extensive, overlapping transcriptional changes primarily in two subtypes of inhibitory neurons: Purkinje neurons and Golgi neurons. Immunohistochemical analysis of a subset of these postmortem cerebellar samples revealed no change to Purkinje neuron soma size but evidence for increased activation of microglia in those children who had experienced inflammation. Maturation-associated and inflammation-associated gene expression changes included genes implicated in neurodevelopmental disorders. A gene regulatory network model integrating cell type-specific gene expression and chromatin accessibility identified seven temporally specific gene networks in Purkinje neurons and suggested that inflammation may be associated with the premature downregulation of developmental gene expression programs.

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Nervous system
Topics
Model Organism Samples, Disease Donors, Open Access Data, Experimental Methods

Spatial Transcriptomics-correlated Electron Microscopy maps transcriptional and ultrastructural responses to brain injury.

Androvic P; Schifferer M; Perez Anderson K; Cantuti-Castelvetri L; Jiang H; Ji H; Liu L; Gouna G; Berghoff SA; Besson-Girard S et al

Nature Communications 2023;14;1;4115

Understanding the complexity of cellular function within a tissue necessitates the combination of multiple phenotypic readouts. Here, we developed a method that links spatially-resolved gene expression of single cells with their ultrastructural morphology by integrating multiplexed error-robust fluorescence in situ hybridization (MERFISH) and large area volume electron microscopy (EM) on adjacent tissue sections. Using this method, we characterized in situ ultrastructural and transcriptional responses of glial cells and infiltrating T-cells after demyelinating brain injury in male mice. We identified a population of lipid-loaded "foamy" microglia located in the center of remyelinating lesion, as well as rare interferon-responsive microglia, oligodendrocytes, and astrocytes that co-localized with T-cells. We validated our findings using immunocytochemistry and lipid staining-coupled single-cell RNA sequencing. Finally, by integrating these datasets, we detected correlations between full-transcriptome gene expression and ultrastructural features of microglia. Our results offer an integrative view of the spatial, ultrastructural, and transcriptional reorganization of single cells after demyelinating brain injury.

Pre-print

Scaling cross-tissue single-cell annotation models.

Fischer F; Fischer DS; Biederstedt E; Villani AC; Theis FJ

bioRxiv 2023

Identifying cellular identities (both novel and well-studied) is one of the key use cases in single-cell transcriptomics. While supervised machine learning has been leveraged to automate cell annotation predictions for some time, there has been relatively little progress both in scaling neural networks to large data sets and in constructing models that generalize well across diverse tissues and biological contexts up to whole organisms. Here, we propose scTab, an automated, feature-attention-based cell type prediction model specific to tabular data, and train it using a novel data augmentation scheme across a large corpus of single-cell RNA-seq observations (22.2 million human cells in total). In addition, scTab leverages deep ensembles for uncertainty quantification. Moreover, we account for ontological relationships between labels in the model evaluation to accommodate for differences in annotation granularity across datasets. On this large-scale corpus, we show that cross-tissue annotation requires nonlinear models and that the performance of scTab scales in terms of training dataset size as well as model size - demonstrating the advantage of scTab over current state-of-the-art linear models in this context. Additionally, we show that the proposed data augmentation schema improves model generalization. In summary, we introduce a de novo cell type prediction model for single-cell RNA-seq data that can be trained across a large-scale collection of curated datasets from a diverse selection of human tissues and demonstrate the benefits of using deep learning methods in this paradigm. Our codebase, training data, and model checkpoints are publicly available at https://github.com/theislab/scTab to further enable rigorous benchmarks of foundation models for single-cell RNA-seq data.

Peer reviewed
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Development, Immune
Topics
Experimental Methods

FIPRESCI: droplet microfluidics based combinatorial indexing for massive-scale 5'-end single-cell RNA sequencing.

Li Y; Huang Z; Zhang Z; Wang Q; Li F; Wang S; Ji X; Shu S; Fang X; Jiang L

Genome biology 2023;24;1;70

Single-cell RNA sequencing methods focusing on the 5'-end of transcripts can reveal promoter and enhancer activity and efficiently profile immune receptor repertoire. However, ultra-high-throughput 5'-end single-cell RNA sequencing methods have not been described. We introduce FIPRESCI, 5'-end single-cell combinatorial indexing RNA-Seq, enabling massive sample multiplexing and increasing the throughput of the droplet microfluidics system by over tenfold. We demonstrate FIPRESCI enables the generation of approximately 100,000 single-cell transcriptomes from E10.5 whole mouse embryos in a single-channel experiment, and simultaneous identification of subpopulation differences and T cell receptor signatures of peripheral blood T cells from 12 cancer patients.

Peer reviewed
Networks
Lung, Organoid

Organoid modeling of human fetal lung alveolar development reveals mechanisms of cell fate patterning and neonatal respiratory disease.

Lim K; Donovan APA; Tang W; Sun D; He P; Pett JP; Teichmann SA; Marioni JC; Meyer KB; Brand AH et al

Cell stem cell 2023;30;1;20-37.e9

Variation in lung alveolar development is strongly linked to disease susceptibility. However, underlying cellular and molecular mechanisms are difficult to study in humans. We have identified an alveolar-fated epithelial progenitor in human fetal lungs, which we grow as self-organizing organoids that model key aspects of cell lineage commitment. Using this system, we have functionally validated cell-cell interactions in the developing human alveolar niche, showing that Wnt signaling from differentiating fibroblasts promotes alveolar-type-2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. We identify a Wnt-NKX2.1 axis controlling alveolar differentiation. Moreover, we show that differential binding of NKX2.1 coordinates alveolar maturation, allowing us to model the effects of human genetic variation in NKX2.1 on alveolar differentiation. Our organoid system recapitulates key aspects of human fetal lung stem cell biology allowing mechanistic experiments to determine the cellular and molecular regulation of human development and disease.

Peer reviewed
Networks
Lung

The COPD GWAS gene ADGRG6 instructs function and injury response in human iPSC-derived type II alveolar epithelial cells.

Werder RB; Berthiaume KA; Merritt C; Gallagher M; Villacorta-Martin C; Wang F; Bawa P; Malik V; Lyons SM; Basil MC et al

American journal of human genetics 2023;110;10;1735-1749

Emphysema and chronic obstructive pulmonary disease (COPD) most commonly result from the effects of environmental exposures in genetically susceptible individuals. Genome-wide association studies have implicated ADGRG6 in COPD and reduced lung function, and a limited number of studies have examined the role of ADGRG6 in cells representative of the airway. However, the ADGRG6 locus is also associated with DLCO/VA, an indicator of gas exchange efficiency and alveolar function. Here, we sought to evaluate the mechanistic contributions of ADGRG6 to homeostatic function and disease in type 2 alveolar epithelial cells. We applied an inducible CRISPR interference (CRISPRi) human induced pluripotent stem cell (iPSC) platform to explore ADGRG6 function in iPSC-derived AT2s (iAT2s). We demonstrate that ADGRG6 exerts pleiotropic effects on iAT2s including regulation of focal adhesions, cytoskeleton, tight junctions, and proliferation. Moreover, we find that ADGRG6 knockdown in cigarette smoke-exposed iAT2s alters cellular responses to injury, downregulating apical complexes in favor of proliferation. Our work functionally characterizes the COPD GWAS gene ADGRG6 in human alveolar epithelium.

Peer reviewed
Networks
Gut, Immune

Identification of a unique subset of tissue-resident memory CD4+ T cells in Crohn's disease.

Yokoi T; Murakami M; Kihara T; Seno S; Arase M; Wing JB; Søndergaard JN; Kuwahara R; Minagawa T; Oguro-Igashira E et al

Proceedings of the National Academy of Sciences of the United States of America 2023;120;1;e2204269120

T cells differentiate into highly diverse subsets and display plasticity depending on the environment. Although lymphocytes are key mediators of inflammation, functional specialization of T cells in inflammatory bowel disease (IBD) has not been effectively described. Here, we performed deep profiling of T cells in the intestinal mucosa of IBD and identified a CD4+ tissue-resident memory T cell (Trm) subset that is increased in Crohn's disease (CD) showing unique inflammatory properties. Functionally and transcriptionally distinct CD4+ Trm subsets are observed in the inflamed gut mucosa, among which a CD-specific CD4+ Trm subset, expressing CD161 and CCR5 along with CD103, displays previously unrecognized pleiotropic signatures of innate and effector activities. These inflammatory features are further enhanced by their spatial proximity to gut epithelial cells. Furthermore, the CD-specific CD4+ Trm subset is the most predominant producer of type 1 inflammatory cytokines upon various stimulations among all CD4+ T cells, suggesting that the accumulation of this T cell subset is a pathological hallmark of CD. Our results provide comprehensive insights into the pathogenesis of IBD, paving the way for decoding of the molecular mechanisms underlying this disease.

Peer reviewed
Networks
Gut, Immune

Assessing Cellular and Transcriptional Diversity of Ileal Mucosa Among Treatment-Naïve and Treated Crohn's Disease.

Maddipatla SC; Kolachala VL; Venkateswaran S; Dodd AF; Pelia RS; Geem D; Yin H; Sun Y; Xu C; Mo A et al

Inflammatory bowel diseases 2023;29;2;274-285

Crohn's disease is a lifelong disease characterized by chronic inflammation of the gastrointestinal tract. Defining the cellular and transcriptional composition of the mucosa at different stages of disease progression is needed for personalized therapy in Crohn's.

Peer reviewed
Networks
Immune, Lung

Activation of CD8+ T Cells in Chronic Obstructive Pulmonary Disease Lung.

Villaseñor-Altamirano AB; Jain D; Jeong Y; Menon JA; Kamiya M; Haider H; Manandhar R; Sheikh MDA; Athar H; Merriam LT et al

American journal of respiratory and critical care medicine 2023;208;11;1177-1195

Rationale: Despite the importance of inflammation in chronic obstructive pulmonary disease (COPD), the immune cell landscape in the lung tissue of patients with mild-moderate disease has not been well characterized at the single-cell and molecular level. Objectives: To define the immune cell landscape in lung tissue from patients with mild-moderate COPD at single-cell resolution. Methods: We performed single-cell transcriptomic, proteomic, and T-cell receptor repertoire analyses on lung tissue from patients with mild-moderate COPD (n = 5, Global Initiative for Chronic Obstructive Lung Disease I or II), emphysema without airflow obstruction (n = 5), end-stage COPD (n = 2), control (n = 6), or donors (n = 4). We validated in an independent patient cohort (N = 929) and integrated with the Hhip+/- murine model of COPD. Measurements and Main Results: Mild-moderate COPD lungs have increased abundance of two CD8+ T cell subpopulations: cytotoxic KLRG1+TIGIT+CX3CR1+ TEMRA (T effector memory CD45RA+) cells, and DNAM-1+CCR5+ T resident memory (TRM) cells. These CD8+ T cells interact with myeloid and alveolar type II cells via IFNG and have hyperexpanded T-cell receptor clonotypes. In an independent cohort, the CD8+KLRG1+ TEMRA cells are increased in mild-moderate COPD lung compared with control or end-stage COPD lung. Human CD8+KLRG1+ TEMRA cells are similar to CD8+ T cells driving inflammation in an aging-related murine model of COPD. Conclusions: CD8+ TEMRA cells are increased in mild-moderate COPD lung and may contribute to inflammation that precedes severe disease. Further study of these CD8+ T cells may have therapeutic implications for preventing severe COPD.
Peer reviewed
Networks
Lung

A Unique Cellular Organization of Human Distal Airways and Its Disarray in Chronic Obstructive Pulmonary Disease.

Rustam S; Hu Y; Mahjour SB; Rendeiro AF; Ravichandran H; Urso A; D'Ovidio F; Martinez FJ; Altorki NK; Richmond B et al

American journal of respiratory and critical care medicine 2023;207;9;1171-1182

Rationale: Remodeling and loss of distal conducting airways, including preterminal and terminal bronchioles (pre-TBs/TBs), underlie progressive airflow limitation in chronic obstructive pulmonary disease (COPD). The cellular basis of these structural changes remains unknown. Objectives: To identify biological changes in pre-TBs/TBs in COPD at single-cell resolution and determine their cellular origin. Methods: We established a novel method of distal airway dissection and performed single-cell transcriptomic profiling of 111,412 cells isolated from different airway regions of 12 healthy lung donors and pre-TBs of 5 patients with COPD. Imaging CyTOF and immunofluorescence analysis of pre-TBs/TBs from 24 healthy lung donors and 11 subjects with COPD were performed to characterize cellular phenotypes at a tissue level. Region-specific differentiation of basal cells isolated from proximal and distal airways was studied using an air-liquid interface model. Measurements and Main Results: The atlas of cellular heterogeneity along the proximal-distal axis of the human lung was assembled and identified region-specific cellular states, including SCGB3A2+ SFTPB+ terminal airway-enriched secretory cells (TASCs) unique to distal airways. TASCs were lost in COPD pre-TBs/TBs, paralleled by loss of region-specific endothelial capillary cells, increased frequency of CD8+ T cells normally enriched in proximal airways, and augmented IFN-γ signaling. Basal cells residing in pre-TBs/TBs were identified as a cellular origin of TASCs. Regeneration of TASCs by these progenitors was suppressed by IFN-γ. Conclusions: Altered maintenance of the unique cellular organization of pre-TBs/TBs, including loss of the region-specific epithelial differentiation in these bronchioles, represents the cellular manifestation and likely the cellular basis of distal airway remodeling in COPD.
Pre-print
Networks
Gut, Immune

High-dimensional profiling demonstrates complexity, tissue imprinting, and lineage-specific precursors within the mononuclear phagocyte compartment of the human intestine

Thomas M. Fenton; Line Wulff; Gareth-Rhys Jones; Julien Vandamme; Peter B. Jørgensen; Calum C. Bain; Julie Lee; Jose MG. Izarzugaza; Kirstine G. Belling; Gwo-Tzer Ho et al

bioRxiv 2023;2021.03.28.437379

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
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Development, Gut, Immune

Concerted changes in the pediatric single-cell intestinal ecosystem before and after anti-TNF blockade

Hengqi Betty Zheng; Benjamin A. Doran; Kyle Kimler; Alison Yu; Victor Tkachev; Veronika Niederlova; Kayla Cribbin; Ryan Fleming; Brandi Bratrude; Kayla Betz et al

medRxiv 2023;2021.09.17.21263540

medRxiv - The Preprint Server for Health Sciences

Peer reviewed
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Development, Gut

Single-cell atlas of the human neonatal small intestine affected by necrotizing enterocolitis.

Egozi A; Olaloye O; Werner L; Silva T; McCourt B; Pierce RW; An X; Wang F; Chen K; Pober JS et al

PLoS biology 2023;21;5;e3002124

Necrotizing enterocolitis (NEC) is a gastrointestinal complication of premature infants with high rates of morbidity and mortality. A comprehensive view of the cellular changes and aberrant interactions that underlie NEC is lacking. This study aimed at filling in this gap. We combine single-cell RNA sequencing (scRNAseq), T-cell receptor beta (TCRβ) analysis, bulk transcriptomics, and imaging to characterize cell identities, interactions, and zonal changes in NEC. We find an abundance of proinflammatory macrophages, fibroblasts, endothelial cells as well as T cells that exhibit increased TCRβ clonal expansion. Villus tip epithelial cells are reduced in NEC and the remaining epithelial cells up-regulate proinflammatory genes. We establish a detailed map of aberrant epithelial-mesenchymal-immune interactions that are associated with inflammation in NEC mucosa. Our analyses highlight the cellular dysregulations of NEC-associated intestinal tissue and identify potential targets for biomarker discovery and therapeutics.

Peer reviewed
Networks
Development, Gut, Immune

Hematopoietic Stem Cell Development in Mammalian Embryos.

Hou S; Liu C; Yao Y; Bai Z; Gong Y; Wang C; He J; You G; Zhang G; Liu B et al

Advances in experimental medicine and biology 2023;1442;1-16

Hematopoietic stem cells (HSCs) are situated at the top of the adult hematopoietic hierarchy in mammals and give rise to the majority of blood cells throughout life. Recently, with the advance of multiple single-cell technologies, researchers have unprecedentedly deciphered the cellular and molecular evolution, the lineage relationships, and the regulatory mechanisms underlying HSC emergence in mammals. In this review, we describe the precise vascular origin of HSCs in mouse and human embryos, emphasizing the conservation in the unambiguous arterial characteristics of the HSC-primed hemogenic endothelial cells (HECs). Serving as the immediate progeny of some HECs, functional pre-HSCs of mouse embryos can now be isolated at single-cell level using defined surface marker combinations. Heterogeneity regrading cell cycle status or lineage differentiation bias within HECs, pre-HSCs, or emerging HSCs in mouse embryos has been figured out. Several epigenetic regulatory mechanisms of HSC generation, including long noncoding RNA, DNA methylation modification, RNA splicing, and layered epigenetic modifications, have also been recently uncovered. In addition to that of HSCs, the cellular and molecular events underlying the development of multiple hematopoietic progenitors in human embryos/fetus have been unraveled with the use of series of single-cell technologies. Specifically, yolk sac-derived myeloid-biased progenitors have been identified as the earliest multipotent hematopoietic progenitors in human embryo, serving as an important origin of fetal liver monocyte-derived macrophages. Moreover, the development of multiple hematopoietic lineages in human embryos such as T and B lymphocytes, innate lymphoid cells, as well as myeloid cells like monocytes, macrophages, erythrocytes, and megakaryocytes has also been depicted and reviewed here.

