Oral and Craniofacial Networks

Oral and Craniofacial Networks - Overview

Neutrophils are the most abundant white blood cells in the human body responsible for fighting viral, bacterial and fungi infections. Out of the 100 billion neutrophils produced daily, it is estimated that 10 % of these cells end up in oral biofluids. Because saliva is a fluid accessible through non-invasive techniques, it is an optimal source of cells and molecule surveillance in health and disease. While neutrophils are abundant in saliva, scientific advancements in neutrophil biology have been hampered likely due to their short life span, inability to divide once terminally differentiated, sensitivity to physical stress, and low RNA content. Here, we devise a protocol aiming to understand neutrophil heterogeneity by improving isolation methods, single-cell RNA extraction, sequencing and bioinformatic pipelines. Advanced flow cytometry 3D analysis, and machine learning validated our gating system model, by including positive neutrophil markers and excluding other immune cells and uncovered neutrophil heterogeneity. Considering specific cell markers, unique mitochondrial content, stringent and less stringent filtering strategies, our transcriptome single cell findings unraveled novel neutrophil subpopulations. Collectively, this methodology accelerates the discovery of salivary immune landscapes, with the promise of improving the understanding of diversification mechanisms, clinical diagnostics in health and disease, and guide targeted therapies.

A Protocol for Revealing Oral Neutrophil Heterogeneity by Single-Cell Immune Profiling in Human Saliva

Rationale: The respiratory tract constitutes an elaborated line of defense that is based on a unique cellular ecosystem. Single-cell profiling methods enable the investigation of cell population distributions and transcriptional changes along the airways.Methods: We have explored the cellular heterogeneity of the human airway epithelium in 10 healthy living volunteers by single-cell RNA profiling. 77,969 cells were collected at 35 distinct locations, from the nose to the 12th division of the airway tree.Results: The resulting atlas is composed of a high percentage of epithelial cells (89.1%), but also immune (6.2%) and stromal (4.7%) cells with distinct cellular proportions in different regions of the airways. It reveals differential gene expression between identical cell types (suprabasal, secretory, and multiciliated cells) from the nose (MUC4, PI3, SIX3) and tracheobronchial (SCGB1A1, TFF3) airways. By contrast, cell-type specific gene expression is stable across all tracheobronchial samples. Our atlas improves the description of ionocytes, pulmonary neuro-endocrine (PNEC) and brush cells, and identifies a related population of NREP-positive cells. We also report the association of KRT13 with dividing cells that are reminiscent of previously described mouse “hillock” cells, and with squamous cells expressing SCEL, SPRR1A/B.Conclusions: Robust characterization of a single-cell cohort in healthy airways establishes a valuable resource for future investigations. The precise description of the continuum existing
from the nasal epithelium to successive divisions of the airways and the stable gene expression profile of these regions better defines conditions under which relevant tracheobronchial proxies of human respiratory diseases can be developed.

A Single-Cell Atlas of the Human Healthy Airways

Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (T<sub>H</sub>2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a T<sub>H</sub>2-dominated interactome in asthmatic lungs.

A cellular census of human lungs identifies novel cell states in health and in asthma

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.

A single cell atlas of human teeth

Barrier tissue dysfunction is a fundamental feature of chronic human inflammatory diseases. Specialized subsets of epithelial cells-including secretory and ciliated cells-differentiate from basal stem cells to collectively protect the upper airway. Allergic inflammation can develop from persistent activation of type 2 immunity in the upper airway, resulting in chronic rhinosinusitis, which ranges in severity from rhinitis to severe nasal polyps. Basal cell hyperplasia is a hallmark of severe disease, but it is not known how these progenitor cells contribute to clinical presentation and barrier tissue dysfunction in humans. Here we profile primary human surgical chronic rhinosinusitis samples (18,036 cells, n = 12) that span the disease spectrum using Seq-Well for massively parallel single-cell RNA sequencing, report transcriptomes for human respiratory epithelial, immune and stromal cell types and subsets from a type 2 inflammatory disease, and map key mediators. By comparison with nasal scrapings (18,704 cells, n = 9), we define signatures of core, healthy, inflamed and polyp secretory cells. We reveal marked differences between the epithelial compartments of the non-polyp and polyp cellular ecosystems, identifying and validating a global reduction in cellular diversity of polyps characterized by basal cell hyperplasia, concomitant decreases in glandular cells, and phenotypic shifts in secretory cell antimicrobial expression. We detect an aberrant basal progenitor differentiation trajectory in polyps, and propose cell-intrinsic, epigenetic and extrinsic factors that lock polyp basal cells into this uncommitted state. Finally, we functionally demonstrate that ex vivo cultured basal cells retain intrinsic memory of IL-4/IL-13 exposure, and test the potential for clinical blockade of the IL-4 receptor α-subunit to modify basal and secretory cell states in vivo. Overall, we find that reduced epithelial diversity stemming from functional shifts in basal cells is a key characteristic of type 2 immune-mediated barrier tissue dysfunction. Our results demonstrate that epithelial stem cells may contribute to the persistence of human disease by serving as repositories for allergic memories.