Pre-print
Networks
Immune, Skin
Topics
Human Subjects, Open Access Data

Cutaneous T cell lymphoma atlas reveals malignant Th2 cells supported by a B cell-rich microenvironment

Ruoyan Li; Johanna Strobl; Elizabeth F.M. Poyner; Fereshteh Torabi; Pasha Mazin; Nana-Jane Chipampe; Emily Stephenson; Louis Gardner; Bayanne Olabi; Rowen Coulthard et al

bioRxiv 2023;2023.11.06.565474

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Networks
Immune, Skin
Topics
Human Subjects, Open Access Data

A human prenatal skin cell atlas reveals immune cell regulation of skin morphogenesis

Nusayhah Hudaa Gopee; Ni Huang; Bayanne Olabi; Chloe Admane; Rachel A. Botting; April Rose Foster; Fereshteh Torabi; Elena Winheim; Dinithi Sumanaweera; Issac Goh et al

bioRxiv 2023;2023.10.12.556307

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Networks
Liver
Topics
Human Subjects, Open Access Data

uniLIVER: a Human Liver Cell Atlas for Data-Driven Cellular State Mapping

Yanhong Wu; Yuhan Fan; Yuxin Miao; Yuman Li; Guifang Du; Zeyu Chen; Jinmei Diao; Yu-Ann Chen; Mingli Ye; Renke You et al

bioRxiv 2023;2023.12.09.570903

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

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Networks
Reproduction
Topics
Human Subjects, Open Access Data

An integrated single-cell reference atlas of the human endometrium

Magda Marečková; Luz Garcia-Alonso; Marie Moullet; Valentina Lorenzi; Robert Petryszak; Carmen Sancho-Serra; Agnes Oszlanczi; Cecilia Icoresi Mazzeo; Sophie Hoffman; Michał Krassowski et al

bioRxiv 2023;2023.11.03.564728

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Networks
Immune
Topics
Human Subjects, Open Access Data

Quantifying Adaptive Evolution of the Human Immune Cell Landscape

Irepan Salvador-Martínez; Jesus Murga-Moreno; Juan C. Nieto; Clara Alsinet; David Enard; Holger Heyn

bioRxiv 2023;2023.10.06.559946

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

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Networks
Breast
Topics
Human Subjects, Open Access Data

Single nuclei chromatin accessibility and transcriptomic map of breast tissues of women of diverse genetic ancestry

Poornima Bhat-Nakshatri; Hongyu Gao; Aditi S. Khatpe; Patrick C. McGuire; Cihat Erdogan; Duojiao Chen; Guanglong Jiang; Felicia New; Rana German; Anna Maria Storniolo et al

bioRxiv 2023;2023.10.04.560911

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Networks
Nervous system
Topics
Human Subjects, Open Access Data

An integrated transcriptomic cell atlas of human neural organoids

Zhisong He; Leander Dony; Jonas Simon Fleck; Artur Szałata; Katelyn X. Li; Irena Slišković; Hsiu-Chuan Lin; Malgorzata Santel; Alexander Atamian; Giorgia Quadrato et al

bioRxiv 2023;2023.10.05.561097

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Topics
Human Subjects, Open Access Data, Experimental Methods, Computational Methods

STEM: A Method for Mapping Single-cell and Spatial Transcriptomics Data with Transfer Learning

Minsheng Hao; Erpai Luo; Yixin Chen; Yanhong Wu; Chen Li; Sijie Chen; Haoxiang Gao; Haiyang Bian; Lei Wei; Xuegong Zhang

bioRxiv 2023;2022.09.23.509186

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Topics
Human Subjects, Open Access Data, Experimental Methods, Computational Methods

Building a learnable universal coordinate system for single-cell atlas with a joint-VAE model

Haoxiang Gao; Kui Hua; Lei Wei; Xinze Wu; Sijie Chen; Qijin Yin; Rui Jiang; Xuegong Zhang

bioRxiv 2023;2021.09.09.459281

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Topics
Human Subjects, Open Access Data, Experimental Methods, Computational Methods

Spatial landmark detection and tissue registration with deep learning

Markus Ekvall; Ludvig Bergenstråhle; Alma Andersson; Paulo Czarnewski; Johannes Olegård; Lukas Käll; Joakim Lundeberg

bioRxiv 2023;2023.08.24.554614

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Nervous system, Organoid

Inferring and perturbing cell fate regulomes in human brain organoids.

Fleck JS; Jansen SMJ; Wollny D; Zenk F; Seimiya M; Jain A; Okamoto R; Santel M; He Z; Camp JG et al

Nature 2023;621;7978;365-372

Self-organizing neural organoids grown from pluripotent stem cells1-3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando-a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology.

Pre-print
Networks
Organoid

An integrated transcriptomic cell atlas of human endoderm-derived organoids

Quan Xu; Lennard Halle; Soroor Hediyeh-zadeh; Merel Kuijs; Umut Kilik; Qianhui Yu; Tristan Frum; Lukas Adam; Shrey Parikh; Manuel Gander et al

bioRxiv 2023;2023.11.20.567825

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Liver, Organoid

A human liver organoid screening platform for DILI risk prediction.

Zhang CJ; Meyer SR; O'Meara MJ; Huang S; Capeling MM; Ferrer-Torres D; Childs CJ; Spence JR; Fontana RJ; Sexton JZ

Journal of hepatology 2023;78;5;998-1006

Drug-induced liver injury (DILI), both intrinsic and idiosyncratic, causes frequent morbidity, mortality, clinical trial failures and post-approval withdrawal. This suggests an unmet need for improved in vitro models for DILI risk prediction that can account for diverse host genetics and other clinical factors. In this study, we evaluated the utility of human liver organoids (HLOs) for high-throughput DILI risk prediction and in an organ-on-chip system.

Peer reviewed
Networks
Immune, Lung

Heme oxygenase-1 determines the cell fate of ferroptotic death of alveolar macrophages in COPD.

Li Y; Yang Y; Guo T; Weng C; Yang Y; Wang Z; Zhang L; Li W

Frontiers in immunology 2023;14;1162087

Despite an increasing understanding of chronic obstructive pulmonary disease (COPD) pathogenesis, the mechanisms of diverse cell populations in the human lung remain unknown. Using single-cell RNA sequencing (scRNA-Seq), we can reveal changes within individual cell populations in COPD that are important for disease pathogenesis and characteristics.

Pre-print
Networks
Development, Immune, Reproduction
Topics
Human Subjects, Healthy Donors, Disease Donors, Open Access Data, Experimental Methods

Early infection response of the first trimester human placenta at single-cell scale

Regina Hoo; Elias R. Ruiz-Morales; Iva Kelava; Carmen Sancho-Serra; Cecilia Icoresi Mazzeo; Sara Chelaghma; Elizabeth Tuck; Alexander V. Predeus; David Fernandez-Antoran; Ross F. Waller et al

bioRxiv 2023;2023.01.02.522155

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Skin
Topics
Human Subjects

A Roadmap for a Consensus Human Skin Cell Atlas and Single-Cell Data Standardization.

Almet AA; Yuan H; Annusver K; Ramos R; Liu Y; Wiedemann J; Sorkin DH; Landén NX; Sonkoly E; Haniffa M et al

The Journal of investigative dermatology 2023;143;9;1667-1677

Single-cell technologies have become essential to driving discovery in both basic and translational investigative dermatology. Despite the multitude of available datasets, a central reference atlas of normal human skin, which can serve as a reference resource for skin cell types, cell states, and their molecular signatures, is still lacking. For any such atlas to receive broad acceptance, participation by many investigators during atlas construction is an essential prerequisite. As part of the Human Cell Atlas project, we have assembled a Skin Biological Network to build a consensus Human Skin Cell Atlas and outline a roadmap toward that goal. We define the drivers of skin diversity to be considered when selecting sequencing datasets for the atlas and list practical hurdles during skin sampling that can result in data gaps and impede comprehensive representation and technical considerations for tissue processing and computational analysis, the accounting for which should minimize biases in cell type enrichments and exclusions and decrease batch effects. By outlining our goals for Atlas 1.0, we discuss how it will uncover new aspects of skin biology.

Press release

Peer reviewed

Impact of the Human Cell Atlas on medicine

Rood, Jennifer E.; Maartens, Aidan; Hupalowska, Anna; Teichmann, Sarah A.; Regev, Aviv

Nature Medicine 2022;1-11

Single-cell atlases promise to provide a ‘missing link’ between genes, diseases and therapies. By identifying the specific cell types, states, programs and contexts where disease-implicated genes act, we will understand the mechanisms of disease at the cellular and tissue levels and can use this understanding to develop powerful disease diagnostics; identify promising new drug targets; predict their efficacy, toxicity and resistance mechanisms; and empower new kinds of therapies, from cancer therapies to regenerative medicine. Here, we lay out a vision for the potential of cell atlases to impact the future of medicine, and describe how advances over the past decade have begun to realize this potential in common complex diseases, infectious diseases (including COVID-19), rare diseases and cancer. This Perspective outlines how cell atlases can provide the missing links between genes, diseases and therapies, with advances already being made in several fields, including COVID-19 and cancer.

Pre-print
Networks
Gut, Immune, Liver
Topics
Human Subjects, Healthy Donors, Disease Donors, Open Access Data, Computational Methods, Benchmarking

Singletrome: A method to analyze and enhance the transcriptome with long noncoding RNAs for single cell analysis

Raza Ur Rahman; Iftikhar Ahmad; Robert Sparks; Amel Ben Saad; Alan Mullen

bioRxiv 2022;2022.10.31.514182

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

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Networks
Nervous system
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Computational Methods

Single-cell genomics reveals region-specific developmental trajectories underlying neuronal diversity in the human hypothalamus

Brian R. Herb; Hannah J. Glover; Aparna Bhaduri; Carlo Colantuoni; Tracy L. Bale; Kimberly Siletti; Sten Linnarsson; Rebecca Hodge; Ed Lein; Arnold R. Kriegstein et al

bioRxiv 2022;2021.07.20.453090

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Pre-print
Networks
Development
Topics
Model Organism Samples, Open Access Data, Experimental Methods, Computational Methods

Raman2RNA: Live-cell label-free prediction of single-cell RNA expression profiles by Raman microscopy

Koseki J. Kobayashi-Kirschvink; Shreya Gaddam; Taylor James-Sorenson; Emanuelle Grody; Johain R. Ounadjela; Baoliang Ge; Ke Zhang; Jeon Woong Kang; Ramnik Xavier; Peter T. C. So et al

bioRxiv 2022;2021.11.30.470655

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Immune
Topics
Human Subjects

Single-cell Atlas of common variable immunodeficiency shows germinal center-associated epigenetic dysregulation in B-cell responses

Rodríguez-Ubreva, Javier; Arutyunyan, Anna; Bonder, Marc Jan; Del Pino-Molina, Lucía; Clark, Stephen J.; de la Calle-Fabregat, Carlos; Garcia-Alonso, Luz; Handfield, Louis-François; Ciudad, Laura; Andrés-León, Eduardo et al

Nature Communications 2022;13;1

Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, displays impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and ample phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. Here, we generate a single-cell epigenomics and transcriptomics census of naïve-to-memory B cell differentiation in a CVID-discordant MZ twin pair. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B-cells mirroring defective cell-cell communication upon activation. These findings are validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naïve-to-memory B-cell transition in CVID and indicate links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, gives insight into future diagnosis and treatments of CVID patients. Common variable immunodeficiency (CVID) is the most prevalent primary immunodeficiency. Here the authors perform single-cell omics analyses in CVID-discordant monozygotic twins and show epigenetic and transcriptional alterations associated with activation in memory B cells.

Press release

Peer reviewed
Networks
Reproduction
Topics
Human Subjects, Healthy Donors, Computational Methods

Single nucleus transcriptome and chromatin accessibility of postmortem human pituitaries reveal diverse stem cell regulatory mechanisms.

Zhang Z; Zamojski M; Smith GR; Willis TL; Yianni V; Mendelev N; Pincas H; Seenarine N; Amper MAS; Vasoya M et al

Cell reports 2022;38;10;110467

Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus RNA-seq and ATAC-seq resource from pediatric, adult, and aged postmortem pituitaries (snpituitaryatlas.princeton.edu) and characterize cell-type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early-life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors that are significantly associated with gene expression in PSCs compared with other cell types and within PSCs. We identify distinct deterministic mechanisms that contribute to heterogeneous marker expression within PSCs. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes and cell type identity.

Pre-print
Networks
Development, Lung
Topics
Human Subjects

Developmental origins of cell heterogeneity in the human lung

Alexandros Sountoulidis; Sergio Marco Salas; Emelie Braun; Christophe Avenel; Joseph Bergenstråhle; Marco Vicari; Paulo Czarnewski; Jonas Theelke; Andreas Liontos; Xesus Abalo et al

bioRxiv 2022

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Kidney
Topics
Human Subjects, Disease Donors

Defining cellular complexity in human autosomal dominant polycystic kidney disease by multimodal single cell analysis.

Muto Y; Dixon EE; Yoshimura Y; Wu H; Omachi K; Ledru N; Wilson PC; King AJ; Eric Olson N; Gunawan MG et al

Nature Communications 2022;13;1;6497

Autosomal dominant polycystic kidney disease (ADPKD) is the leading genetic cause of end stage renal disease characterized by progressive expansion of kidney cysts. To better understand the cell types and states driving ADPKD progression, we analyze eight ADPKD and five healthy human kidney samples, generating single cell multiomic atlas consisting of ~100,000 single nucleus transcriptomes and ~50,000 single nucleus epigenomes. Activation of proinflammatory, profibrotic signaling pathways are driven by proximal tubular cells with a failed repair transcriptomic signature, proinflammatory fibroblasts and collecting duct cells. We identify GPRC5A as a marker for cyst-lining collecting duct cells that exhibits increased transcription factor binding motif availability for NF-κB, TEAD, CREB and retinoic acid receptors. We identify and validate a distal enhancer regulating GPRC5A expression containing these motifs. This single cell multiomic analysis of human ADPKD reveals previously unrecognized cellular heterogeneity and provides a foundation to develop better diagnostic and therapeutic approaches.

Peer reviewed
Networks
Nervous system, Organoid

Proper acquisition of cell class identity in organoids allows definition of fate specification programs of the human cerebral cortex.

Uzquiano A; Kedaigle AJ; Pigoni M; Paulsen B; Adiconis X; Kim K; Faits T; Nagaraja S; Antón-Bolaños N; Gerhardinger C et al

Cell 2022;185;20;3770-3788.e27

Realizing the full utility of brain organoids to study human development requires understanding whether organoids precisely replicate endogenous cellular and molecular events, particularly since acquisition of cell identity in organoids can be impaired by abnormal metabolic states. We present a comprehensive single-cell transcriptomic, epigenetic, and spatial atlas of human cortical organoid development, comprising over 610,000 cells, from generation of neural progenitors through production of differentiated neuronal and glial subtypes. We show that processes of cellular diversification correlate closely to endogenous ones, irrespective of metabolic state, empowering the use of this atlas to study human fate specification. We define longitudinal molecular trajectories of cortical cell types during organoid development, identify genes with predicted human-specific roles in lineage establishment, and uncover early transcriptional diversity of human callosal neurons. The findings validate this comprehensive atlas of human corticogenesis in vitro as a resource to prime investigation into the mechanisms of human cortical development.

Peer reviewed
Networks
Gut, Organoid

Systematic evaluation of colorectal cancer organoid system by single-cell RNA-Seq analysis.

Wang R; Mao Y; Wang W; Zhou X; Wang W; Gao S; Li J; Wen L; Fu W; Tang F

Genome biology 2022;23;1;106

Patient-derived organoid culture is a powerful system for studying the molecular mechanisms of cancers, especially colorectal cancer (CRC), one of the most prevalent cancers worldwide. There are two main types of 3D culture methods for colonic cells, but the similarities and differences between gene expression patterns in different culture media remain largely unexplored.

Peer reviewed
Networks
Eye

Cell-specific cis-regulatory elements and mechanisms of non-coding genetic disease in human retina and retinal organoids.

Thomas ED; Timms AE; Giles S; Harkins-Perry S; Lyu P; Hoang T; Qian J; Jackson VE; Bahlo M; Blackshaw S et al

Developmental cell 2022;57;6;820-836.e6

Cis-regulatory elements (CREs) play a critical role in the development and disease-states of all human cell types. In the retina, CREs have been implicated in several inherited disorders. To better characterize human retinal CREs, we performed single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) and single-nucleus RNA sequencing (snRNA-seq) on the developing and adult human retina and on induced pluripotent stem cell (iPSC)-derived retinal organoids. These analyses identified developmentally dynamic, cell-class-specific CREs, enriched transcription-factor-binding motifs, and putative target genes. CREs in the retina and organoids are highly correlated at the single-cell level, and this supports the use of organoids as a model for studying disease-associated CREs. As a proof of concept, we disrupted a disease-associated CRE at 5q14.3, confirming its principal target gene as the miR-9-2 primary transcript and demonstrating its role in neurogenesis and gene regulation in mature glia. This study provides a resource for characterizing human retinal CREs and showcases organoids as a model to study the function of CREs that influence development and disease.

Peer reviewed
Networks
Breast
Topics
Human Subjects

Transcriptional changes in the mammary gland during lactation revealed by single cell sequencing of cells from human milk.

Twigger AJ; Engelbrecht LK; Bach K; Schultz-Pernice I; Pensa S; Stenning J; Petricca S; Scheel CH; Khaled WT

Nature Communications 2022;13;1;562

Under normal conditions, the most significant expansion and differentiation of the adult mammary gland occurs in response to systemic reproductive hormones during pregnancy and lactation to enable milk synthesis and secretion to sustain the offspring. However, human mammary tissue remodelling that takes place during pregnancy and lactation remains poorly understood due to the challenge of acquiring samples. We report here single-cell transcriptomic analysis of 110,744 viable breast cells isolated from human milk or non-lactating breast tissue, isolated from nine and seven donors, respectively. We found that human milk largely contains epithelial cells belonging to the luminal lineage and a repertoire of immune cells. Further transcriptomic analysis of the milk cells identified two distinct secretory cell types that shared similarities with luminal progenitors, but no populations comparable to hormone-responsive cells. Taken together, our data offers a reference map and a window into the cellular dynamics that occur during human lactation and may provide further insights on the interplay between pregnancy, lactation and breast cancer.

Peer reviewed
Networks
Gut

Single-cell analysis of gastric pre-cancerous and cancer lesions reveals cell lineage diversity and intratumoral heterogeneity.

Kim J; Park C; Kim KH; Kim EH; Kim H; Woo JK; Seong JK; Nam KT; Lee YC; Cho SY

NPJ precision oncology 2022;6;1;9

Single-cell transcriptomic profiles analysis has proposed new insights for understanding the behavior of human gastric cancer (GC). GC offers a unique model of intratumoral heterogeneity. However, the specific classes of cells involved in carcinogenetic passage, and the tumor microenvironment of stromal cells was poorly understood. We characterized the heterogeneous cell population of precancerous lesions and gastric cancer at the single-cell resolution by RNA sequencing. We identified 10 gastric cell subtypes and showed the intestinal and diffuse-type cancer were characterized by different cell population. We found that the intestinal and diffuse-type cancer cells have the differential metaplastic cell lineages: intestinal-type cancer cells differentiated along the intestinal metaplasia lineage while diffuse-type cancer cells resemble de novo pathway. We observed an enriched CCND1 mutation in premalignant disease state and discovered cancer-associated fibroblast cells harboring pro-stemness properties. In particular, tumor cells could be categorized into previously proposed molecular subtypes and harbored specific subtype of malignant cell with high expression level of epithelial-myofibroblast transition which was correlated with poor clinical prognosis. In addition to intratumoral heterogeneity, the analysis revealed different cellular lineages were responsible for potential carcinogenetic pathways. Single-cell transcriptomes analysis of gastric pre-cancerous lesions and cancer may provide insights for understanding GC cell behavior, suggesting potential targets for the diagnosis and treatment of GC.

Peer reviewed
Networks
Lung, Organoid

Human alveolar progenitors generate dual lineage bronchioalveolar organoids.

Hoffmann K; Obermayer B; Hönzke K; Fatykhova D; Demir Z; Löwa A; Alves LGT; Wyler E; Lopez-Rodriguez E; Mieth M et al

Communications biology 2022;5;1;875

Mechanisms of epithelial renewal in the alveolar compartment remain incompletely understood. To this end, we aimed to characterize alveolar progenitors. Single-cell RNA-sequencing (scRNA-seq) analysis of the HTII-280+/EpCAM+ population from adult human lung revealed subclusters enriched for adult stem cell signature (ASCS) genes. We found that alveolar progenitors in organoid culture in vitro show phenotypic lineage plasticity as they can yield alveolar or bronchial cell-type progeny. The direction of the differentiation is dependent on the presence of the GSK-3β inhibitor, CHIR99021. By RNA-seq profiling of GSK-3β knockdown organoids we identified additional candidate target genes of the inhibitor, among others FOXM1 and EGF. This gives evidence of Wnt pathway independent regulatory mechanisms of alveolar specification. Following influenza A virus (IAV) infection organoids showed a similar response as lung tissue explants which confirms their suitability for studies of sequelae of pathogen-host interaction.

Peer reviewed
Networks
Lung

Characterization of the COPD alveolar niche using single-cell RNA sequencing.

Sauler M; McDonough JE; Adams TS; Kothapalli N; Barnthaler T; Werder RB; Schupp JC; Nouws J; Robertson MJ; Coarfa C et al

Nature Communications 2022;13;1;494

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide, however our understanding of cell specific mechanisms underlying COPD pathobiology remains incomplete. Here, we analyze single-cell RNA sequencing profiles of explanted lung tissue from subjects with advanced COPD or control lungs, and we validate findings using single-cell RNA sequencing of lungs from mice exposed to 10 months of cigarette smoke, RNA sequencing of isolated human alveolar epithelial cells, functional in vitro models, and in situ hybridization and immunostaining of human lung tissue samples. We identify a subpopulation of alveolar epithelial type II cells with transcriptional evidence for aberrant cellular metabolism and reduced cellular stress tolerance in COPD. Using transcriptomic network analyses, we predict capillary endothelial cells are inflamed in COPD, particularly through increased CXCL-motif chemokine signaling. Finally, we detect a high-metallothionein expressing macrophage subpopulation enriched in advanced COPD. Collectively, these findings highlight cell-specific mechanisms involved in the pathobiology of advanced COPD.

Peer reviewed
Networks
Development, Immune
Topics
Human Subjects, Healthy Donors, Open Access Data, Experimental Methods

Robust temporal map of human in vitro myelopoiesis using single-cell genomics.

Alsinet C; Primo MN; Lorenzi V; Bello E; Kelava I; Jones CP; Vilarrasa-Blasi R; Sancho-Serra C; Knights AJ; Park JE et al

Nature Communications 2022;13;1;2885

Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities.

Peer reviewed
Networks
Gut, Musculoskeletal

The myogenesis program drives clonal selection and drug resistance in rhabdomyosarcoma.

Patel AG; Chen X; Huang X; Clay MR; Komorova N; Krasin MJ; Pappo A; Tillman H; Orr BA; McEvoy J et al

Developmental cell 2022;57;10;1226-1240.e8

Rhabdomyosarcoma (RMS) is a pediatric cancer with features of skeletal muscle; patients with unresectable or metastatic RMS fare poorly due to high rates of disease recurrence. Here, we use single-cell and single-nucleus RNA sequencing to show that RMS tumors recapitulate the spectrum of embryonal myogenesis. Using matched patient samples from a clinical trial and orthotopic patient-derived xenografts (O-PDXs), we show that chemotherapy eliminates the most proliferative component with features of myoblasts within embryonal RMS; after treatment, the immature population with features of paraxial mesoderm expands to reconstitute the developmental hierarchy of the original tumor. We discovered that this paraxial mesoderm population is dependent on EGFR signaling and is sensitive to EGFR inhibitors. Taken together, these data serve as a proof of concept that targeting each developmental state in embryonal RMS is an effective strategy for improving outcomes by preventing disease recurrence.

Peer reviewed
Networks
Gut, Immune
Topics
Human Subjects, Healthy Donors, Disease Donors

Single cell transcriptomic analysis of the immune cell compartment in the human small intestine and in Celiac disease.

Atlasy N; Bujko A; Bækkevold ES; Brazda P; Janssen-Megens E; Lundin KEA; Jahnsen J; Jahnsen FL; Stunnenberg HG

Nature Communications 2022;13;1;4920

Celiac disease is an autoimmune disorder in which ingestion of dietary gluten triggers an immune reaction in the small intestine leading to destruction of the lining epithelium. Current treatment focusses on lifelong adherence to a gluten-free diet. Gluten-specific CD4+ T cells and cytotoxic intraepithelial CD8+ T cells have been proposed to be central in disease pathogenesis. Here we use unbiased single-cell RNA-sequencing and explore the heterogeneity of CD45+ immune cells in the human small intestine. We show altered myeloid cell transcriptomes present in active celiac lesions. CD4+ and CD8+ T cells transcriptomes show extensive changes and we define a natural intraepithelial lymphocyte population that is reduced in celiac disease. We show that the immune landscape in Celiac patients on a gluten-free diet is only partially restored compared to control samples. Altogether, we provide a single cell transcriptomic resource that can inform the immune landscape of the small intestine during Celiac disease.

Peer reviewed
Networks
Kidney, Skin
Topics
Human Subjects, Disease Donors

Single-cell transcriptomics reveals distinct effector profiles of infiltrating T cells in lupus skin and kidney.

Dunlap GS; Billi AC; Xing X; Ma F; Maz MP; Tsoi LC; Wasikowski R; Hodgin JB; Gudjonsson JE; Kahlenberg JM et al

JCI insight 2022;7;8

Cutaneous lupus is commonly present in patients with systemic lupus erythematosus (SLE). T cells have been strongly suspected to contribute to the pathology of cutaneous lupus; however, our understanding of the relevant T cell phenotypes and functions remains incomplete. Here, we present a detailed single-cell RNA-Seq profile of T and NK cell populations present within lesional and nonlesional skin biopsies of patients with cutaneous lupus. T cells across clusters from lesional and nonlesional skin biopsies expressed elevated levels of IFN-simulated genes (ISGs). Compared with T cells from control skin, however, T cells from cutaneous lupus lesions did not show elevated expression profiles of activation, cytotoxicity, or exhaustion. Integrated analyses indicated that skin lymphocytes appeared less activated and lacked the expanded cytotoxic populations prominent in lupus nephritis kidney T/NK cells. Comparison of skin T cells from lupus and systemic sclerosis skin biopsies further revealed an elevated ISG signature specific to cells from lupus biopsies. Overall, these data represent the first detailed transcriptomic analysis to our knowledge of the T and NK cells in cutaneous lupus at the single-cell level and have enabled a cross-tissue comparison that highlights stark differences in composition and activation of T/NK cells in distinct tissues in lupus.

Peer reviewed
Networks
Gut, Immune, Oral & Craniofacial

Salivary ZG16B expression loss follows exocrine gland dysfunction related to oral chronic graft-versus-host disease.

Costa-da-Silva AC; Aure MH; Dodge J; Martin D; Dhamala S; Cho M; Rose JJ; Bassim CW; Ambatipudi K; Hakim FT et al

iScience 2022;25;1;103592

Chronic graft-versus-host disease (cGVHD) targets include the oral mucosa and salivary glands after allogeneic hematopoietic stem cell transplant (HSCT). Without incisional biopsy, no diagnostic test exists to confirm oral cGVHD. Consequently, therapy is often withheld until severe manifestations develop. This proteomic study examined saliva and human salivary gland for a biomarker profile at first onset of oral cGVHD prior to initiation of topical steroid therapy. Whole saliva collected at onset of biopsy-proven oral GVHD was assessed using liquid chromatography-coupled tandem mass spectrometry with identification of 569 proteins, of which 77 significantly changed in abundance. ZG16B, a secretory lectin protein, was reduced 2-fold in oral cGVHD saliva (p <0.05), and significantly decreased in salivary gland secretory cells affected by cGVHD. Single-cell RNA-seq analysis of healthy MSG localized ZG16B expression to two discrete acinar cell populations. Reduced ZG16B expression may indicate specific cGVHD activity and possibly general salivary gland dysfunction.

Peer reviewed
Networks
Gut, Immune

The immune cell atlas of human neuroblastoma.

Verhoeven BM; Mei S; Olsen TK; Gustafsson K; Valind A; Lindström A; Gisselsson D; Fard SS; Hagerling C; Kharchenko PV et al

Cell reports. Medicine 2022;3;6;100657

Understanding the complete immune cell composition of human neuroblastoma (NB) is crucial for the development of immunotherapeutics. Here, we perform single-cell RNA sequencing (scRNA-seq) on 19 human NB samples coupled with multiplex immunohistochemistry, survival analysis, and comparison with normal fetal adrenal gland data. We provide a comprehensive immune cell landscape and characterize cell-state changes from normal tissue to NB. Our analysis reveals 27 immune cell subtypes, including distinct subpopulations of myeloid, NK, B, and T cells. Several different cell types demonstrate a survival benefit. In contrast to adult cancers and previous NB studies, we show an increase in inflammatory monocyte cell state when contrasting normal and tumor tissue, while no differences in cytotoxicity and exhaustion score for T cells, nor in Treg activity, are observed. Our receptor-ligand interaction analysis reveals a highly complex interactive network of the NB microenvironment from which we highlight several interactions that we suggest for future therapeutic studies.

Peer reviewed
Networks
Lung
Topics
Human Subjects, Healthy Donors, Open Access Data

A spatially resolved atlas of the human lung characterizes a gland-associated immune niche.

Madissoon E; Oliver AJ; Kleshchevnikov V; Wilbrey-Clark A; Polanski K; Richoz N; Ribeiro Orsi A; Mamanova L; Bolt L; Elmentaite R et al

Nature Genetics 2022

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.

Press release

Peer reviewed
Networks
Gut, Immune

Single-cell atlas of diverse immune populations in the advanced biliary tract cancer microenvironment.

Shi X; Li Z; Yao R; Cheng Q; Li W; Wu R; Xie Z; Zhu Y; Qiu X; Yang S et al

NPJ precision oncology 2022;6;1;58

Immunotherapies have been explored in treating solid tumors, albeit with disparate clinical effects in distinct cancer types. Systematic interrogation of immune cells in the tumor microenvironment (TME) is vital to the prediction of immunotherapy response and the development of innovative immunotherapeutics. To comprehensively characterize the immune microenvironment in advanced biliary tract cancer (BTC), we utilized single-cell RNA sequencing in unselected viable cells from 16 matched samples, and identified nineteen cell subsets from a total of 45,851 cells, in which exhausted CD8+ T cells, macrophages, and dendritic cells (DCs) in BTC were shown to augment and communicate within the TME. Transcriptional profiles coupled with T cell receptor (TCR) sequences revealed that exhausted CD8+ T cells retained clonal expansion and high proliferation in the TME, and some of them highly expressed the endoplasmic reticulum stress (ER) response gene, XBP1, indicating the role of ER stress in remodeling TME. Functional assays demonstrated that XBP1 and common immune checkpoints (PD1, TIGIT) were significantly upregulated in CD8+ T cells cocultured within the TME of BTC cells (GBC-SD, HCCC-9810). When treating the coculture groups with the specific inhibitor of IRE1α-XBP1 (4μ8C), the downregulation of TIGIT was observed in the treatment group. Collectively, comprehensive transcriptome profiling provides deep insights into the immune atlas in advanced BTC, which might be instrumental in exploring innovative immunotherapy strategies.

Peer reviewed
Networks
Lung, Organoid

Development of human alveolar epithelial cell models to study distal lung biology and disease.

Tran E; Shi T; Li X; Chowdhury AY; Jiang D; Liu Y; Wang H; Yan C; Wallace WD; Lu R et al

iScience 2022;25;2;103780

Many acute and chronic diseases affect the distal lung alveoli. Alveolar epithelial cell (AEC) lines are needed to better model these diseases. We used de-identified human remnant transplant lungs to develop a method to establish AEC lines. The lines grow well in 2-dimensional (2D) culture as epithelial monolayers expressing lung progenitor markers. In 3-dimensional (3D) culture with fibroblasts, Matrigel, and specific media conditions, the cells form alveolar-like organoids expressing mature AEC markers including aquaporin 5 (AQP5), G-protein-coupled receptor class C group 5 member A (GPRC5A), and surface marker HTII280. Single-cell RNA sequencing of an AEC line in 2D versus 3D culture revealed increased cellular heterogeneity and induction of cytokine and lipoprotein signaling in 3D organoids. Our approach yields lung progenitor lines that retain the ability to differentiate along the alveolar cell lineage despite long-term expansion and provides a valuable system to model and study the distal lung in vitro.

Peer reviewed
Networks
Organoid

Transplantable human thyroid organoids generated from embryonic stem cells to rescue hypothyroidism.

Romitti M; Tourneur A; de Faria da Fonseca B; Doumont G; Gillotay P; Liao XH; Eski SE; Van Simaeys G; Chomette L; Lasolle H et al

Nature Communications 2022;13;1;7057

The thyroid gland captures iodide in order to synthesize hormones that act on almost all tissues and are essential for normal growth and metabolism. Low plasma levels of thyroid hormones lead to hypothyroidism, which is one of the most common disorder in humans and is not always satisfactorily treated by lifelong hormone replacement. Therefore, in addition to the lack of in vitro tractable models to study human thyroid development, differentiation and maturation, functional human thyroid organoids could pave the way to explore new therapeutic approaches. Here we report the generation of transplantable thyroid organoids derived from human embryonic stem cells capable of restoring plasma thyroid hormone in athyreotic mice as a proof of concept for future therapeutic development.

Peer reviewed
Networks
Kidney
Topics
Human Subjects, Disease Donors

Mapping single-cell transcriptomes in the intra-tumoral and associated territories of kidney cancer.

Li R; Ferdinand JR; Loudon KW; Bowyer GS; Laidlaw S; Muyas F; Mamanova L; Neves JB; Bolt L; Fasouli ES et al

Cancer cell 2022

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8 T cell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.

Peer reviewed
Networks
Gut, Organoid

Suspension culture promotes serosal mesothelial development in human intestinal organoids.

Capeling MM; Huang S; Childs CJ; Wu JH; Tsai YH; Wu A; Garg N; Holloway EM; Sundaram N; Bouffi C et al

Cell reports 2022;38;7;110379

Pluripotent-stem-cell-derived human intestinal organoids (HIOs) model some aspects of intestinal development and disease, but current culture methods do not fully recapitulate the diverse cell types and complex organization of the human intestine and are reliant on 3D extracellular matrix or hydrogel systems, which limit experimental control and translational potential for regenerative medicine. We describe suspension culture as a simple, low-maintenance method for culturing HIOs and for promoting in vitro differentiation of an organized serosal mesothelial layer that is similar to primary human intestinal serosal mesothelium based on single-cell RNA sequencing and histological analysis. Functionally, HIO serosal mesothelium has the capacity to differentiate into smooth-muscle-like cells and exhibits fibrinolytic activity. An inhibitor screen identifies Hedgehog and WNT signaling as regulators of human serosal mesothelial differentiation. Collectively, suspension HIOs represent a three-dimensional model to study the human serosal mesothelium.

Peer reviewed
Networks
Lung
Topics
COVID-19

Increased SARS-CoV-2 Infection, Protease, and Inflammatory Responses in Chronic Obstructive Pulmonary Disease Primary Bronchial Epithelial Cells Defined with Single-Cell RNA Sequencing.

Johansen MD; Mahbub RM; Idrees S; Nguyen DH; Miemczyk S; Pathinayake P; Nichol K; Hansbro NG; Gearing LJ; Hertzog PJ et al

American journal of respiratory and critical care medicine 2022;206;6;712-729

Rationale: Patients with chronic obstructive pulmonary disease (COPD) develop more severe coronavirus disease (COVID-19); however, it is unclear whether they are more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and what mechanisms are responsible for severe disease. Objectives: To determine whether SARS-CoV-2 inoculated primary bronchial epithelial cells (pBECs) from patients with COPD support greater infection and elucidate the effects and mechanisms involved. Methods: We performed single-cell RNA sequencing analysis on differentiated pBECs from healthy subjects and patients with COPD 7 days after SARS-CoV-2 inoculation. We correlated changes with viral titers, proinflammatory responses, and IFN production. Measurements and Main Results: Single-cell RNA sequencing revealed that COPD pBECs had 24-fold greater infection than healthy cells, which was supported by plaque assays. Club/goblet and basal cells were the predominant populations infected and expressed mRNAs involved in viral replication. Proteases involved in SARS-CoV-2 entry/infection (TMPRSS2 and CTSB) were increased, and protease inhibitors (serpins) were downregulated more so in COPD. Inflammatory cytokines linked to COPD exacerbations and severe COVID-19 were increased, whereas IFN responses were blunted. Coexpression analysis revealed a prominent population of club/goblet cells with high type 1/2 IFN responses that were important drivers of immune responses to infection in both healthy and COPD pBECs. Therapeutic inhibition of proteases and inflammatory imbalances reduced viral titers and cytokine responses, particularly in COPD pBECs. Conclusions: COPD pBECs are more susceptible to SARS-CoV-2 infection because of increases in coreceptor expression and protease imbalances and have greater inflammatory responses. A prominent cluster of IFN-responsive club/goblet cells emerges during infection, which may be important drivers of immunity. Therapeutic interventions suppress SARS-CoV-2 replication and consequent inflammation.
Peer reviewed
Topics
Computational Methods

SCAFE: a software suite for analysis of transcribed cis-regulatory elements in single cells.

Moody J; Kouno T; Chang JC; Ando Y; Carninci P; Shin JW; Hon CC

Bioinformatics (Oxford, England) 2022;38;22;5126-5128

Cell type-specific activities of cis-regulatory elements (CRE) are central to understanding gene regulation and disease predisposition. Single-cell RNA 5'end sequencing (sc-end5-seq) captures the transcription start sites (TSS) which can be used as a proxy to measure the activity of transcribed CREs (tCREs). However, a substantial fraction of TSS identified from sc-end5-seq data may not be genuine due to various artifacts, hindering the use of sc-end5-seq for de novo discovery of tCREs.

Peer reviewed
Networks
Gut

Single-nucleus cross-tissue molecular reference maps toward understanding disease gene function.

Eraslan G; Drokhlyansky E; Anand S; Fiskin E; Subramanian A; Slyper M; Wang J; Van Wittenberghe N; Rouhana JM; Waldman J et al

Science 2022;376;6594;eabl4290

Understanding gene function and regulation in homeostasis and disease requires knowledge of the cellular and tissue contexts in which genes are expressed. Here, we applied four single-nucleus RNA sequencing methods to eight diverse, archived, frozen tissue types from 16 donors and 25 samples, generating a cross-tissue atlas of 209,126 nuclei profiles, which we integrated across tissues, donors, and laboratory methods with a conditional variational autoencoder. Using the resulting cross-tissue atlas, we highlight shared and tissue-specific features of tissue-resident cell populations; identify cell types that might contribute to neuromuscular, metabolic, and immune components of monogenic diseases and the biological processes involved in their pathology; and determine cell types and gene modules that might underlie disease mechanisms for complex traits analyzed by genome-wide association studies.