Allergic inflammatory memory in human respiratory epithelial progenitor cells

The adult human brain likely comprises more than a thousand kinds of neurons, and an unknown number of glial cell types, but how cellular diversity arises during early brain development is not known. Here, in order to reveal the precise sequence of events during early brain development, we used single-cell RNA sequencing and spatial transcriptomics to uncover cell states and trajectories in human brains at 5 – 14 post-conceptional weeks (p.c.w.). We identified twelve major classes and over 600 distinct cell states, which mapped to precise spatial anatomical domains at 5 p.c.w. We uncovered detailed differentiation trajectories of the human forebrain, and a surprisingly large number of region-specific glioblasts maturing into distinct pre-astrocytes and pre-oligodendrocyte precursor cells (pre-OPCs). Our findings reveal the emergence of cell types during the critical first trimester of human brain development.

Comprehensive cell atlas of the first-trimester developing human brain

Human oral soft tissues provide the first barrier of defence against chronic inflammatory disease and hold a remarkable scarless wounding phenotype. Tissue homeostasis requires coordinated actions of epithelial, mesenchymal, and immune cells. However, the extent of heterogeneity within the human oral mucosa and how tissue cell types are affected during the course of disease progression is unknown. Using single-cell transcriptome profiling we reveal a striking remodelling of the epithelial and mesenchymal niches with a decrease in functional populations that are linked to the aetiology of the disease. Analysis of ligand-receptor interaction pairs identify potential intercellular hubs driving the inflammatory component of the disease. Our work establishes a reference map of the human oral mucosa in health and disease, and a framework for the development of new therapeutic strategies.

Defining human mesenchymal and epithelial heterogeneity in response to oral inflammatory disease

Injuries that heal by fibrosis can compromise organ function and increase patient morbidity. The oral mucosal barrier has a high regenerative capacity with minimal scarring, but the cellular mechanisms remain elusive. Here, we identify distinct postnatal paired-related homeobox-1+ (Prx1+) cells as a critical fibroblast subpopulation that expedites mucosal healing by facilitating early immune response. Using transplantation and genetic ablation model in mice, we show that oral mucosa enriched with Prx1+ cells heals faster than those that lack Prx1+ cells. Lineage tracing and scRNA-seq reveal that Prx1+ fibroblasts exhibit progenitor signatures in physiologic and injured conditions. Mechanistically, Prx1+ progenitors accelerate wound healing by differentiating into immunomodulatory SCA1+ fibroblasts, which prime macrophage recruitment through CCL2 as a key part of pro-wound healing response. Furthermore, human Prx1+ fibroblasts share similar gene and spatial profiles compared to their murine counterpart. Thus, our data suggest that Prx1+ fibroblasts may provide a valuable source in regenerative procedures for the treatment of corneal wounds and enteropathic fibrosis.