Peer reviewed
Collection

Cross-Tissue Analysis - Science

Networks
Development, Gut, Immune
Topics
Human Subjects, Healthy Donors, Open Access Data

Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Domínguez Conde C; Xu C; Jarvis LB; Rainbow DB; Wells SB; Gomes T; Howlett SK; Suchanek O; Polanski K; King HW et al

Science 2022;376;6594;eabl5197

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. We surveyed the immune compartment of 16 tissues from 12 adult donors by single-cell RNA sequencing and VDJ sequencing generating a dataset of ~360,000 cells. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of finely phenotyped immune cell types, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. Our multitissue 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.

Editorial Press release Press release

Peer reviewed
Collection

Cross-Tissue Analysis - Science

Topics
Human Subjects, Healthy Donors, Open Access Data, Experimental Methods, Computational Methods

The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans.

; Jones RC; Karkanias J; Krasnow MA; Pisco AO; Quake SR; Salzman J; Yosef N; Bulthaup B; Brown P et al

Science 2022;376;6594;eabl4896

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

Editorial Press release Press release

Peer reviewed
Collection

Cross-Tissue Analysis - Science

Topics
Human Subjects, Healthy Donors, Open Access Data, Experimental Methods, Computational Methods

Single-nucleus cross-tissue molecular reference maps toward understanding disease gene function.

Eraslan G; Drokhlyansky E; Anand S; Fiskin E; Subramanian A; Slyper M; Wang J; Van Wittenberghe N; Rouhana JM; Waldman J et al

Science 2022;376;6594;eabl4290

Understanding gene function and regulation in homeostasis and disease requires knowledge of the cellular and tissue contexts in which genes are expressed. Here, we applied four single-nucleus RNA sequencing methods to eight diverse, archived, frozen tissue types from 16 donors and 25 samples, generating a cross-tissue atlas of 209,126 nuclei profiles, which we integrated across tissues, donors, and laboratory methods with a conditional variational autoencoder. Using the resulting cross-tissue atlas, we highlight shared and tissue-specific features of tissue-resident cell populations; identify cell types that might contribute to neuromuscular, metabolic, and immune components of monogenic diseases and the biological processes involved in their pathology; and determine cell types and gene modules that might underlie disease mechanisms for complex traits analyzed by genome-wide association studies.

Editorial Press release Press release

Peer reviewed
Networks
Gut

Refining colorectal cancer classification and clinical stratification through a single-cell atlas.

Khaliq AM; Erdogan C; Kurt Z; Turgut SS; Grunvald MW; Rand T; Khare S; Borgia JA; Hayden DM; Pappas SG et al

Genome biology 2022;23;1;113

Colorectal cancer (CRC) consensus molecular subtypes (CMS) have different immunological, stromal cell, and clinicopathological characteristics. Single-cell characterization of CMS subtype tumor microenvironments is required to elucidate mechanisms of tumor and stroma cell contributions to pathogenesis which may advance subtype-specific therapeutic development. We interrogate racially diverse human CRC samples and analyze multiple independent external cohorts for a total of 487,829 single cells enabling high-resolution depiction of the cellular diversity and heterogeneity within the tumor and microenvironmental cells.

Peer reviewed
Networks
Lung, Organoid
Topics
COVID-19

The establishment of COPD organoids to study host-pathogen interaction reveals enhanced viral fitness of SARS-CoV-2 in bronchi.

Chan LLY; Anderson DE; Cheng HS; Ivan FX; Chen S; Kang AEZ; Foo R; Gamage AM; Tiew PY; Koh MS et al

Nature Communications 2022;13;1;7635

Chronic obstructive pulmonary disease (COPD) is characterised by airflow limitation and infective exacerbations, however, in-vitro model systems for the study of host-pathogen interaction at the individual level are lacking. Here, we describe the establishment of nasopharyngeal and bronchial organoids from healthy individuals and COPD that recapitulate disease at the individual level. In contrast to healthy organoids, goblet cell hyperplasia and reduced ciliary beat frequency were observed in COPD organoids, hallmark features of the disease. Single-cell transcriptomics uncovered evidence for altered cellular differentiation trajectories in COPD organoids. SARS-CoV-2 infection of COPD organoids revealed more productive replication in bronchi, the key site of infection in severe COVID-19. Viral and bacterial exposure of organoids induced greater pro-inflammatory responses in COPD organoids. In summary, we present an organoid model that recapitulates the in vivo physiological lung microenvironment at the individual level and is amenable to the study of host-pathogen interaction and emerging infectious disease.

Peer reviewed
Networks
Kidney
Topics
Human Subjects, Healthy Donors

Single-cell profiling of healthy human kidney reveals features of sex-based transcriptional programs and tissue-specific immunity.

McEvoy CM; Murphy JM; Zhang L; Clotet-Freixas S; Mathews JA; An J; Karimzadeh M; Pouyabahar D; Su S; Zaslaver O et al

Nature Communications 2022;13;1;7634

Knowledge of the transcriptional programs underpinning the functions of human kidney cell populations at homeostasis is limited. We present a single-cell perspective of healthy human kidney from 19 living donors, with equal contribution from males and females, profiling the transcriptome of 27677 cells to map human kidney at high resolution. Sex-based differences in gene expression within proximal tubular cells were observed, specifically, increased anti-oxidant metallothionein genes in females and aerobic metabolism-related genes in males. Functional differences in metabolism were confirmed in proximal tubular cells, with male cells exhibiting higher oxidative phosphorylation and higher levels of energy precursor metabolites. We identified kidney-specific lymphocyte populations with unique transcriptional profiles indicative of kidney-adapted functions. Significant heterogeneity in myeloid cells was observed, with a MRC1+LYVE1+FOLR2+C1QC+ population representing a predominant population in healthy kidney. This study provides a detailed cellular map of healthy human kidney, and explores the complexity of parenchymal and kidney-resident immune cells.

Peer reviewed
Networks
Skin
Topics
Human Subjects, Disease Donors

Single cell transcriptomic landscape of diabetic foot ulcers.

Theocharidis G; Thomas BE; Sarkar D; Mumme HL; Pilcher WJR; Dwivedi B; Sandoval-Schaefer T; Sîrbulescu RF; Kafanas A; Mezghani I et al

Nature Communications 2022;13;1;181

Diabetic foot ulceration (DFU) is a devastating complication of diabetes whose pathogenesis remains incompletely understood. Here, we profile 174,962 single cells from the foot, forearm, and peripheral blood mononuclear cells using single-cell RNA sequencing. Our analysis shows enrichment of a unique population of fibroblasts overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 and increased M1 macrophage polarization in the DFU patients with healing wounds. Further, analysis of spatially separated samples from the same patient and spatial transcriptomics reveal preferential localization of these healing associated fibroblasts toward the wound bed as compared to the wound edge or unwounded skin. Spatial transcriptomics also validates our findings of higher abundance of M1 macrophages in healers and M2 macrophages in non-healers. Our analysis provides deep insights into the wound healing microenvironment, identifying cell types that could be critical in promoting DFU healing, and may inform novel therapeutic approaches for DFU treatment.

Peer reviewed
Networks
Development, Lung

A single-cell regulatory map of postnatal lung alveologenesis in humans and mice.

Duong TE; Wu Y; Sos BC; Dong W; Limaye S; Rivier LH; Myers G; Hagood JS; Zhang K

Cell genomics 2022;2;3

Ex-utero regulation of the lungs' responses to breathing air and continued alveolar development shape adult respiratory health. Applying single-cell transposome hypersensitive site sequencing (scTHS-seq) to over 80,000 cells, we assembled the first regulatory atlas of postnatal human and mouse lung alveolar development. We defined regulatory modules and elucidated new mechanistic insights directing alveolar septation, including alveolar type 1 and myofibroblast cell signaling and differentiation, and a unique human matrix fibroblast population. Incorporating GWAS, we mapped lung function causal variants to myofibroblasts and identified a pathogenic regulatory unit linked to lineage marker FGF18, demonstrating the utility of chromatin accessibility data to uncover disease mechanism targets. Our regulatory map and analysis model provide valuable new resources to investigate age-dependent and species-specific control of critical developmental processes. Furthermore, these resources complement existing atlas efforts to advance our understanding of lung health and disease across the human lifespan.

Peer reviewed
Networks
Development, Lung
Topics
Human Subjects, Open Access Data

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

He P; Lim K; Sun D; Pett JP; Jeng Q; Polanski K; Dong Z; Bolt L; Richardson L; Mamanova L et al

Cell 2022;185;25;4841-4860.e25

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.

Press release

Peer reviewed
Networks
Oral & Craniofacial, Organoid

Salivary gland organoid culture maintains distinct glandular properties of murine and human major salivary glands.

Yoon YJ; Kim D; Tak KY; Hwang S; Kim J; Sim NS; Cho JM; Choi D; Ji Y; Hur JK et al

Nature Communications 2022;13;1;3291

Salivary glands that produce and secrete saliva, which is essential for lubrication, digestion, immunity, and oral homeostasis, consist of diverse cells. The long-term maintenance of diverse salivary gland cells in organoids remains problematic. Here, we establish long-term murine and human salivary gland organoid cultures. Murine and human salivary gland organoids express gland-specific genes and proteins of acinar, myoepithelial, and duct cells, and exhibit gland functions when stimulated with neurotransmitters. Furthermore, human salivary gland organoids are established from isolated basal or luminal cells, retaining their characteristics. Single-cell RNA sequencing also indicates that human salivary gland organoids contain heterogeneous cell types and replicate glandular diversity. Our protocol also enables the generation of tumoroid cultures from benign and malignant salivary gland tumor types, in which tumor-specific gene signatures are well-conserved. In this study, we provide an experimental platform for the exploration of precision medicine in the era of tissue regeneration and anticancer treatment.

Peer reviewed
Networks
Gut, Organoid

Helicobacter pylori shows tropism to gastric differentiated pit cells dependent on urea chemotaxis.

Aguilar C; Pauzuolis M; Pompaiah M; Vafadarnejad E; Arampatzi P; Fischer M; Narres D; Neyazi M; Kayisoglu Ö; Sell T et al

Nature Communications 2022;13;1;5878

The human gastric epithelium forms highly organized gland structures with different subtypes of cells. The carcinogenic bacterium Helicobacter pylori can attach to gastric cells and subsequently translocate its virulence factor CagA, but the possible host cell tropism of H. pylori is currently unknown. Here, we report that H. pylori preferentially attaches to differentiated cells in the pit region of gastric units. Single-cell RNA-seq shows that organoid-derived monolayers recapitulate the pit region, while organoids capture the gland region of the gastric units. Using these models, we show that H. pylori preferentially attaches to highly differentiated pit cells, marked by high levels of GKN1, GKN2 and PSCA. Directed differentiation of host cells enable enrichment of the target cell population and confirm H. pylori preferential attachment and CagA translocation into these cells. Attachment is independent of MUC5AC or PSCA expression, and instead relies on bacterial TlpB-dependent chemotaxis towards host cell-released urea, which scales with host cell size.

Peer reviewed
Networks
Liver, Organoid

Human branching cholangiocyte organoids recapitulate functional bile duct formation.

Roos FJM; van Tienderen GS; Wu H; Bordeu I; Vinke D; Albarinos LM; Monfils K; Niesten S; Smits R; Willemse J et al

Cell stem cell 2022;29;5;776-794.e13

Human cholangiocyte organoids show great promise for regenerative therapies and in vitro modeling of bile duct development and diseases. However, the cystic organoids lack the branching morphology of intrahepatic bile ducts (IHBDs). Here, we report establishing human branching cholangiocyte organoid (BRCO) cultures. BRCOs self-organize into complex tubular structures resembling the IHBD architecture. Single-cell transcriptomics and functional analysis showed high similarity to primary cholangiocytes, and importantly, the branching growth mimics aspects of tubular development and is dependent on JAG1/NOTCH2 signaling. When applied to cholangiocarcinoma tumor organoids, the morphology changes to an in vitro morphology like primary tumors. Moreover, these branching cholangiocarcinoma organoids (BRCCAOs) better match the transcriptomic profile of primary tumors and showed increased chemoresistance to gemcitabine and cisplatin. In conclusion, BRCOs recapitulate a complex process of branching morphogenesis in vitro. This provides an improved model to study tubular formation, bile duct functionality, and associated biliary diseases.

Peer reviewed
Collection

Cross-Tissue Analysis - Science

Networks
Development
Topics
Human Subjects, Healthy Donors, Open Access Data

Mapping the developing human immune system across organs.

Suo C; Dann E; Goh I; Jardine L; Kleshchevnikov V; Park JE; Botting RA; Stephenson E; Engelbert J; Tuong ZK et al

Science 2022;376;6597;eabo0510

Single-cell genomics studies have decoded the immune cell composition of several human prenatal organs but were limited in describing the developing immune system as a distributed network across tissues. We profiled nine prenatal tissues combining single-cell RNA sequencing, antigen-receptor sequencing, and spatial transcriptomics to reconstruct the developing human immune system. This revealed the late acquisition of immune-effector functions by myeloid and lymphoid cell subsets and the maturation of monocytes and T cells before peripheral tissue seeding. Moreover, we uncovered system-wide blood and immune cell development beyond primary hematopoietic organs, characterized human prenatal B1 cells, and shed light on the origin of unconventional T cells. Our atlas provides both valuable data resources and biological insights that will facilitate cell engineering, regenerative medicine, and disease understanding.

Editorial Press release Press release

Peer reviewed
Networks
Gut

Single-Cell Atlas of Lineage States, Tumor Microenvironment, and Subtype-Specific Expression Programs in Gastric Cancer.

Kumar V; Ramnarayanan K; Sundar R; Padmanabhan N; Srivastava S; Koiwa M; Yasuda T; Koh V; Huang KK; Tay ST et al

Cancer discovery 2022;12;3;670-691

Gastric cancer heterogeneity represents a barrier to disease management. We generated a comprehensive single-cell atlas of gastric cancer (>200,000 cells) comprising 48 samples from 31 patients across clinical stages and histologic subtypes. We identified 34 distinct cell-lineage states including novel rare cell populations. Many lineage states exhibited distinct cancer-associated expression profiles, individually contributing to a combined tumor-wide molecular collage. We observed increased plasma cell proportions in diffuse-type tumors associated with epithelial-resident KLF2 and stage-wise accrual of cancer-associated fibroblast subpopulations marked by high INHBA and FAP coexpression. Single-cell comparisons between patient-derived organoids (PDO) and primary tumors highlighted inter- and intralineage similarities and differences, demarcating molecular boundaries of PDOs as experimental models. We complemented these findings by spatial transcriptomics, orthogonal validation in independent bulk RNA-sequencing cohorts, and functional demonstration using in vitro and in vivo models. Our results provide a high-resolution molecular resource of intra- and interpatient lineage states across distinct gastric cancer subtypes.

Peer reviewed
Networks
Gut, Skin

Genome-wide DNA hypermethylation opposes healing in patients with chronic wounds by impairing epithelial-mesenchymal transition.

Singh K; Rustagi Y; Abouhashem AS; Tabasum S; Verma P; Hernandez E; Pal D; Khona DK; Mohanty SK; Kumar M et al

The Journal of clinical investigation 2022;132;17

An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. An unbiased whole-genome methylome was studied in the wound-edge tissue of patients with chronic wounds. A total of 4,689 differentially methylated regions (DMRs) were identified in chronic wound-edge skin compared with unwounded human skin. Hypermethylation was more frequently observed (3,661 DMRs) in the chronic wound-edge tissue compared with hypomethylation (1,028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial-mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA-Seq analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified 4 genes, ADAM17, NOTCH, TWIST1, and SMURF1, that functionally represent the EMT pathway. Single-cell RNA-Seq studies revealed that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces wound-edge gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the wound edge was achieved by a tissue nanotransfection-based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene silencing represents a productive therapeutic strategy to improve wound closure.

Peer reviewed
Networks
Gut, Organoid

BMP gradient along the intestinal villus axis controls zonated enterocyte and goblet cell states.

Beumer J; Puschhof J; Yengej FY; Zhao L; Martinez-Silgado A; Blotenburg M; Begthel H; Boot C; van Oudenaarden A; Chen YG et al

Cell reports 2022;38;9;110438

Intestinal epithelial cells derive from stem cells at the crypt base and travel along the crypt-villus axis to die at the villus tip. The two dominant villus epithelial cell types, absorptive enterocytes and mucous-secreting goblet cells, are mature when they exit crypts. Murine enterocytes switch functional cell states during migration along the villus. Here, we ask whether this zonation is driven by the bone morphogenetic protein (BMP) gradient, which increases toward the villus. Using human intestinal organoids, we show that BMP signaling controls the expression of zonated genes in enterocytes. We find that goblet cells display similar zonation involving antimicrobial genes. Using an inducible Bmpr1a knockout mouse model, we confirm that BMP controls these zonated genes in vivo. Our findings imply that local manipulation of BMP signal strength may be used to reset the enterocyte "rheostat" of carbohydrate versus lipid uptake and to control the antimicrobial response through goblet cells.

Peer reviewed
Networks
Nervous system, Organoid

Gruffi: an algorithm for computational removal of stressed cells from brain organoid transcriptomic datasets.

Vértesy Á; Eichmüller OL; Naas J; Novatchkova M; Esk C; Balmaña M; Ladstaetter S; Bock C; von Haeseler A; Knoblich JA

The EMBO journal 2022;41;17;e111118

Organoids enable in vitro modeling of complex developmental processes and disease pathologies. Like most 3D cultures, organoids lack sufficient oxygen supply and therefore experience cellular stress. These negative effects are particularly prominent in complex models, such as brain organoids, and can affect lineage commitment. Here, we analyze brain organoid and fetal single-cell RNA sequencing (scRNAseq) data from published and new datasets, totaling about 190,000 cells. We identify a unique stress signature in the data from all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined subpopulation of cells that is unique to organoids and does not affect neuronal specification or maturation. We have developed a computational algorithm, Gruffi, which uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. We validated our method using six additional datasets from different organoid protocols and early brains, and show its usefulness to other organoid systems including retinal organoids. Our data show that the adverse effects of cell stress can be corrected by bioinformatic analysis for improved delineation of developmental trajectories and resemblance to in vivo data.

Peer reviewed
Networks
Gut, Immune, Organoid

Optimized human intestinal organoid model reveals interleukin-22-dependency of paneth cell formation.

He GW; Lin L; DeMartino J; Zheng X; Staliarova N; Dayton T; Begthel H; van de Wetering WJ; Bodewes E; van Zon J et al

Cell stem cell 2022;29;9;1333-1345.e6

Opposing roles have been proposed for IL-22 in intestinal pathophysiology. We have optimized human small intestinal organoid (hSIO) culturing, constitutively generating all differentiated cell types while maintaining an active stem cell compartment. IL-22 does not promote the expansion of stem cells but rather slows the growth of hSIOs. In hSIOs, IL-22 is required for formation of Paneth cells, the prime producers of intestinal antimicrobial peptides (AMPs). Introduction of inflammatory bowel disease (IBD)-associated loss-of-function mutations in the IL-22 co-receptor gene IL10RB resulted in abolishment of Paneth cells in hSIOs. Moreover, IL-22 induced expression of host defense genes (such as REG1A, REG1B, and DMBT1) in enterocytes, goblet cells, Paneth cells, Tuft cells, and even stem cells. Thus, IL-22 does not directly control the regenerative capacity of crypt stem cells but rather boosts Paneth cell numbers, as well as the expression of AMPs in all cell types.

Peer reviewed
Networks
Lung

The ZIP8/SIRT1 axis regulates alveolar progenitor cell renewal in aging and idiopathic pulmonary fibrosis.

Liang J; Huang G; Liu X; Taghavifar F; Liu N; Wang Y; Deng N; Yao C; Xie T; Kulur V et al

The Journal of clinical investigation 2022;132;11

Type 2 alveolar epithelial cells (AEC2s) function as progenitor cells in the lung. We have shown previously that failure of AEC2 regeneration results in progressive lung fibrosis in mice and is a cardinal feature of idiopathic pulmonary fibrosis (IPF). In this study, we identified deficiency of a specific zinc transporter, SLC39A8 (ZIP8), in AEC2s from both IPF lungs and lungs of old mice. Loss of ZIP8 expression was associated with impaired renewal capacity of AEC2s and enhanced lung fibrosis. ZIP8 regulation of AEC2 progenitor function was dependent on SIRT1. Replenishment with exogenous zinc and SIRT1 activation promoted self-renewal and differentiation of AEC2s from lung tissues of IPF patients and old mice. Deletion of Zip8 in AEC2s in mice resulted in impaired AEC2 renewal, increased susceptibility to bleomycin injury, and development of spontaneous lung fibrosis. Therapeutic strategies to restore zinc metabolism and appropriate SIRT1 signaling could improve AEC2 progenitor function and mitigate ongoing fibrogenesis.

Peer reviewed
Networks
Gut

Single-cell analyses define a continuum of cell state and composition changes in the malignant transformation of polyps to colorectal cancer.

Becker WR; Nevins SA; Chen DC; Chiu R; Horning AM; Guha TK; Laquindanum R; Mills M; Chaib H; Ladabaum U et al

Nature Genetics 2022;54;7;985-995

To chart cell composition and cell state changes that occur during the transformation of healthy colon to precancerous adenomas to colorectal cancer (CRC), we generated single-cell chromatin accessibility profiles and single-cell transcriptomes from 1,000 to 10,000 cells per sample for 48 polyps, 27 normal tissues and 6 CRCs collected from patients with or without germline APC mutations. A large fraction of polyp and CRC cells exhibit a stem-like phenotype, and we define a continuum of epigenetic and transcriptional changes occurring in these stem-like cells as they progress from homeostasis to CRC. Advanced polyps contain increasing numbers of stem-like cells, regulatory T cells and a subtype of pre-cancer-associated fibroblasts. In the cancerous state, we observe T cell exhaustion, RUNX1-regulated cancer-associated fibroblasts and increasing accessibility associated with HNF4A motifs in epithelia. DNA methylation changes in sporadic CRC are strongly anti-correlated with accessibility changes along this continuum, further identifying regulatory markers for molecular staging of polyps.

Peer reviewed
Networks
Gut, Immune

Ulcerative colitis is characterized by a plasmablast-skewed humoral response associated with disease activity.

Uzzan M; Martin JC; Mesin L; Livanos AE; Castro-Dopico T; Huang R; Petralia F; Magri G; Kumar S; Zhao Q et al

Nature Medicine 2022;28;4;766-779

B cells, which are critical for intestinal homeostasis, remain understudied in ulcerative colitis (UC). In this study, we recruited three cohorts of patients with UC (primary cohort, n = 145; validation cohort 1, n = 664; and validation cohort 2, n = 143) to comprehensively define the landscape of B cells during UC-associated intestinal inflammation. Using single-cell RNA sequencing, single-cell IgH gene sequencing and protein-level validation, we mapped the compositional, transcriptional and clonotypic landscape of mucosal and circulating B cells. We found major perturbations within the mucosal B cell compartment, including an expansion of naive B cells and IgG+ plasma cells with curtailed diversity and maturation. Furthermore, we isolated an auto-reactive plasma cell clone targeting integrin αvβ6 from inflamed UC intestines. We also identified a subset of intestinal CXCL13-expressing TFH-like T peripheral helper cells that were associated with the pathogenic B cell response. Finally, across all three cohorts, we confirmed that changes in intestinal humoral immunity are reflected in circulation by the expansion of gut-homing plasmablasts that correlates with disease activity and predicts disease complications. Our data demonstrate a highly dysregulated B cell response in UC and highlight a potential role of B cells in disease pathogenesis.

Peer reviewed
Networks
Gut, Immune, Reproduction

Single-cell analysis of endometriosis reveals a coordinated transcriptional programme driving immunotolerance and angiogenesis across eutopic and ectopic tissues.

Tan Y; Flynn WF; Sivajothi S; Luo D; Bozal SB; Davé M; Luciano AA; Robson P; Luciano DE; Courtois ET

Nature Cell biology 2022;24;8;1306-1318

Endometriosis is characterized by the growth of endometrial-like tissue outside the uterus. It affects many women during their reproductive age, causing years of pelvic pain and potential infertility. Its pathophysiology remains largely unknown, which limits early diagnosis and treatment. We characterized peritoneal and ovarian lesions at single-cell transcriptome resolution and compared them to matched eutopic endometrium, unaffected endometrium and organoids derived from these tissues, generating data on over 122,000 cells across 14 individuals. We spatially localized many of the cell types using imaging mass cytometry. We identify a perivascular mural cell specific to the peritoneal lesions, with dual roles in angiogenesis promotion and immune cell trafficking. We define an immunotolerant peritoneal niche, fundamental differences in eutopic endometrium and between lesion microenvironments and an unreported progenitor-like epithelial cell subpopulation. Altogether, this study provides a holistic view of the endometriosis microenvironment that represents a comprehensive cell atlas of the disease in individuals undergoing hormonal treatment, providing essential information for future therapeutics and diagnostics.

Pre-print
Networks
Musculoskeletal
Topics
Human Subjects, Open Access Data

An integrated single-cell atlas of the skeleton from development through adulthood

Tim Herpelinck; Liesbeth Ory; Gabriele Nasello; Mojtaba Barzegari; Johanna Bolander; Frank P. Luyten; Przemko Tylzanowski; Liesbet Geris

bioRxiv 2022;2022.03.14.484345

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Networks
Nervous system, Organoid

Androgens increase excitatory neurogenic potential in human brain organoids.

Kelava I; Chiaradia I; Pellegrini L; Kalinka AT; Lancaster MA

Nature 2022;602;7895;112-116

The biological basis of male-female brain differences has been difficult to elucidate in humans. The most notable morphological difference is size, with male individuals having on average a larger brain than female individuals1,2, but a mechanistic understanding of how this difference arises remains unknown. Here we use brain organoids3 to show that although sex chromosomal complement has no observable effect on neurogenesis, sex steroids-namely androgens-lead to increased proliferation of cortical progenitors and an increased neurogenic pool. Transcriptomic analysis and functional studies demonstrate downstream effects on histone deacetylase activity and the mTOR pathway. Finally, we show that androgens specifically increase the neurogenic output of excitatory neuronal progenitors, whereas inhibitory neuronal progenitors are not increased. These findings reveal a role for androgens in regulating the number of excitatory neurons and represent a step towards understanding the origin of sex-related brain differences in humans.

Peer reviewed
Networks
Nervous system, Organoid

Autism genes converge on asynchronous development of shared neuron classes.

Paulsen B; Velasco S; Kedaigle AJ; Pigoni M; Quadrato G; Deo AJ; Adiconis X; Uzquiano A; Sartore R; Yang SM et al

Nature 2022;602;7896;268-273

Genetic risk for autism spectrum disorder (ASD) is associated with hundreds of genes spanning a wide range of biological functions1-6. The alterations in the human brain resulting from mutations in these genes remain unclear. Furthermore, their phenotypic manifestation varies across individuals7,8. Here we used organoid models of the human cerebral cortex to identify cell-type-specific developmental abnormalities that result from haploinsufficiency in three ASD risk genes-SUV420H1 (also known as KMT5B), ARID1B and CHD8-in multiple cell lines from different donors, using single-cell RNA-sequencing (scRNA-seq) analysis of more than 745,000 cells and proteomic analysis of individual organoids, to identify phenotypic convergence. Each of the three mutations confers asynchronous development of two main cortical neuronal lineages-γ-aminobutyric-acid-releasing (GABAergic) neurons and deep-layer excitatory projection neurons-but acts through largely distinct molecular pathways. Although these phenotypes are consistent across cell lines, their expressivity is influenced by the individual genomic context, in a manner that is dependent on both the risk gene and the developmental defect. Calcium imaging in intact organoids shows that these early-stage developmental changes are followed by abnormal circuit activity. This research uncovers cell-type-specific neurodevelopmental abnormalities that are shared across ASD risk genes and are finely modulated by human genomic context, finding convergence in the neurobiological basis of how different risk genes contribute to ASD pathology.

Peer reviewed
Networks
Nervous system, Organoid

Lineage recording in human cerebral organoids.

He Z; Maynard A; Jain A; Gerber T; Petri R; Lin HC; Santel M; Ly K; Dupré JS; Sidow L et al

Nature Methods 2022;19;1;90-99

Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.

Peer reviewed
Networks
Liver, Organoid

Single-cell atlas of human liver development reveals pathways directing hepatic cell fates.

Wesley BT; Ross ADB; Muraro D; Miao Z; Saxton S; Tomaz RA; Morell CM; Ridley K; Zacharis ED; Petrus-Reurer S et al

Nature Cell biology 2022;24;10;1487-1498

The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances have been hampered by the lack of knowledge concerning human hepatic development. Here, we addressed this limitation by describing the developmental trajectories of different cell types that make up the human liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing essential roles during organogenesis. We utilized this information to derive bipotential hepatoblast organoids and then exploited this model system to validate the importance of signalling pathways in hepatocyte and cholangiocyte specification. Further insights into hepatic maturation also enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a platform to investigate the basic mechanisms directing human liver development and to produce cell types for clinical applications.

Peer reviewed
Networks
Lung, Organoid

Human distal lung maps and lineage hierarchies reveal a bipotent progenitor.

Kadur Lakshminarasimha Murthy P; Sontake V; Tata A; Kobayashi Y; Macadlo L; Okuda K; Conchola AS; Nakano S; Gregory S; Miller LA et al

Nature 2022;604;7904;111-119

Mapping the spatial distribution and molecular identity of constituent cells is essential for understanding tissue dynamics in health and disease. We lack a comprehensive map of human distal airways, including the terminal and respiratory bronchioles (TRBs), which are implicated in respiratory diseases1-4. Here, using spatial transcriptomics and single-cell profiling of microdissected distal airways, we identify molecularly distinct TRB cell types that have not-to our knowledge-been previously characterized. These include airway-associated LGR5+ fibroblasts and TRB-specific alveolar type-0 (AT0) cells and TRB secretory cells (TRB-SCs). Connectome maps and organoid-based co-cultures reveal that LGR5+ fibroblasts form a signalling hub in the airway niche. AT0 cells and TRB-SCs are conserved in primates and emerge dynamically during human lung development. Using a non-human primate model of lung injury, together with human organoids and tissue specimens, we show that alveolar type-2 cells in regenerating lungs transiently acquire an AT0 state from which they can differentiate into either alveolar type-1 cells or TRB-SCs. This differentiation programme is distinct from that identified in the mouse lung5-7. Our study also reveals mechanisms that drive the differentiation of the bipotent AT0 cell state into normal or pathological states. In sum, our findings revise human lung cell maps and lineage trajectories, and implicate an epithelial transitional state in primate lung regeneration and disease.

Peer reviewed
Networks
Lung

Human distal airways contain a multipotent secretory cell that can regenerate alveoli.

Basil MC; Cardenas-Diaz FL; Kathiriya JJ; Morley MP; Carl J; Brumwell AN; Katzen J; Slovik KJ; Babu A; Zhou S et al

Nature 2022;604;7904;120-126

The human lung differs substantially from its mouse counterpart, resulting in a distinct distal airway architecture affected by disease pathology in chronic obstructive pulmonary disease. In humans, the distal branches of the airway interweave with the alveolar gas-exchange niche, forming an anatomical structure known as the respiratory bronchioles. Owing to the lack of a counterpart in mouse, the cellular and molecular mechanisms that govern respiratory bronchioles in the human lung remain uncharacterized. Here we show that human respiratory bronchioles contain a unique secretory cell population that is distinct from cells in larger proximal airways. Organoid modelling reveals that these respiratory airway secretory (RAS) cells act as unidirectional progenitors for alveolar type 2 cells, which are essential for maintaining and regenerating the alveolar niche. RAS cell lineage differentiation into alveolar type 2 cells is regulated by Notch and Wnt signalling. In chronic obstructive pulmonary disease, RAS cells are altered transcriptionally, corresponding to abnormal alveolar type 2 cell states, which are associated with smoking exposure in both humans and ferrets. These data identify a distinct progenitor in a region of the human lung that is not found in mouse that has a critical role in maintaining the gas-exchange compartment and is altered in chronic lung disease.

Peer reviewed
Networks
Lung

Anomalous Epithelial Variations and Ectopic Inflammatory Response in Chronic Obstructive Pulmonary Disease.

Watanabe N; Fujita Y; Nakayama J; Mori Y; Kadota T; Hayashi Y; Shimomura I; Ohtsuka T; Okamoto K; Araya J et al

American journal of respiratory cell and molecular biology 2022;67;6;708-719

Phenotypic alterations in the lung epithelium have been widely implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, but the precise mechanisms orchestrating this persistent inflammatory process remain unknown because of the complexity of lung parenchymal and mesenchymal architecture. To identify cell type-specific mechanisms and cell-cell interactions among the multiple lung resident cell types and inflammatory cells that contribute to COPD progression, we profiled 57,918 cells from lungs of patients with COPD, smokers without COPD, and never-smokers using single-cell RNA sequencing technology. We predicted pseudotime of cell differentiation and cell-to-cell interaction networks in COPD. Although epithelial components in never-smokers were relatively uniform, smoker groups represent extensive heterogeneity in epithelial cells, particularly in alveolar type 2 (AT2) clusters. Among AT2 cells, which are generally regarded as alveolar progenitors, we identified a unique subset that increased in patients with COPD and specifically expressed a series of chemokines including CXCL1 and CXCL8. A trajectory analysis revealed that the inflammatory AT2 cell subpopulation followed a unique differentiation path, and a prediction model of cell-to-cell interactions inferred significantly increased intercellular networks of inflammatory AT2 cells. Our results identify previously unidentified cell subsets and provide an insight into the biological and clinical characteristics of COPD pathogenesis.

Peer reviewed
Networks
Gut
Topics
Human Subjects, Healthy Donors, Open Access Data

A Proximal-to-Distal Survey of Healthy Adult Human Small Intestine and Colon Epithelium by Single-Cell Transcriptomics.

Burclaff J; Bliton RJ; Breau KA; Ok MT; Gomez-Martinez I; Ranek JS; Bhatt AP; Purvis JE; Woosley JT; Magness ST

Cellular and molecular gastroenterology and hepatology 2022;13;5;1554-1589

Single-cell transcriptomics offer unprecedented resolution of tissue function at the cellular level, yet studies analyzing healthy adult human small intestine and colon are sparse. Here, we present single-cell transcriptomics covering the duodenum, jejunum, ileum, and ascending, transverse, and descending colon from 3 human beings.

Peer reviewed
Networks
Skin
Topics
Human Subjects

Anatomically distinct fibroblast subsets determine skin autoimmune patterns.

Xu Z; Chen D; Hu Y; Jiang K; Huang H; Du Y; Wu W; Wang J; Sui J; Wang W et al

Nature 2022;601;7891;118-124

The skin serves as a physical barrier and an immunological interface that protects the body from the external environment1-3. Aberrant activation of immune cells can induce common skin autoimmune diseases such as vitiligo, which are often characterized by bilateral symmetric lesions in certain anatomic regions of the body4-6. Understanding what orchestrates the activities of cutaneous immune cells at an organ level is necessary for the treatment of autoimmune diseases. Here we identify subsets of dermal fibroblasts that are responsible for driving patterned autoimmune activity, by using a robust mouse model of vitiligo that is based on the activation of endogenous auto-reactive CD8+ T cells that target epidermal melanocytes. Using a combination of single-cell analysis of skin samples from patients with vitiligo, cell-type-specific genetic knockouts and engraftment experiments, we find that among multiple interferon-γ (IFNγ)-responsive cell types in vitiligo-affected skin, dermal fibroblasts are uniquely required to recruit and activate CD8+ cytotoxic T cells through secreted chemokines. Anatomically distinct human dermal fibroblasts exhibit intrinsic differences in the expression of chemokines in response to IFNγ. In mouse models of vitiligo, regional IFNγ-resistant fibroblasts determine the autoimmune pattern of depigmentation in the skin. Our study identifies anatomically distinct fibroblasts with permissive or repressive IFNγ responses as the key determinant of body-level patterns of lesions in vitiligo, and highlights mesenchymal subpopulations as therapeutic targets for treating autoimmune diseases.

Peer reviewed
Networks
Gut
Topics
Human Subjects, Healthy Donors, Disease Donors, Experimental Methods, Computational Methods

An Integrated Taxonomy for Monogenic Inflammatory Bowel Disease.

Bolton C; Smillie CS; Pandey S; Elmentaite R; Wei G; Argmann C; Aschenbrenner D; James KR; McGovern DPB; Macchi M et al

Gastroenterology 2022;162;3;859-876

Monogenic forms of inflammatory bowel disease (IBD) illustrate the essential roles of individual genes in pathways and networks safeguarding immune tolerance and gut homeostasis.

Peer reviewed
Networks
Oral & Craniofacial
Topics
Human Subjects, Healthy Donors, Disease Donors, Open Access Data

A Roadmap for the Human Oral and Craniofacial Cell Atlas.