Distinct fibroblast progenitor subpopulation expedites regenerative mucosal healing by immunomodulation

Head and neck squamous cell carcinoma (HNSCC) arises through exposure to environmental carcinogens or malignant transformation by human papillomavirus (HPV). Here, we assessed the transcriptional profiles of 131,224 single cells from peripheral and intra-tumoral CD45+ immune populations from HPV– and HPV+ HNSCC and healthy donors. A spectrum of transcriptional signatures in immune lineages ranging from similar to highly divergent was present in the tumor microenvironments (TME) in HPV– and HPV+ HNSCC. CD8+ and regulatory CD4+ T cells were similar in HPV– and HPV+ HNSCC, compared to significant differences between helper CD4+ T cells, B cells and myeloid cells. Further dissection of the major immune lineages identified unique cell states and differentiation trajectories. We contextualized our results by performing multispectral immunofluorescence and evaluating putative cell-cell communication based on spatial proximity, and found longer progression free survival in patients with enrichment of a CD4+ T follicular helper cell signature. Overall design: Total of 63 samples were analyzed by scRNAseq. Of these, 26 samples were paired peripheral blood mononuclear cells and tumor infiltrating immune cells from HNSCC patients (18 HPV- and 8 HPV+), 6 were peripheral blood mononuclear cells from healthy donors, and 5 were tissue resident immune cells from healthy donor tonsils.

Immune landscape of viral- and carcinogen-drived head and neck cancer

The germinal centre (GC) response is critical for both effective adaptive immune responses and establishing peripheral tolerance by limiting auto-reactive B cells. To understand the gene regulatory principles underlying B cell maturation and the GC response we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied, representative lymphoid tissue. We identify diverse immune cell subsets to help us map dynamic transcription factor activity during B cell activation, GC formation and plasma cell differentiation. We subsequently leverage cell type-specific transcriptomic and epigenomic maps to interpret the regulatory potential of fine-mapped autoimmune genetic variants. This reveals that many autoimmune disease-associated variants exhibit the greatest regulatory potential in GC-associated immune cell populations, providing a valuable cell-type resolved resource to understanding cellular and genetic causes underpinning autoimmune disease. Overall design: Tonsil samples were collected from children and adults undergoing routine tonsillectomy. Tonsillar immune cells were loaded on to the 10X Genomics Chromium according to the manufacturer’s protocol using either the single-cell 3' kit (vX) or the single-cell ATAC kit (v1). Library preparation for both assays was performed according to the manufacturer’s protocol prior to sequencing on either the Illumina NovaSeq 6000 or NextSeq 500 platforms.

Integrated single-cell transcriptomics and epigenomics reveals strong germinal center–associated etiology of autoimmune risk loci

Single-cell RNA-sequencing of the nasal mucosa across the lifespan Overall design: Single-cell RNA-sequencing of the nasal mucosa across the lifespan

Lifespan charactarization of the nasal mucosa

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes subdivided into ILC1s, ILC2s and ILC3s based on core regulatory programs and signature cytokines secreted. ILCs exhibit functional plasticity. Using flow cytometry, mass cytometry and scRNAseq, here we found that ILC3s and ILC1s occupy opposite ends of a spectrum including discrete subsets in human tonsils. The human intestine also exhibited an ILC3–ILC1 transitional population. Overall design: ILCs were sorted from human tonsils and human intestine and analysed by scRNA-seq and bulk RNA-seq. The sc-RNAseq sequencing data is available upon reasonable request to the authors.

RNA-Seq from human innate lymphoid cells (ILCs) transitional populations.

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.

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

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) which causes the disease COVID-19. SARS-CoV-2 spike (S)-protein binds ACE2, and in concert with host proteases, principally TMPRSS2, promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues, and the factors that regulate ACE2 expression, remain unknown. Here, we leverage human and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 amongst tissue-resident cell subsets. Overall design: Samples from 8 datasets are included: single-cell RNA-Seq studies: 2 human adult donors of inferior turbinate scrapings with two replicates each. Four mouse donors of nasal epithelial cells, two stimulated with IFNA and two controls with two replicates from each animal. 13 human ileal small intestine donors. 8 human lung tissue donors with mTB infection, mTB infection and HIV, or no mTB infection. Bulk RNA-seq samples: Two human donors stimulated with three replicates of each stimulation with IFNA, IFNG, IL17A, or IL4, each at increasing doses (0, 0.1 ,0.5, 1, 2, 5, 10 ng/mL) and 12 replicates of unstimulated samples. One mouse donor of cells from the trachea with three replicates of each stimulation with IFNA, IFNB, or IFNG, each at increasing doses (0, 0.1 ,0.5, 1, 2, 5, 10 ng/mL) and 12 replicates of unstimulated samples. One human cell line stimulated with three replicates of each stimulation with IFNA, IFNG, IL17A, or IL4, each at increasing doses (0, 0.1 ,0.5, 1, 2, 5, 10 ng/mL) and 12 replicates of unstimulated samples.

SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues.

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.

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

Innate lymphoid cells (ILCs) are increasingly appreciated as important participants in homeostasis and inflammation. Substantial plasticity and heterogeneity among ILC populations have been reported. Here we have delineated the heterogeneity of human ILCs through single-cell RNA sequencing of several hundreds of individual tonsil CD127+ ILCs and natural killer (NK) cells. Unbiased transcriptional clustering revealed four distinct populations, corresponding to ILC1 cells, ILC2 cells, ILC3 cells and NK cells, with their respective transcriptomes recapitulating known as well as unknown transcriptional profiles. The single-cell resolution additionally divulged three transcriptionally and functionally diverse subpopulations of ILC3 cells. Our systematic comparison of single-cell transcriptional variation within and between ILC populations provides new insight into ILC biology during homeostasis, with additional implications for dysregulation of the immune system.

Single cell RNA-sequencing of human tonsil Innate lymphoid cells (ILCs)

We present a collection of single-cell transcriptomic profiles of 6,810 pulpal cells isolated from a sound human maxillary third molars and carious teeth at different stages. We showed that the presence of deep, but not enamel caries, altered the immune cell compositions of the dental pulp. Differential expression analysis further revealed that the pro-inflammatory, anti-inflammatory and mineralization-related genes were upregulated in immune and stromal cells in deep dental caries. Cell-cell interaction prediction showed potential interactions between immune and stromal cells during homeostasis, and enhanced interactions between different cell types with macrophage during deep dental caries. Taken together, our study serves as a comprehensive survey of human pulpal cell heterogeneity, as well as provides novel molecular insights into dental pulps in health and disease. Overall design: The single-cell transcriptomic profiles from cells residing in dental pulp from a sound tooth, enamel caries and deep dental caries were compared. Cell type frequency and differential gene expression were analyzed. Cell-cell interactions were predicted from the expression level of ligands and receptors.

Single-Cell Transcriptomic Profiling of Human Dental Pulp in Sound and Carious Teeth: A Pilot Study

Understanding on pathogenesis of COVID-19 is rapidly growing, but primary target cells of SARS-CoV-2 infection is still not known. Here, we performed single cell RNA sequencing on human nasal swab from healthy donors to investigate the expression patterns of host cell entry factors of SARS-CoV-2. Overall design: Single cells from healthy donors' nasal swabs were isolated and single-cell transcriptome libraries were generated

Single-cell RNA sequencing of human nasal swab from healthy donors

Protective humoral memory forms in secondary lymphoid organs where B cells undergo affinity maturation and differentiation into memory or plasma cells. Here, we provide a comprehensive roadmap of human B cell maturation with single-cell transcriptomics matched with bulk and single-cell antibody repertoires to define gene expression, antibody repertoires, and clonal sharing of B cell states at single-cell resolution, including memory B cell heterogeneity that reflects diverse functional and signaling states. We reconstruct gene expression dynamics during B cell activation to reveal a pre-germinal center state primed to undergo class switch recombination and dissect how antibody class-dependent gene expression in germinal center and memory B cells is linked with a distinct transcriptional wiring with potential to influence their fate and function. Our analyses reveal the dynamic cellular states that shape human B cell-mediated immunity and highlight how antibody isotype may play a role during their antibody-based selection.

Single-cell analysis of human B cell maturation predicts how antibody class switching shapes selection dynamics