Caetano AJ; ; Sequeira I; Byrd KM

Journal of dental research 2022;101;11;1274-1288

Oral and craniofacial tissues are uniquely adapted for continuous and intricate functioning, including breathing, feeding, and communication. To achieve these vital processes, this complex is supported by incredible tissue diversity, variously composed of epithelia, vessels, cartilage, bone, teeth, ligaments, and muscles, as well as mesenchymal, adipose, and peripheral nervous tissue. Recent single cell and spatial multiomics assays-specifically, genomics, epigenomics, transcriptomics, proteomics, and metabolomics-have annotated known and new cell types and cell states in human tissues and animal models, but these concepts remain limitedly explored in the human postnatal oral and craniofacial complex. Here, we highlight the collaborative and coordinated efforts of the newly established Oral and Craniofacial Bionetwork as part of the Human Cell Atlas, which aims to leverage single cell and spatial multiomics approaches to first understand the cellular and molecular makeup of human oral and craniofacial tissues in health and to then address common and rare diseases. These powerful assays have already revealed the cell types that support oral tissues, and they will unravel cell types and molecular networks utilized across development, maintenance, and aging as well as those affected in diseases of the craniofacial complex. This level of integration and cell annotation with partner laboratories across the globe will be critical for understanding how multiple variables, such as age, sex, race, and ancestry, influence these oral and craniofacial niches. Here, we 1) highlight these recent collaborative efforts to employ new single cell and spatial approaches to resolve our collective biology at a higher resolution in health and disease, 2) discuss the vision behind the Oral and Craniofacial Bionetwork, 3) outline the stakeholders who contribute to and will benefit from this network, and 4) outline directions for creating the first Human Oral and Craniofacial Cell Atlas.

Peer reviewed
Networks
Development, Reproduction
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Computational Methods

Single-cell roadmap of human gonadal development.

Garcia-Alonso L; Lorenzi V; Mazzeo CI; Alves-Lopes JP; Roberts K; Sancho-Serra C; Engelbert J; Marečková M; Gruhn WH; Botting RA et al

Nature 2022;607;7919;540-547

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. 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.

Peer reviewed
Networks
Lung
Topics
Human Subjects, Healthy Donors, Disease Donors, Experimental Methods, Computational Methods, Benchmarking

The discovAIR project: a roadmap towards the Human Lung Cell Atlas.

Luecken MD; Zaragosi LE; Madissoon E; Sikkema L; Firsova AB; De Domenico E; Kümmerle L; Saglam A; Berg M; Gay ACA et al

The European respiratory journal 2022;60;2

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.

Peer reviewed
Topics
Human Subjects, Healthy Donors, COVID-19

Local and systemic responses to SARS-CoV-2 infection in children and adults.

Yoshida M; Worlock KB; Huang N; Lindeboom RGH; Butler CR; Kumasaka N; Dominguez Conde C; Mamanova L; Bolt L; Richardson L et al

Nature 2022;602;7896;321-327

It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children.

Press release

Peer reviewed
Networks
Heart, Immune, Kidney, Liver, Lung, Skin
Topics
Human Subjects, Healthy Donors

Anatomical structures, cell types and biomarkers of the Human Reference Atlas

Börner, Katy; Teichmann, Sarah A.; Quardokus, Ellen M.; Gee, James C.; Browne, Kristen; Osumi-Sutherland, David; Herr, Bruce W.; Bueckle, Andreas; Paul, Hrishikesh; Haniffa, Muzlifah et al

Nature Cell Biology 2021;23;11

The Human Reference Atlas (HRA) aims to map all of the cells of the human body to advance biomedical research and clinical practice. This Perspective presents collaborative work by members of 16 international consortia on two essential and interlinked parts of the HRA: (1) three-dimensional representations of anatomy that are linked to (2) tables that name and interlink major anatomical structures, cell types, plus biomarkers (ASCT+B). We discuss four examples that demonstrate the practical utility of the HRA. In this Perspective, Börner et al. discuss initiatives by 16 consortia to construct a Human Reference Atlas (HRA) comprising reference organs linked to tables that name major anatomical structures, cell types, plus biomarkers (ASCT+B) and present examples of HRA usage.

Editorial

Peer reviewed

Cell type ontologies of the Human Cell Atlas

Osumi-Sutherland, David; Xu, Chuan; Keays, Maria; Levine, Adam P.; Kharchenko, Peter V.; Regev, Aviv; Lein, Ed; Teichmann, Sarah A.

Nature Cell Biology 2021;23;11

Massive single-cell profiling efforts have accelerated our discovery of the cellular composition of the human body while at the same time raising the need to formalize this new knowledge. Here, we discuss current efforts to harmonize and integrate different sources of annotations of cell types and states into a reference cell ontology. We illustrate with examples how a unified ontology can consolidate and advance our understanding of cell types across scientific communities and biological domains. In this Perspective, Teichmann and colleagues present ongoing efforts from consortia of the Human Cell Atlas to harmonize and integrate data sources into a reference cell ontology and the contributions of cell ontologies to discovery.

Editorial

Pre-print
Networks
Development, Eye, Gut, Heart, Immune, Kidney, Liver, Lung, Musculoskeletal, Nervous system, Pancreas, Reproduction
Topics
Human Subjects, Healthy Donors

Comprehensive identification of fetal cis-regulatory elements in the human genome by single-cell multi-omics analysis

Hao Yu; Na Ai; Ping Peng; Yuwen Ke; Xuepeng Chen; Yun Li; Ting Zhao; Shan Jiang; Jiang Liu; Lan Jiang

bioRxiv 2021

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Topics
Computational Methods

DUBStepR is a scalable correlation-based feature selection method for accurately clustering single-cell data

Ranjan, Bobby; Sun, Wenjie; Park, Jinyu; Mishra, Kunal; Schmidt, Florian; Xie, Ronald; Alipour, Fatemeh; Singhal, Vipul; Joanito, Ignasius; Honardoost, Mohammad Amin et al

Nature Communications 2021;12;1

Feature selection (marker gene selection) is widely believed to improve clustering accuracy, and is thus a key component of single cell clustering pipelines. Existing feature selection methods perform inconsistently across datasets, occasionally even resulting in poorer clustering accuracy than without feature selection. Moreover, existing methods ignore information contained in gene-gene correlations. Here, we introduce DUBStepR (Determining the Underlying Basis using Stepwise Regression), a feature selection algorithm that leverages gene-gene correlations with a novel measure of inhomogeneity in feature space, termed the Density Index (DI). Despite selecting a relatively small number of genes, DUBStepR substantially outperformed existing single-cell feature selection methods across diverse clustering benchmarks. Additionally, DUBStepR was the only method to robustly deconvolve T and NK heterogeneity by identifying disease-associated common and rare cell types and subtypes in PBMCs from rheumatoid arthritis patients. DUBStepR is scalable to over a million cells, and can be straightforwardly applied to other data types such as single-cell ATAC-seq. We propose DUBStepR as a general-purpose feature selection solution for accurately clustering single-cell data. Cell-type-specific genes are often strongly correlated in expression - an informative yet underexplored property of single-cell data. Here, the authors leverage gene expression correlations to develop DUBStepR, a feature selection method for accurately clustering single-cell data.

Peer reviewed
Networks
Development

A roadmap for the Human Developmental Cell Atlas

Haniffa, Muzlifah; Taylor, Deanne; Linnarsson, Sten; Aronow, Bruce J.; Bader, Gary D.; Barker, Roger A.; Camara, Pablo G.; Camp, J. Gray; Chédotal, Alain; Copp, Andrew et al

Nature 2021;597;7875;196-205

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. This Perspective outlines the Human Developmental Cell Atlas initiative, which uses state-of-the-art technologies to map and model human development across gestation, and discusses the early milestones that have been achieved.

Press release

Peer reviewed

RCA2: a scalable supervised clustering algorithm that reduces batch effects in scRNA-seq data.

Schmidt F; Ranjan B; Lin QXX; Krishnan V; Joanito I; Honardoost MA; Nawaz Z; Venkatesh PN; Tan J; Rayan NA et al

Nucleic acids research 2021;49;15;8505-8519

The transcriptomic diversity of cell types in the human body can be analysed in unprecedented detail using single cell (SC) technologies. Unsupervised clustering of SC transcriptomes, which is the default technique for defining cell types, is prone to group cells by technical, rather than biological, variation. Compared to de-novo (unsupervised) clustering, we demonstrate using multiple benchmarks that supervised clustering, which uses reference transcriptomes as a guide, is robust to batch effects and data quality artifacts. Here, we present RCA2, the first algorithm to combine reference projection (batch effect robustness) with graph-based clustering (scalability). In addition, RCA2 provides a user-friendly framework incorporating multiple commonly used downstream analysis modules. RCA2 also provides new reference panels for human and mouse and supports generation of custom panels. Furthermore, RCA2 facilitates cell type-specific QC, which is essential for accurate clustering of data from heterogeneous tissues. We demonstrate the advantages of RCA2 on SC data from human bone marrow, healthy PBMCs and PBMCs from COVID-19 patients. Scalable supervised clustering methods such as RCA2 will facilitate unified analysis of cohort-scale SC datasets.

Peer reviewed
Topics
Human Subjects, Healthy Donors, Disease Donors

Pre-activated antiviral innate immunity in the upper airways controls early SARS-CoV-2 infection in children

Loske, J.; Röhmel, J.; Lukassen, S.; Stricker, S.; Magalhães, V. G.; Liebig, J.; Chua, R. L.; Thürmann, L.; Messingschlager, M.; Seegebarth, A. et al

Nature Biotechnology 2021

Children have reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection rates and a substantially lower risk for developing severe coronavirus disease 2019 compared with adults. However, the molecular mechanisms underlying protection in younger age groups remain unknown. Here we characterize the single-cell transcriptional landscape in the upper airways of SARS-CoV-2-negative (n = 18) and age-matched SARS-CoV-2-positive (n = 24) children and corresponding samples from adults (n = 44), covering an age range of 4 weeks to 77 years. Children displayed higher basal expression of relevant pattern recognition receptors such as MDA5 (IFIH1) and RIG-I (DDX58) in upper airway epithelial cells, macrophages and dendritic cells, resulting in stronger innate antiviral responses upon SARS-CoV-2 infection than in adults. We further detected distinct immune cell subpopulations including KLRC1 (NKG2A)+ cytotoxic T cells and a CD8+ T cell population with a memory phenotype occurring predominantly in children. Our study provides evidence that the airway immune cells of children are primed for virus sensing, resulting in a stronger early innate antiviral response to SARS-CoV-2 infection than in adults. Single-cell sequencing reveals pre-activated immunity as important for milder COVID-19 symptoms in children.

Peer reviewed
Networks
Lung
Topics
Healthy Donors, Disease Donors

Chronic lung diseases are associated with gene expression programs favoring SARS-CoV-2 entry and severity.

Bui LT; Winters NI; Chung MI; Joseph C; Gutierrez AJ; Habermann AC; Adams TS; Schupp JC; Poli S; Peter LM et al

Nature Communications 2021;12;1;4314

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyze the transcriptomes of 611,398 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observe a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD AT2 cells express higher levels of genes linked directly to the efficiency of viral replication and the innate immune response. Additionally, we identify basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection.

Peer reviewed

Towards a Human Cell Atlas: Taking Notes from the Past.

Lindeboom RGH; Regev A; Teichmann SA

Trends in genetics 2021;37;7;625-630

Comprehensively characterizing the cellular composition and organization of tissues has been a long-term scientific challenge that has limited our ability to study fundamental and clinical aspects of human physiology. The Human Cell Atlas (HCA) is a global collaborative effort to create a reference map of all human cells as a basis for both understanding human health and diagnosing, monitoring, and treating disease. Many aspects of the HCA are analogous to the Human Genome Project (HGP), whose completion presents a major milestone in modern biology. To commemorate the HGP's 20-year anniversary of completion, we discuss the launch of the HCA in light of the HGP, and highlight recent progress by the HCA consortium.

Peer reviewed
Topics
Model Organism Samples, Healthy Donors, Computational Methods

Cell segmentation-free inference of cell types from in situ transcriptomics data.

Park J; Choi W; Tiesmeyer S; Long B; Borm LE; Garren E; Nguyen TN; Tasic B; Codeluppi S; Graf T et al

Nature Communications 2021;12;1;3545

Multiplexed fluorescence in situ hybridization techniques have enabled cell-type identification, linking transcriptional heterogeneity with spatial heterogeneity of cells. However, inaccurate cell segmentation reduces the efficacy of cell-type identification and tissue characterization. Here, we present a method called Spot-based Spatial cell-type Analysis by Multidimensional mRNA density estimation (SSAM), a robust cell segmentation-free computational framework for identifying cell-types and tissue domains in 2D and 3D. SSAM is applicable to a variety of in situ transcriptomics techniques and capable of integrating prior knowledge of cell types. We apply SSAM to three mouse brain tissue images: the somatosensory cortex imaged by osmFISH, the hypothalamic preoptic region by MERFISH, and the visual cortex by multiplexed smFISH. Here, we show that SSAM detects regions occupied by known cell types that were previously missed and discovers new cell types.

Peer reviewed
Topics
Computational Methods

Tutorial: guidelines for annotating single-cell transcriptomic maps using automated and manual methods

Clarke, Zoe A.; Andrews, Tallulah S.; Atif, Jawairia; Pouyabahar, Delaram; Innes, Brendan T.; MacParland, Sonya A.; Bader, Gary D.

Nature Protocols 2021;16;6;2749-2764

Single-cell transcriptomics can profile thousands of cells in a single experiment and identify novel cell types, states and dynamics in a wide variety of tissues and organisms. Standard experimental protocols and analysis workflows have been developed to create single-cell transcriptomic maps from tissues. This tutorial focuses on how to interpret these data to identify cell types, states and other biologically relevant patterns with the objective of creating an annotated map of cells. We recommend a three-step workflow including automatic cell annotation (wherever possible), manual cell annotation and verification. Frequently encountered challenges are discussed, as well as strategies to address them. Guiding principles and specific recommendations for software tools and resources that can be used for each step are covered, and an R notebook is included to help run the recommended workflow. Basic familiarity with computer software is assumed, and basic knowledge of programming (e.g., in the R language) is recommended. This tutorial provides guidelines for interpreting single-cell transcriptomic maps to identify cell types, states and other biologically relevant patterns.

Peer reviewed
Networks
Reproduction
Topics
Model Organism Samples, Healthy Donors, Computational Methods

Single nucleus multi-omics regulatory landscape of the murine pituitary.

Ruf-Zamojski F; Zhang Z; Zamojski M; Smith GR; Mendelev N; Liu H; Nudelman G; Moriwaki M; Pincas H; Castanon RG et al

Nature Communications 2021;12;1;2677

To provide a multi-omics resource and investigate transcriptional regulatory mechanisms, we profile the transcriptome, chromatin accessibility, and methylation status of over 70,000 single nuclei (sn) from adult mouse pituitaries. Paired snRNAseq and snATACseq datasets from individual animals highlight a continuum between developmental epigenetically-encoded cell types and transcriptionally-determined transient cell states. Co-accessibility analysis-based identification of a putative Fshb cis-regulatory domain that overlaps the fertility-linked rs11031006 human polymorphism, followed by experimental validation illustrate the use of this resource for hypothesis generation. We also identify transcriptional and chromatin accessibility programs distinguishing each major cell type. Regulons, which are co-regulated gene sets sharing binding sites for a common transcription factor driver, recapitulate cell type clustering. We identify both cell type-specific and sex-specific regulons that are highly correlated with promoter accessibility, but not with methylation state, supporting the centrality of chromatin accessibility in shaping cell-defining transcriptional programs. The sn multi-omics atlas is accessible at snpituitaryatlas.princeton.edu.

Peer reviewed
Networks
Oral & Craniofacial
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data, Experimental Methods, Computational Methods, COVID-19

SARS-CoV-2 infection of the oral cavity and saliva.

Huang N; Pérez P; Kato T; Mikami Y; Okuda K; Gilmore RC; Conde CD; Gasmi B; Stein S; Beach M et al

Nature Medicine 2021;27;5;892-903

Despite signs of infection-including taste loss, dry mouth and mucosal lesions such as ulcerations, enanthema and macules-the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly understood. To address this, we generated and analyzed two single-cell RNA sequencing datasets of the human minor salivary glands and gingiva (9 samples, 13,824 cells), identifying 50 cell clusters. Using integrated cell normalization and annotation, we classified 34 unique cell subpopulations between glands and gingiva. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry factors such as ACE2 and TMPRSS members were broadly enriched in epithelial cells of the glands and oral mucosae. Using orthogonal RNA and protein expression assessments, we confirmed SARS-CoV-2 infection in the glands and mucosae. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting ACE2 and TMPRSS expression and sustained SARS-CoV-2 infection. Acellular and cellular salivary fractions from asymptomatic individuals were found to transmit SARS-CoV-2 ex vivo. Matched nasopharyngeal and saliva samples displayed distinct viral shedding dynamics, and salivary viral burden correlated with COVID-19 symptoms, including taste loss. Upon recovery, this asymptomatic cohort exhibited sustained salivary IgG antibodies against SARS-CoV-2. Collectively, these data show that the oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.

Peer reviewed
Topics
COVID-19

Single-cell multi-omics analysis of the immune response in COVID-19.

Stephenson E; Reynolds G; Botting RA; Calero-Nieto FJ; Morgan MD; Tuong ZK; Bach K; Sungnak W; Worlock KB; Yoshida M et al

Nature Medicine 2021;27;5;904-916

Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16C1QA/B/C) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34 hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8 T cells and an increased ratio of CD8 effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy.

Peer reviewed
Topics
COVID-19

SARS-CoV-2 infection of the oral cavity and saliva.

Huang N; Pérez P; Kato T; Mikami Y; Okuda K; Gilmore RC; Conde CD; Gasmi B; Stein S; Beach M et al

Nature Medicine 2021;27;5;892-903

Despite signs of infection-including taste loss, dry mouth and mucosal lesions such as ulcerations, enanthema and macules-the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly understood. To address this, we generated and analyzed two single-cell RNA sequencing datasets of the human minor salivary glands and gingiva (9 samples, 13,824 cells), identifying 50 cell clusters. Using integrated cell normalization and annotation, we classified 34 unique cell subpopulations between glands and gingiva. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry factors such as ACE2 and TMPRSS members were broadly enriched in epithelial cells of the glands and oral mucosae. Using orthogonal RNA and protein expression assessments, we confirmed SARS-CoV-2 infection in the glands and mucosae. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting ACE2 and TMPRSS expression and sustained SARS-CoV-2 infection. Acellular and cellular salivary fractions from asymptomatic individuals were found to transmit SARS-CoV-2 ex vivo. Matched nasopharyngeal and saliva samples displayed distinct viral shedding dynamics, and salivary viral burden correlated with COVID-19 symptoms, including taste loss. Upon recovery, this asymptomatic cohort exhibited sustained salivary IgG antibodies against SARS-CoV-2. Collectively, these data show that the oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.

Pre-print
Topics
Human Subjects, Healthy Donors, Open Access Data, Computational Methods

Profiling of transcribed cis-regulatory elements in single cells

Jonathan Moody; Tsukasa Kouno; Akari Suzuki; Youtaro Shibayama; Chikashi Terao; Jen-Chien Chang; Fernando López-Redondo; Chi Wai Yip; Jessica Severin; Hiroyuki Suetsugu et al

bioRxiv 2021;2021.04.04.438388

bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution

Peer reviewed
Topics
Human Subjects, Healthy Donors, Disease Donors, Open Access Data, Experimental Methods, Computational Methods, COVID-19

Shotgun transcriptome, spatial omics, and isothermal profiling of SARS-CoV-2 infection reveals unique host responses, viral diversification, and drug interactions.

Butler D; Mozsary C; Meydan C; Foox J; Rosiene J; Shaiber A; Danko D; Afshinnekoo E; MacKay M; Sedlazeck FJ et al

Nature Communications 2021;12;1;1660

In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin-angiotensin-aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.

Peer reviewed
Networks
Musculoskeletal
Topics
Healthy Donors, Open Access Data, Experimental Methods, Computational Methods

Mapping the musculoskeletal system one cell at a time

Baldwin, Mathew J.; Cribbs, Adam P.; Guilak, Farshid; Snelling, Sarah J. B.

Nature Reviews Rheumatology 2021;17;5;247-248

The Human Cell Atlas (HCA) project aims to map tissues and organs during development, maturation and pathology at single cell resolution. The musculoskeletal HCA network is a community for fostering collaboration and shared expertise to help develop the therapeutic approaches needed to address the high global burden of musculoskeletal disorders.

Peer reviewed
Topics
Human Subjects, Healthy Donors

Single-Nucleus and In Situ RNA-Sequencing Reveal Cell Topographies in the Human Pancreas.

Tosti L; Hang Y; Debnath O; Tiesmeyer S; Trefzer T; Steiger K; Ten FW; Lukassen S; Ballke S; Kühl AA et al

Gastroenterology 2021;160;4;1330-1344.e11

Molecular evidence of cellular heterogeneity in the human exocrine pancreas has not been yet established because of the local concentration and cascade of hydrolytic enzymes that can rapidly degrade cells and RNA upon pancreatic resection. We sought to better understand the heterogeneity and cellular composition of the pancreas in neonates and adults in healthy and diseased conditions using single-cell sequencing approaches.

Peer reviewed
Networks
Gut, Immune

Spatiotemporal analysis of human intestinal development at single-cell resolution.

Fawkner-Corbett D; Antanaviciute A; Parikh K; Jagielowicz M; Gerós AS; Gupta T; Ashley N; Khamis D; Fowler D; Morrissey E et al

Cell 2021;184;3;810-826.e23

Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.

Peer reviewed
Topics
Human Subjects, Open Access Data

Spatiotemporal analysis of human intestinal development at single-cell resolution.

Fawkner-Corbett D; Antanaviciute A; Parikh K; Jagielowicz M; Gerós AS; Gupta T; Ashley N; Khamis D; Fowler D; Morrissey E et al

Cell 2021;184;3;810-826.e23

Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.

Peer reviewed
Topics
Human Subjects, Healthy Donors, Disease Donors, Experimental Methods

Building a high-quality Human Cell Atlas

Rozenblatt-Rosen, Orit; Shin, Jay W.; Rood, Jennifer E.; Hupalowska, Anna; Regev, Aviv; Heyn, Holger

Nature Biotechnology 2021;39;2;149-153

Peer reviewed
Topics
Human Subjects, Healthy Donors, Disease Donors, Open Access Data, Experimental Methods

Developmental cell programs are co-opted in inflammatory skin disease.

Reynolds G; Vegh P; Fletcher J; Poyner EFM; Stephenson E; Goh I; Botting RA; Huang N; Olabi B; Dubois A et al

Science 2021;371;6527

The skin confers biophysical and immunological protection through a complex cellular network established early in embryonic development. We profiled the transcriptomes of more than 500,000 single cells from developing human fetal skin, healthy adult skin, and adult skin with atopic dermatitis and psoriasis. We leveraged these datasets to compare cell states across development, homeostasis, and disease. Our analysis revealed an enrichment of innate immune cells in skin during the first trimester and clonal expansion of disease-associated lymphocytes in atopic dermatitis and psoriasis. We uncovered and validated in situ a reemergence of prenatal vascular endothelial cell and macrophage cellular programs in atopic dermatitis and psoriasis lesional skin. These data illustrate the dynamism of cutaneous immunity and provide opportunities for targeting pathological developmental programs in inflammatory skin diseases.

Press release

Peer reviewed
Networks
Organoid
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Disease Donors, Open Access Data, Experimental Methods, Computational Methods

The Organoid Cell Atlas.

Bock C; Boutros M; Camp JG; Clarke L; Clevers H; Knoblich JA; Liberali P; Regev A; Rios AC; Stegle O et al

Nature Biotechnology 2021;39;1;13-17

Peer reviewed
Networks
Skin
Topics
Human Subjects, Disease Donors

Single-cell RNA sequencing reveals markers of disease progression in primary cutaneous T-cell lymphoma.

Rindler K; Jonak C; Alkon N; Thaler FM; Kurz H; Shaw LE; Stingl G; Weninger W; Halbritter F; Bauer WM et al

Molecular cancer 2021;20;1;124

In early-stage mycosis fungoides (MF), the most common primary cutaneous T-cell lymphoma, limited skin involvement with patches and plaques is associated with a favorable prognosis. Nevertheless, approximately 20-30% of cases progress to tumors or erythroderma, resulting in poor outcome. At present, factors contributing to this switch from indolent to aggressive disease are only insufficiently understood.

Peer reviewed
Networks
Kidney
Topics
Human Subjects, Disease Donors

Single-cell protein activity analysis identifies recurrence-associated renal tumor macrophages.

Obradovic A; Chowdhury N; Haake SM; Ager C; Wang V; Vlahos L; Guo XV; Aggen DH; Rathmell WK; Jonasch E et al

Cell 2021;184;11;2988-3005.e16

Clear cell renal carcinoma (ccRCC) is a heterogeneous disease with a variable post-surgical course. To assemble a comprehensive ccRCC tumor microenvironment (TME) atlas, we performed single-cell RNA sequencing (scRNA-seq) of hematopoietic and non-hematopoietic subpopulations from tumor and tumor-adjacent tissue of treatment-naive ccRCC resections. We leveraged the VIPER algorithm to quantitate single-cell protein activity and validated this approach by comparison to flow cytometry. The analysis identified key TME subpopulations, as well as their master regulators and candidate cell-cell interactions, revealing clinically relevant populations, undetectable by gene-expression analysis. Specifically, we uncovered a tumor-specific macrophage subpopulation characterized by upregulation of TREM2/APOE/C1Q, validated by spatially resolved, quantitative multispectral immunofluorescence. In a large clinical validation cohort, these markers were significantly enriched in tumors from patients who recurred following surgery. The study thus identifies TREM2/APOE/C1Q-positive macrophage infiltration as a potential prognostic biomarker for ccRCC recurrence, as well as a candidate therapeutic target.

Peer reviewed
Topics
Human Subjects, Model Organism Samples, Healthy Donors, Open Access Data

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

Schupp JC; Adams TS; Cosme C; Raredon MSB; Yuan Y; Omote N; Poli S; Chioccioli M; Rose KA; Manning EP et al

Circulation 2021;144;4;286-302

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.

Peer reviewed
Networks
Gut, Immune

Single-cell analyses of Crohn's disease tissues reveal intestinal intraepithelial T cells heterogeneity and altered subset distributions.

Jaeger N; Gamini R; Cella M; Schettini JL; Bugatti M; Zhao S; Rosadini CV; Esaulova E; Di Luccia B; Kinnett B et al

Nature Communications 2021;12;1;1921

Crohn's disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Here, we profile, via multiple single-cell technologies, T cells purified from the intestinal epithelium and lamina propria (LP) from terminal ileum resections of adult severe CD cases. We find that intraepithelial lymphocytes (IEL) contain several unique T cell subsets, including NKp30+γδT cells expressing RORγt and producing IL-26 upon NKp30 engagement. Further analyses comparing tissues from non-inflamed and inflamed regions of patients with CD versus healthy controls show increased activated TH17 but decreased CD8+T, γδT, TFH and Treg cells in inflamed tissues. Similar analyses of LP find increased CD8+, as well as reduced CD4+T cells with an elevated TH17 over Treg/TFH ratio. Our analyses of CD tissues thus suggest a potential link, pending additional validations, between transmural inflammation, reduced IEL γδT cells and altered spatial distribution of IEL and LP T cell subsets.

Peer reviewed
Networks
Gut

A single-cell atlas of chromatin accessibility in the human genome.

Zhang K; Hocker JD; Miller M; Hou X; Chiou J; Poirion OB; Qiu Y; Li YE; Gaulton KJ; Wang A et al

Cell 2021;184;24;5985-6001.e19

Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of gene regulatory elements in diverse cell types and tissues in the human body, we applied single-cell chromatin accessibility assays to 30 adult human tissue types from multiple donors. We integrated these datasets with previous single-cell chromatin accessibility data from 15 fetal tissue types to reveal the status of open chromatin for ∼1.2 million candidate cis-regulatory elements (cCREs) in 222 distinct cell types comprised of >1.3 million nuclei. We used these chromatin accessibility maps to delineate cell-type-specificity of fetal and adult human cCREs and to systematically interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues, life stages, and organ systems.

Peer reviewed
Networks
Gut

Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps.

Chen B; Scurrah CR; McKinley ET; Simmons AJ; Ramirez-Solano MA; Zhu X; Markham NO; Heiser CN; Vega PN; Rolong A et al

Cell 2021;184;26;6262-6280.e26

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

Peer reviewed
Networks
Liver, Organoid

A human multi-lineage hepatic organoid model for liver fibrosis.

Guan Y; Enejder A; Wang M; Fang Z; Cui L; Chen SY; Wang J; Tan Y; Wu M; Chen X et al

Nature Communications 2021;12;1;6138

To investigate the pathogenesis of a congenital form of hepatic fibrosis, human hepatic organoids were engineered to express the most common causative mutation for Autosomal Recessive Polycystic Kidney Disease (ARPKD). Here we show that these hepatic organoids develop the key features of ARPKD liver pathology (abnormal bile ducts and fibrosis) in only 21 days. The ARPKD mutation increases collagen abundance and thick collagen fiber production in hepatic organoids, which mirrors ARPKD liver tissue pathology. Transcriptomic and other analyses indicate that the ARPKD mutation generates cholangiocytes with increased TGFβ pathway activation, which are actively involved stimulating myofibroblasts to form collagen fibers. There is also an expansion of collagen-producing myofibroblasts with markedly increased PDGFRB protein expression and an activated STAT3 signaling pathway. Moreover, the transcriptome of ARPKD organoid myofibroblasts resemble those present in commonly occurring forms of liver fibrosis. PDGFRB pathway involvement was confirmed by the anti-fibrotic effect observed when ARPKD organoids were treated with PDGFRB inhibitors. Besides providing insight into the pathogenesis of congenital (and possibly acquired) forms of liver fibrosis, ARPKD organoids could also be used to test the anti-fibrotic efficacy of potential anti-fibrotic therapies.

Peer reviewed
Networks
Development, Lung
Topics
Human Subjects, Healthy Donors, Disease Donors, Computational Methods, COVID-19

An interactive single cell web portal identifies gene and cell networks in COVID-19 host responses.

Jin K; Bardes EE; Mitelpunkt A; Wang JY; Bhatnagar S; Sengupta S; Krummel DP; Rothenberg ME; Aronow BJ

iScience 2021;24;10;103115

Numerous studies have provided single-cell transcriptome profiles of host responses to SARS-CoV-2 infection. Critically lacking however is a data mine that allows users to compare and explore cell profiles to gain insights and develop new hypotheses. To accomplish this, we harmonized datasets from COVID-19 and other control condition blood, bronchoalveolar lavage, and tissue samples, and derived a compendium of gene signature modules per cell type, subtype, clinical condition, and compartment. We demonstrate approaches to interacting with, exploring, and functional evaluating these modules via a new interactive web portal ToppCell (http://toppcell.cchmc.org/). As examples, we develop three hypotheses: (1) alternatively-differentiated monocyte-derived macrophages form a multicelllar signaling cascade that drives T cell recruitment and activation; (2) COVID-19-generated platelet subtypes exhibit dramatically altered potential to adhere, coagulate, and thrombose; and (3) extrafollicular B maturation is driven by a multilineage cell activation network that expresses an ensemble of genes strongly associated with risk for developing post-viral autoimmunity.

Peer reviewed
Topics
Human Subjects, Healthy Donors, Disease Donors

Human oral mucosa cell atlas reveals a stromal-neutrophil axis regulating tissue immunity.

Williams DW; Greenwell-Wild T; Brenchley L; Dutzan N; Overmiller A; Sawaya AP; Webb S; Martin D; ; Hajishengallis G et al

Cell 2021;184;15;4090-4104.e15

The oral mucosa remains an understudied barrier tissue. This is a site of rich exposure to antigens and commensals, and a tissue susceptible to one of the most prevalent human inflammatory diseases, periodontitis. To aid in understanding tissue-specific pathophysiology, we compile a single-cell transcriptome atlas of human oral mucosa in healthy individuals and patients with periodontitis. We uncover the complex cellular landscape of oral mucosal tissues and identify epithelial and stromal cell populations with inflammatory signatures that promote antimicrobial defenses and neutrophil recruitment. Our findings link exaggerated stromal cell responsiveness with enhanced neutrophil and leukocyte infiltration in periodontitis. Our work provides a resource characterizing the role of tissue stroma in regulating mucosal tissue homeostasis and disease pathogenesis.

Peer reviewed
Networks
Gut, Oral & Craniofacial

A single-cell atlas of human teeth.

Pagella P; de Vargas Roditi L; Stadlinger B; Moor AE; Mitsiadis TA

iScience 2021;24;5;102405

Teeth exert fundamental functions related to mastication and speech. Despite their great biomedical importance, an overall picture of their cellular and molecular composition is still missing. In this study, we have mapped the transcriptional landscape of the various cell populations that compose human teeth at single-cell resolution, and we analyzed in deeper detail their stem cell populations and their microenvironment. Our study identified great cellular heterogeneity in the dental pulp and the periodontium. Unexpectedly, we found that the molecular signatures of the stem cell populations were very similar, while their respective microenvironments strongly diverged. Our findings suggest that the microenvironmental specificity is a potential source for functional differences between highly similar stem cells located in the various tooth compartments and open new perspectives toward cell-based dental therapeutic approaches.

Peer reviewed
Networks
Liver, Organoid

Gene Regulatory Network Analysis and Engineering Directs Development and Vascularization of Multilineage Human Liver Organoids.

Velazquez JJ; LeGraw R; Moghadam F; Tan Y; Kilbourne J; Maggiore JC; Hislop J; Liu S; Cats D; Chuva de Sousa Lopes SM et al

Cell systems 2021;12;1;41-55.e11

Pluripotent stem cell (PSC)-derived organoids have emerged as novel multicellular models of human tissue development but display immature phenotypes, aberrant tissue fates, and a limited subset of cells. Here, we demonstrate that integrated analysis and engineering of gene regulatory networks (GRNs) in PSC-derived multilineage human liver organoids direct maturation and vascular morphogenesis in vitro. Overexpression of PROX1 and ATF5, combined with targeted CRISPR-based transcriptional activation of endogenous CYP3A4, reprograms tissue GRNs and improves native liver functions, such as FXR signaling, CYP3A4 enzymatic activity, and stromal cell reactivity. The engineered tissues possess superior liver identity when compared with other PSC-derived liver organoids and show the presence of hepatocyte, biliary, endothelial, and stellate-like cell populations in single-cell RNA-seq analysis. Finally, they show hepatic functions when studied in vivo. Collectively, our approach provides an experimental framework to direct organogenesis in vitro by systematically probing molecular pathways and transcriptional networks that promote tissue development.

Peer reviewed
Networks
Nervous system, Organoid

ELAVL4, splicing, and glutamatergic dysfunction precede neuron loss in MAPT mutation cerebral organoids.

Bowles KR; Silva MC; Whitney K; Bertucci T; Berlind JE; Lai JD; Garza JC; Boles NC; Mahali S; Strang KH et al

Cell 2021;184;17;4547-4563.e17

Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.

Peer reviewed
Networks
Liver, Organoid

Cholangiocyte organoids can repair bile ducts after transplantation in the human liver.

Sampaziotis F; Muraro D; Tysoe OC; Sawiak S; Beach TE; Godfrey EM; Upponi SS; Brevini T; Wesley BT; Garcia-Bernardo J et al

Science 2021;371;6531;839-846

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium.

Peer reviewed
Networks
Skin
Topics
Human Subjects

Myofibroblast transcriptome indicates SFRP2hi fibroblast progenitors in systemic sclerosis skin.

Tabib T; Huang M; Morse N; Papazoglou A; Behera R; Jia M; Bulik M; Monier DE; Benos PV; Chen W et al

Nature Communications 2021;12;1;4384

Skin and lung fibrosis in systemic sclerosis (SSc) is driven by myofibroblasts, alpha-smooth muscle actin expressing cells. The number of myofibroblasts in SSc skin correlates with the modified Rodnan skin score, the most widely used clinical measure of skin disease severity. Murine fibrosis models indicate that myofibroblasts can arise from a variety of different cell types, but their origin in SSc skin has remained uncertain. Utilizing single cell RNA-sequencing, we define different dermal fibroblast populations and transcriptome changes, comparing SSc to healthy dermal fibroblasts. Here, we show that SSc dermal myofibroblasts arise in two steps from an SFRP2hi/DPP4-expressing progenitor fibroblast population. In the first step, SSc fibroblasts show globally upregulated expression of transcriptome markers, such as PRSS23 and THBS1. A subset of these cells shows markers indicating that they are proliferating. Only a fraction of SFRP2hi SSc fibroblasts differentiate into myofibroblasts, as shown by expression of additional markers, SFRP4 and FNDC1. Bioinformatics analysis of the SSc fibroblast transcriptomes implicated upstream transcription factors, including FOSL2, RUNX1, STAT1, FOXP1, IRF7 and CREB3L1, as well as SMAD3, driving SSc myofibroblast differentiation.

Peer reviewed
Networks
Lung, Organoid

Differentiation of human pluripotent stem cells into functional airway basal stem cells.

Suzuki S; Hawkins FJ; Barillà C; Beermann ML; Kotton DN; Davis BR

STAR protocols 2021;2;3;100683

Airway basal cells play an essential role in the maintenance of the airway epithelium. Here, we provide a detailed directed differentiation protocol to generate ''induced basal cells (iBCs)'' from human pluripotent stem cells. iBCs recapitulate biological and functional properties of airway basal cells including mucociliary differentiation in vitro or in vivo in tracheal xenografts, facilitating the study of inherited and acquired diseases of the airway, as well as potential use in regenerative medicine. For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2021).

Peer reviewed
Networks
Eye

Gene regulatory networks controlling temporal patterning, neurogenesis, and cell-fate specification in mammalian retina.