The oral mucosa remains an understudied barrier tissue rich in exposure to antigens, commensals and pathogens. Moreover, it is the tissue where one of the most prevalent human microbe-triggered inflammatory diseases, periodontitis, occurs. To understand this complex environment at the cellular level, we assemble herein a human single-cell transcriptome atlas of oral mucosal tissues in health and periodontitis. Our work reveals transcriptional diversity of stromal and immune cell populations, predicts intercellular communication and uncovers an altered immune responsiveness of stromal cells participating in tissue homeostasis and disease at the gingival mucosa. In health, we define unique populations of CXCL1,2,8- expressing epithelial cells and fibroblasts mediating immune homeostasis primarily through the recruitment of neutrophils. In disease, we further observe stromal, particularly fibroblast hyper-responsiveness linked to recruitment of leukocytes and neutrophil populations. Our work suggests a stromal-neutrophil axis as a key regulator of mucosal immunity. Pursuant to these findings, most Mendelian forms of periodontitis were shown to be linked to genetic mutations in neutrophil-expressed genes. Moreover, we document previously unappreciated expression of known pattern- and damage- recognition receptors on stromal cell populations in the setting of periodontitis, suggesting avenues for triggering of stromal responsiveness. This comprehensive atlas offers an important reference for in-depth understanding of oral mucosal homeostasis and inflammation and reveals unique stromal–immune interactions implicated in tissue immunity. Overall design: Examination of human oral mucosa including healthy and diseased gingival mucosa and healthy buccal mucosa.

Single-cell atlas of human oral mucosa reveals a stromal-neutrophil axis in tissue immunity regulation

Chronic apical periodontitis (CAP) is a unique dynamic interaction between microbial invasions and host defense mechanisms, resulting in bone absorption, infiltration of immune cells and sporadic periapical granuloma. In this study, we constituted a single-cell atlas for 26,737 high-quality cells from hyperplastic periapical tissue using single-cell RNA sequencing. Identifying cell types and signatures at the single-cell level might generate novel insights into the clinical pathogenesis of CAP. A histological analysis to verify the gene signatures of nonimmune cells was combined with immunohistochemistry staining. We then discovered the diversity and heterogeneity of nonimmune cells in regional CAP lesions. The temporal profiling of genomic alterations from common CAP to typical periapical granuloma provided predictions for key transcription factors and biological processes. Our study also inferred that the marked shift of inflammatory cytokines, chemokines, proteases and growth factors enables the initiation of polymorphic cell differentiation, lymphangiogenesis and angiogenesis during CAP. Overall design: Three samples from extracted residual molars with chronic apical periodontitis

Single-cell sequencing unveils the heterogeneity of nonimmune cells in chronic apical periodontitis

Oral squamous cell carcinoma of the gingivo-buccal region (OSCC-GB) is the most common cancer among men in India, and is associated with poor prognosis and frequent recurrence. Cellular heterogeneity in OSCC-GB was investigated by single-cell RNA sequencing of tumors derived from the oral cavity of 12 OSCC-GB patients, 3 of whom had concomitant presence of a precancerous lesion (oral submucous fibrosis [OSMF]). Unique malignant cell types, features, and phenotypic shifts in the stromal cell population were identified in oral tumors with associated submucous fibrosis. Expression levels of FOS, ATP1A, and DUSP1 provided robust discrimination between tumors with or without the concomitant presence of OSMF. Malignant cell populations shared between tumors with and without OSMF were enriched with the expression of partial epithelial-mesenchymal transition (pEMT) or fetal cell type signatures indicative of two dominant cellular programs in OSCC-GB-pEMT and fetal cellular reprogramming. Malignant cells exhibiting fetal cellular and pEMT programs were enriched with the expression of immune-related pathway genes known to be involved in antitumor immune response. In the tumor microenvironment, higher infiltration of immune cells than the stromal cells was observed. The T cell population was large in tumors and diverse subtypes of T cells with varying levels of infiltration were found. We also detected double-negative PLCG2+ T cells and cells with intermediate M1-M2 macrophage polarization. Our findings shed light on unique aspects of cellular heterogeneity and cell states in OSCC-GB.

Single-cell transcriptomic analysis of gingivo-buccal oral cancer reveals two dominant cellular programs

We have created a compendium of single cell transcriptome data from the model
organism Mus musculus comprising more than 100,000 cells from 20 organs and
tissues. These data represent a new resource for cell biology, revealing gene
expression in poorly characterized cell populations and allowing for direct and
controlled comparison of gene expression in cell types shared between tissues, such
as T-lymphocytes and endothelial cells from distinct anatomical locations. Two
distinct technical approaches were used for most tissues: one approach, microfluidic
droplet-based 3’-end counting, enabled the survey of thousands of cells at relatively
low coverage, while the other, FACS-based full length transcript analysis, enabled
characterization of cell types with high sensitivity and coverage. The cumulative
data provide the foundation for an atlas of transcriptomic cell biology.