Lyu P; Hoang T; Santiago CP; Thomas ED; Timms AE; Appel H; Gimmen M; Le N; Jiang L; Kim DW et al

Cell reports 2021;37;7;109994

Gene regulatory networks (GRNs), consisting of transcription factors and their target sites, control neurogenesis and cell-fate specification in the developing central nervous system. In this study, we use integrated single-cell RNA and single-cell ATAC sequencing (scATAC-seq) analysis in developing mouse and human retina to identify multiple interconnected, evolutionarily conserved GRNs composed of cell-type-specific transcription factors that both activate genes within their own network and inhibit genes in other networks. These GRNs control temporal patterning in primary progenitors, regulate transition from primary to neurogenic progenitors, and drive specification of each major retinal cell type. We confirm that NFI transcription factors selectively activate expression of genes promoting late-stage temporal identity in primary retinal progenitors and identify other transcription factors that regulate rod photoreceptor specification in postnatal retina. This study inventories cis- and trans-acting factors that control retinal development and can guide cell-based therapies aimed at replacing retinal neurons lost to disease.

Peer reviewed
Networks
Nervous system

Single-cell transcriptomics captures features of human midbrain development and dopamine neuron diversity in brain organoids.

Fiorenzano A; Sozzi E; Birtele M; Kajtez J; Giacomoni J; Nilsson F; Bruzelius A; Sharma Y; Zhang Y; Mattsson B et al

Nature Communications 2021;12;1;7302

Three-dimensional brain organoids have emerged as a valuable model system for studies of human brain development and pathology. Here we establish a midbrain organoid culture system to study the developmental trajectory from pluripotent stem cells to mature dopamine neurons. Using single cell RNA sequencing, we identify the presence of three molecularly distinct subtypes of human dopamine neurons with high similarity to those in developing and adult human midbrain. However, despite significant advancements in the field, the use of brain organoids can be limited by issues of reproducibility and incomplete maturation which was also observed in this study. We therefore designed bioengineered ventral midbrain organoids supported by recombinant spider-silk microfibers functionalized with full-length human laminin. We show that silk organoids reproduce key molecular aspects of dopamine neurogenesis and reduce inter-organoid variability in terms of cell type composition and dopamine neuron formation.

Peer reviewed
Networks
Immune, Lung

Airspace Macrophages and Monocytes Exist in Transcriptionally Distinct Subsets in Healthy Adults.

Mould KJ; Moore CM; McManus SA; McCubbrey AL; McClendon JD; Griesmer CL; Henson PM; Janssen WJ

American journal of respiratory and critical care medicine 2021;203;8;946-956

Rationale: Macrophages are the most abundant immune cell in the alveoli and small airways and are traditionally viewed as a homogeneous population during health. Whether distinct subsets of airspace macrophages are present in healthy humans is unknown. Single-cell RNA sequencing allows for examination of transcriptional heterogeneity between cells and between individuals. Understanding the conserved repertoire of airspace macrophages during health is essential to understanding cellular programing during disease.Objectives: We sought to determine the transcriptional heterogeneity of human cells obtained from BAL of healthy adults.Methods: Ten subjects underwent bronchoscopy with BAL. Cells from lavage were subjected to single-cell RNA sequencing. Unique cell populations and putative functions were identified. Transcriptional profiles were compared across individuals.Measurements and Main Results: We identify two novel subgroups of resident airspace macrophages-defined by proinflammatory and metallothionein gene expression profiles. We define subsets of monocyte-like cells and compare them with peripheral blood mononuclear cells. Finally, we compare global macrophage and monocyte programing between males and females.Conclusions: Healthy human airspaces contain multiple populations of myeloid cells that are highly conserved between individuals and between sexes. Resident macrophages make up the largest population and include novel subsets defined by inflammatory and metal-binding profiles. Monocyte-like cells within the airspaces are transcriptionally aligned with circulating blood cells and include a rare population defined by expression of cell-matrix interaction genes. This study is the first to delineate the conserved heterogeneity of airspace immune cells during health and identifies two previously unrecognized macrophage subsets.
Peer reviewed
Networks
Lung

Single-cell analysis of human non-small cell lung cancer lesions refines tumor classification and patient stratification.

Leader AM; Grout JA; Maier BB; Nabet BY; Park MD; Tabachnikova A; Chang C; Walker L; Lansky A; Le Berichel J et al

Cancer cell 2021;39;12;1594-1609.e12

Immunotherapy is a mainstay of non-small cell lung cancer (NSCLC) management. While tumor mutational burden (TMB) correlates with response to immunotherapy, little is known about the relationship between the baseline immune response and tumor genotype. Using single-cell RNA sequencing, we profiled 361,929 cells from 35 early-stage NSCLC lesions. We identified a cellular module consisting of PDCD1+CXCL13+ activated T cells, IgG+ plasma cells, and SPP1+ macrophages, referred to as the lung cancer activation module (LCAMhi). We confirmed LCAMhi enrichment in multiple NSCLC cohorts, and paired CITE-seq established an antibody panel to identify LCAMhi lesions. LCAM presence was found to be independent of overall immune cell content and correlated with TMB, cancer testis antigens, and TP53 mutations. High baseline LCAM scores correlated with enhanced NSCLC response to immunotherapy even in patients with above median TMB, suggesting that immune cell composition, while correlated with TMB, may be a nonredundant biomarker of response to immunotherapy.

Peer reviewed
Networks
Immune, Lung
Topics
COVID-19

Immune signatures underlying post-acute COVID-19 lung sequelae.

Cheon IS; Li C; Son YM; Goplen NP; Wu Y; Cassmann T; Wang Z; Wei X; Tang J; Li Y et al

Science immunology 2021;6;65;eabk1741

Severe coronavirus disease 2019 (COVID-19) pneumonia survivors often exhibit long-term pulmonary sequelae, but the underlying mechanisms or associated local and systemic immune correlates are not known. Here, we have performed high-dimensional characterization of the pathophysiological and immune traits of aged COVID-19 convalescents, and correlated the local and systemic immune profiles with pulmonary function and lung imaging. We found that chronic lung impairment was accompanied by persistent respiratory immune alterations. We showed that functional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific memory T and B cells were enriched at the site of infection compared with those of blood. Detailed evaluation of the lung immune compartment revealed that dysregulated respiratory CD8+ T cell responses were associated with the impaired lung function after acute COVID-19. Single-cell transcriptomic analysis identified the potential pathogenic subsets of respiratory CD8+ T cells contributing to persistent tissue conditions after COVID-19. Our results have revealed pathophysiological and immune traits that may support the development of lung sequelae after SARS-CoV-2 pneumonia in older individuals, with implications for the treatment of chronic COVID-19 symptoms.

Peer reviewed
Topics
Open Access Data, Computational Methods

scConsensus: combining supervised and unsupervised clustering for cell type identification in single-cell RNA sequencing data.

Ranjan B; Schmidt F; Sun W; Park J; Honardoost MA; Tan J; Arul Rayan N; Prabhakar S

BMC bioinformatics 2021;22;1;186

Clustering is a crucial step in the analysis of single-cell data. Clusters identified in an unsupervised manner are typically annotated to cell types based on differentially expressed genes. In contrast, supervised methods use a reference panel of labelled transcriptomes to guide both clustering and cell type identification. Supervised and unsupervised clustering approaches have their distinct advantages and limitations. Therefore, they can lead to different but often complementary clustering results. Hence, a consensus approach leveraging the merits of both clustering paradigms could result in a more accurate clustering and a more precise cell type annotation.

Peer reviewed
Networks
Liver, Organoid

Generation of functional ciliated cholangiocytes from human pluripotent stem cells.

Ogawa M; Jiang JX; Xia S; Yang D; Ding A; Laselva O; Hernandez M; Cui C; Higuchi Y; Suemizu H et al

Nature Communications 2021;12;1;6504

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) provides a model for studying the pathogenesis of cholangiopathies and for developing therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of cystic fibrosis transmembrane conductance regulator (CFTR) and the presence of primary cilia capable of sensing flow. With this level of maturation, these cholangiocytes are amenable for testing the efficacy of cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies show that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice indicating that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease.

Peer reviewed
Networks
Gut, Organoid

Induced organoids derived from patients with ulcerative colitis recapitulate colitic reactivity.

Sarvestani SK; Signs S; Hu B; Yeu Y; Feng H; Ni Y; Hill DR; Fisher RC; Ferrandon S; DeHaan RK et al

Nature Communications 2021;12;1;262

The pathogenesis of ulcerative colitis (UC), a major type of inflammatory bowel disease, remains unknown. No model exists that adequately recapitulates the complexity of clinical UC. Here, we take advantage of induced pluripotent stem cells (iPSCs) to develop an induced human UC-derived organoid (iHUCO) model and compared it with the induced human normal organoid model (iHNO). Notably, iHUCOs recapitulated histological and functional features of primary colitic tissues, including the absence of acidic mucus secretion and aberrant adherens junctions in the epithelial barrier both in vitro and in vivo. We demonstrate that the CXCL8/CXCR1 axis was overexpressed in iHUCO but not in iHNO. As proof-of-principle, we show that inhibition of CXCL8 receptor by the small-molecule non-competitive inhibitor repertaxin attenuated the progression of UC phenotypes in vitro and in vivo. This patient-derived organoid model, containing both epithelial and stromal compartments, will generate new insights into the underlying pathogenesis of UC while offering opportunities to tailor interventions to the individual patient.

Peer reviewed
Networks
Lung

Deciphering cell lineage specification of human lung adenocarcinoma with single-cell RNA sequencing.

Wang Z; Li Z; Zhou K; Wang C; Jiang L; Zhang L; Yang Y; Luo W; Qiao W; Wang G et al

Nature Communications 2021;12;1;6500

Lung adenocarcinomas (LUAD) arise from precancerous lesions such as atypical adenomatous hyperplasia, which progress into adenocarcinoma in situ and minimally invasive adenocarcinoma, then finally into invasive adenocarcinoma. The cellular heterogeneity and molecular events underlying this stepwise progression remain unclear. In this study, we perform single-cell RNA sequencing of 268,471 cells collected from 25 patients in four histologic stages of LUAD and compare them to normal cell types. We detect a group of cells closely resembling alveolar type 2 cells (AT2) that emerged during atypical adenomatous hyperplasia and whose transcriptional profile began to diverge from that of AT2 cells as LUAD progressed, taking on feature characteristic of stem-like cells. We identify genes related to energy metabolism and ribosome synthesis that are upregulated in early stages of LUAD and may promote progression. MDK and TIMP1 could be potential biomarkers for understanding LUAD pathogenesis. Our work shed light on the underlying transcriptional signatures of distinct histologic stages of LUAD progression and our findings may facilitate early diagnosis.

Peer reviewed
Networks
Gut, Organoid

Charting human development using a multi-endodermal organ atlas and organoid models.

Yu Q; Kilik U; Holloway EM; Tsai YH; Harmel C; Wu A; Wu JH; Czerwinski M; Childs CJ; He Z et al

Cell 2021;184;12;3281-3298.e22

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.

Peer reviewed
Networks
Gut, Immune

Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution.

Busslinger GA; Weusten BLA; Bogte A; Begthel H; Brosens LAA; Clevers H

Cell reports 2021;34;10;108819

The upper gastrointestinal tract, consisting of the esophagus, stomach, and duodenum, controls food transport, digestion, nutrient uptake, and hormone production. By single-cell analysis of healthy epithelia of these human organs, we molecularly define their distinct cell types. We identify a quiescent COL17A1high KRT15high stem/progenitor cell population in the most basal cell layer of the esophagus and detect substantial gene expression differences between identical cell types of the human and mouse stomach. Selective expression of BEST4, CFTR, guanylin, and uroguanylin identifies a rare duodenal cell type, referred to as BCHE cell, which likely mediates high-volume fluid secretion because of continual activation of the CFTR channel by guanylin/uroguanylin-mediated autocrine signaling. Serotonin-producing enterochromaffin cells in the antral stomach significantly differ in gene expression from duodenal enterochromaffin cells. We, furthermore, discover that the histamine-producing enterochromaffin-like cells in the oxyntic stomach express the luteinizing hormone, yet another member of the enteroendocrine hormone family.

Peer reviewed

Integrative analysis of cell state changes in lung fibrosis with peripheral protein biomarkers.

Mayr CH; Simon LM; Leuschner G; Ansari M; Schniering J; Geyer PE; Angelidis I; Strunz M; Singh P; Kneidinger N et al

EMBO molecular medicine 2021;13;4;e12871

The correspondence of cell state changes in diseased organs to peripheral protein signatures is currently unknown. Here, we generated and integrated single-cell transcriptomic and proteomic data from multiple large pulmonary fibrosis patient cohorts. Integration of 233,638 single-cell transcriptomes (n = 61) across three independent cohorts enabled us to derive shifts in cell type proportions and a robust core set of genes altered in lung fibrosis for 45 cell types. Mass spectrometry analysis of lung lavage fluid (n = 124) and plasma (n = 141) proteomes identified distinct protein signatures correlated with diagnosis, lung function, and injury status. A novel SSTR2+ pericyte state correlated with disease severity and was reflected in lavage fluid by increased levels of the complement regulatory factor CFHR1. We further discovered CRTAC1 as a biomarker of alveolar type-2 epithelial cell health status in lavage fluid and plasma. Using cross-modal analysis and machine learning, we identified the cellular source of biomarkers and demonstrated that information transfer between modalities correctly predicts disease status, suggesting feasibility of clinical cell state monitoring through longitudinal sampling of body fluid proteomes.

Pre-print
Topics
Human Subjects, Healthy Donors, Disease Donors, Experimental Methods, Computational Methods, COVID-19

Systemic Tissue and Cellular Disruption from SARS-CoV-2 Infection revealed in COVID-19 Autopsies and Spatial Omics Tissue Maps.

Park J; Foox J; Hether T; Danko D; Warren S; Kim Y; Reeves J; Butler DJ; Mozsary C; Rosiene J et al

bioRxiv 2021

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has infected over 115 million people and caused over 2.5 million deaths worldwide. Yet, the molecular mechanisms underlying the clinical manifestations of COVID-19, as well as what distinguishes them from common seasonal influenza virus and other lung injury states such as Acute Respiratory Distress Syndrome (ARDS), remains poorly understood. To address these challenges, we combined transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues, matched with spatial protein and expression profiling (GeoMx) across 357 tissue sections. These results define both body-wide and tissue-specific (heart, liver, lung, kidney, and lymph nodes) damage wrought by the SARS-CoV-2 infection, evident as a function of varying viral load (high vs. low) during the course of infection and specific, transcriptional dysregulation in splicing isoforms, T cell receptor expression, and cellular expression states. In particular, cardiac and lung tissues revealed the largest degree of splicing isoform switching and cell expression state loss. Overall, these findings reveal a systemic disruption of cellular and transcriptional pathways from COVID-19 across all tissues, which can inform subsequent studies to combat the mortality of COVID-19, as well to better understand the molecular dynamics of lethal SARS-CoV-2 infection and other viruses.

Peer reviewed
Networks
Gut

Mitogen-activated protein kinase activity drives cell trajectories in colorectal cancer.

Uhlitz F; Bischoff P; Peidli S; Sieber A; Trinks A; Lüthen M; Obermayer B; Blanc E; Ruchiy Y; Sell T et al

EMBO molecular medicine 2021;13;10;e14123

In colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue lacking visible organization. We sought to define transcriptional states of colorectal cancer cells and signals controlling their development by performing single-cell transcriptome analysis of tumors and matched non-cancerous tissues of twelve colorectal cancer patients. We defined patient-overarching colorectal cancer cell clusters characterized by differential activities of oncogenic signaling pathways such as mitogen-activated protein kinase and oncogenic traits such as replication stress. RNA metabolic labeling and assessment of RNA velocity in patient-derived organoids revealed developmental trajectories of colorectal cancer cells organized along a mitogen-activated protein kinase activity gradient. This was in contrast to normal colon organoid cells developing along graded Wnt activity. Experimental targeting of EGFR-BRAF-MEK in cancer organoids affected signaling and gene expression contingent on predictive KRAS/BRAF mutations and induced cell plasticity overriding default developmental trajectories. Our results highlight directional cancer cell development as a driver of non-genetic cancer cell heterogeneity and re-routing of trajectories as a response to targeted therapy.

Peer reviewed
Networks
Gut, Immune, Liver

A single-cell atlas of liver metastases of colorectal cancer reveals reprogramming of the tumor microenvironment in response to preoperative chemotherapy.

Che LH; Liu JW; Huo JP; Luo R; Xu RM; He C; Li YQ; Zhou AJ; Huang P; Chen YY et al

Cell discovery 2021;7;1;80

Metastasis is the primary cause of cancer-related mortality in colorectal cancer (CRC) patients. How to improve therapeutic options for patients with metastatic CRC is the core question for CRC treatment. However, the complexity and diversity of stromal context of the tumor microenvironment (TME) in liver metastases of CRC have not been fully understood, and the influence of stromal cells on response to chemotherapy is unclear. Here we performed an in-depth analysis of the transcriptional landscape of primary CRC, matched liver metastases and blood at single-cell resolution, and a systematic examination of transcriptional changes and phenotypic alterations of the TME in response to preoperative chemotherapy (PC). Based on 111,292 single-cell transcriptomes, our study reveals that TME of treatment-naïve tumors is characterized by the higher abundance of less-activated B cells and higher heterogeneity of tumor-associated macrophages (TAMs). By contrast, in tumors treated with PC, we found activation of B cells, lower diversity of TAMs with immature and less activated phenotype, lower abundance of both dysfunctional T cells and ECM-remodeling cancer-associated fibroblasts, and an accumulation of myofibroblasts. Our study provides a foundation for future investigation of the cellular mechanisms underlying liver metastasis of CRC and its response to PC, and opens up new possibilities for the development of therapeutic strategies for CRC.

Pre-print
Networks
Lung, Organoid
Topics
COVID-19

Self-organized stem cell-derived human lung buds with proximo-distal patterning and novel targets of SARS-CoV-2.

Rosado-Olivieri EA; Razooky B; Hoffmann HH; De Santis R; Rice CM; Brivanlou AH

bioRxiv 2021

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic and the lack of therapeutics hinders pandemic control1-2. Although lung disease is the primary clinical outcome in COVID-19 patients1-3, how SARS-CoV-2 induces tissue pathology in the lung remains elusive. Here we describe a high-throughput platform to generate tens of thousands of self-organizing, nearly identical, and genetically matched human lung buds derived from human pluripotent stem cells (hPSCs) cultured on micropatterned substrates. Strikingly, in vitro-derived human lung buds resemble fetal human lung tissue and display in vivo-like proximo-distal coordination of alveolar and airway tissue differentiation whose 3D epithelial self-organization is directed by the levels of KGF. Single-cell transcriptomics unveiled the cellular identities of airway and alveolar tissue and the differentiation of WNThi cycling alveolar stem cells, a human-specific lung cell type4. These synthetic human lung buds are susceptible to infection by SARS-CoV-2 and endemic coronaviruses and can be used to track cell type-dependent susceptibilities to infection, intercellular transmission and cytopathology in airway and alveolar tissue in individual lung buds. Interestingly, we detected an increased susceptibility to infection in alveolar cells and identified cycling alveolar stem cells as targets of SARS-CoV-2. We used this platform to test neutralizing antibodies isolated from convalescent plasma that efficiently blocked SARS-CoV-2 in