Tabula Muris: Transcriptomic characterization of 20 organs and tissues from Mus musculus at single cell resolution

In recent years there has been tremendous progress towards deep molecular characterization of cell types using single cell transcriptome sequencing. Here we report a single cell transcriptomic atlas comprising nearly 500,000 cells from 24 different human tissues and organs. In several instances multiple organs were analyzed from the same donor. Analyzing organs from the same individual controls for genetic background, age, environment, and epigenetic effects, and enables a detailed comparison of cell types that are shared between tissues. This resource provides a rich molecular characterization of more than 400 cell types, their distribution across tissues, and detailed information about tissue specific variation in gene expression. We have used the fact that multiple tissues came from the same donor to study the clonal distribution of T cells between tissues, to understand the tissue specific mutation rate in B cells, and to analyze the cell cycle state and proliferative potential of shared cell types across tissues. Finally, we have also used this data to characterize cell type specific RNA splicing and how such splicing varies across tissues within an individual.

The Tabula Sapiens: a single cell transcriptomic atlas of multiple organs from individual human donors

While a substantial proportion of adults infected with SARS-CoV-2 progress to develop severe disease, children rarely manifest respiratory complications. Therefore, understanding differences in the local and systemic response to SARS-CoV-2 infection between children and adults may provide important clues about the pathogenesis of SARS-CoV-2 infection. To address this, we first generated a healthy reference multi-omics single cell data set from children (n=30) in whom we have profiled triple matched samples: nasal and tracheal brushings and PBMCs, where we track the developmental changes for 42 airway and 31 blood cell populations from infancy, through childhood to adolescence. This has revealed the presence of naive B and T lymphocytes in neonates and infants with a unique gene expression signature bearing hallmarks of innate immunity. We then contrast the healthy reference with equivalent data from severe paediatric and adult COVID-19 patients (total n=27), from the same three types of samples: upper and lower airways and blood. We found striking differences: children with COVID-19 as opposed to adults had a higher proportion of innate lymphoid and non-clonally expanded naive T cells in peripheral blood, and a limited interferon-response signature. In the airway epithelium, we found the highest viral load in goblet and ciliated cells and describe a novel inflammatory epithelial cell population. These cells represent a transitional regenerative state between secretory and ciliated cells; they were found in healthy children and were enriched in paediatric and adult COVID-19 patients. Epithelial cells display an antiviral and neutrophil-recruiting gene signature that is weaker in severe paediatric versus adult COVID-19. Our matched blood and airway samples allowed us to study the spatial dynamics of infection. Lastly, we provide a user-friendly interface for this data 1 as a highly granular reference for the study of immune responses in airways and blood in children.

The local and systemic response to SARS-CoV-2 infection in children and adults

Rhabdomyosarcoma (RMS) is the most soft tissue sarcoma of childhood and histologically mimics developing skeletal muscle. To better understand RMS, we performed single-cell RNA-seq of one embryonal subtype RMS (ERMS) and one alveolar subtype RMS (ARMS). We expanded our observations by performing single-nucleus RNA-seq of 18 patient tumors (12 ERMS, 6 ARMS). All 18 of these tumors were used to generate orthotopic patient-derived xenografts, and we performed single-cell RNA-seq of all 18 O-PDXs to compare heterogeneity between the original tumor and the patient-derived model. 15 of the O-PDXs underwent lentiviral barcoding, and lineage could be tracked using a separate dial-out PCR step during the single-cell RNA-seq workflow. We performed single-cell RNA-seq of an ex vivo organoid, showing that we can preserve heterogeneity in this model. We expanded our study to the epigenetic level by using single-cell ATAC-sequencing of 7 O-PDXs. Finally, we longitudinally tracked shifts in heterogeneity in an O-PDX model treated with chemotherapy. Overall design: We performed single-cell RNA-seq from 2 patient RMS tumors, 18 O-PDXs, and 1 organoid. Additionally, we performed single-nucleus RNA-seq from 18 patient RMS tumors and single-cell ATAC-seq from 7 O-PDXs. We also provide dialout PCR data from 15 RMS O-PDX that were used for lentiviral barcode experiments to track clonal lineages within O-PDXs.

Oral and Craniofacial Networks

Network Description

Oral and Craniofacial Network atlases are still under development.

Network Coordinators