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

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 The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

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

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.

Single-cell roadmap of human gonadal development.

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.

Mapping the developing human immune system across organs.

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.

MultiMAP: dimensionality reduction and integration of multimodal data.

Multimodal data is rapidly growing in many fields of science and engineering, including single-cell biology. We introduce MultiMAP, a novel algorithm for dimensionality reduction and integration. MultiMAP can integrate any number of datasets, leverages features not present in all datasets, is not restricted to a linear mapping, allows the user to specify the influence of each dataset, and is extremely scalable to large datasets. We apply MultiMAP to single-cell transcriptomics, chromatin accessibility, methylation, and spatial data and show that it outperforms current approaches. On a new thymus dataset, we use MultiMAP to integrate cells along a temporal trajectory. This enables quantitative comparison of transcription factor expression and binding site accessibility over the course of T cell differentiation, revealing patterns of expression versus binding site opening kinetics.

Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2.

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNß or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

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

Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11-12 weeks after conception1,2, yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6-7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21).

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

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Human anogenital monocyte-derived dendritic cells and langerin+cDC2 are major HIV target cells.

Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).

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

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 (CD16+C1QA/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.

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

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.

A cell atlas of human thymic development defines T cell repertoire formation.

The thymus provides a nurturing environment for the differentiation and selection of T cells, a process orchestrated by their interaction with multiple thymic cell types. We used single-cell RNA sequencing to create a cell census of the human thymus across the life span and to reconstruct T cell differentiation trajectories and T cell receptor (TCR) recombination kinetics. Using this approach, we identified and located in situ CD8αα+ T cell populations, thymic fibroblast subtypes, and activated dendritic cell states. In addition, we reveal a bias in TCR recombination and selection, which is attributed to genomic position and the kinetics of lineage commitment. Taken together, our data provide a comprehensive atlas of the human thymus across the life span with new insights into human T cell development.

Spatiotemporal immune zonation of the human kidney.

Tissue-resident immune cells are important for organ homeostasis and defense. The epithelium may contribute to these functions directly or by cross-talk with immune cells. We used single-cell RNA sequencing to resolve the spatiotemporal immune topology of the human kidney. We reveal anatomically defined expression patterns of immune genes within the epithelial compartment, with antimicrobial peptide transcripts evident in pelvic epithelium in the mature, but not fetal, kidney. A network of tissue-resident myeloid and lymphoid immune cells was evident in both fetal and mature kidney, with postnatal acquisition of transcriptional programs that promote infection-defense capabilities. Epithelial-immune cross-talk orchestrated localization of antibacterial macrophages and neutrophils to the regions of the kidney most susceptible to infection. Overall, our study provides a global overview of how the immune landscape of the human kidney is zonated to counter the dominant immunological challenge.

Decoding human fetal liver haematopoiesis.

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.

Identification of HIV transmitting CD11c+ human epidermal dendritic cells.

Langerhans cells (LC) are thought to be the only mononuclear phagocyte population in the epidermis where they detect pathogens. Here, we show that CD11c+ dendritic cells (DCs) are also present. These cells are transcriptionally similar to dermal cDC2 but are more efficient antigen-presenting cells. Compared to LCs, epidermal CD11c+ DCs are enriched in anogenital tissues where they preferentially interact with HIV, express the higher levels of HIV entry receptor CCR5, support the higher levels of HIV uptake and replication and are more efficient at transmitting the virus to CD4 T cells. Importantly, these findings are observed using both a lab-adapted and transmitted/founder strain of HIV. We also describe a CD33low cell population, which is transcriptionally similar to LCs but does not appear to function as antigen-presenting cells or acts as HIV target cells. Our findings reveal that epidermal DCs in anogenital tissues potentially play a key role in sexual transmission of HIV.

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

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

Single-cell transcriptomes from human kidneys reveal the cellular identity of renal tumors.

Messenger RNA encodes cellular function and phenotype. In the context of human cancer, it defines the identities of malignant cells and the diversity of tumor tissue. We studied 72,501 single-cell transcriptomes of human renal tumors and normal tissue from fetal, pediatric, and adult kidneys. We matched childhood Wilms tumor with specific fetal cell types, thus providing evidence for the hypothesis that Wilms tumor cells are aberrant fetal cells. In adult renal cell carcinoma, we identified a canonical cancer transcriptome that matched a little-known subtype of proximal convoluted tubular cell. Analyses of the tumor composition defined cancer-associated normal cells and delineated a complex vascular endothelial growth factor (VEGF) signaling circuit. Our findings reveal the precise cellular identities and compositions of human kidney tumors.

Mechanism of Interferon-Stimulated Gene Induction in HIV-1-Infected Macrophages.

Viruses manipulate the complex interferon and interferon-stimulated gene (ISG) system in different ways. We have previously shown that HIV inhibits type I and III interferons in its key target cells but directly stimulates a subset of >20 ISGs in macrophages and dendritic cells, many of which are antiviral. Here, we examine the mechanism of induction of ISGs and show this occurs in two phases. The first phase was transient (0 to 24 h postinfection [hpi]), induced mainly by extracellular vesicles and one of its component proteins, HSP90α, contained within the HIV inoculum. The second, dominant, and persistent phase (>48 hpi) was induced via newly transcribed HIV RNA and sensed via RIGI, as shown by the reduction in ISG expression after the knockdown of the RIGI adaptor, MAVS, by small interfering RNA (siRNA) and the inhibition of both the initiation and elongation of HIV transcription by short hairpin RNA (shRNA) transcriptional silencing. We further define the induction pathway, showing sequential HIV RNA stimulation via Tat, RIGI, MAVS, IRF1, and IRF7, also identified by siRNA knockdown. IRF1 also plays a key role in the first phase. We also show that the ISGs IFIT1 to -3 inhibit HIV production, measured as extracellular infectious virus. All induced antiviral ISGs probably lead to restriction of HIV replication in macrophages, contributing to a persistent, noncytopathic infection, while the inhibition of interferon facilitates spread to adjacent cells. Both may influence the size of macrophage HIV reservoirs in vivo Elucidating the mechanisms of ISG induction may help in devising immunotherapeutic strategies to limit the size of these reservoirs.IMPORTANCE HIV, like other viruses, manipulates the antiviral interferon and interferon-stimulated gene (ISG) system to facilitate its initial infection and establishment of viral reservoirs. HIV specifically inhibits all type I and III interferons in its target cells, including macrophages, dendritic cells, and T cells. It also induces a subset of over 20 ISGs of differing compositions in each cell target. This occurs in two temporal phases in macrophages. Extracellular vesicles contained within the inoculum induce the first, transient phase of ISGs. Newly transcribed HIV RNA induce the second, dominant ISG phase, and here, the full induction pathway is defined. Therefore, HIV nucleic acids, which are potent inducers of interferon and ISGs, are initially concealed, and antiviral ISGs are not fully induced until replication is well established. These antiviral ISGs may contribute to persistent infection in macrophages and to the establishment of viral reservoirs in vivo.

Phenotypic and functional consequences of different isolation protocols on skin mononuclear phagocytes.

Mononuclear phagocytes are present in skin and mucosa and represent one of the first lines of defense against invading pathogens, which they detect via an array of pathogen-binding receptors expressed on their surface. However, their extraction from tissue is difficult, and the isolation technique used has functional consequences on the cells obtained. Here, we compare mononuclear phagocytes isolated from human skin using either enzymatic digestion or spontaneous migration. Cells isolated via enzymatic digestion are in an immature state, and all subsets are easily defined. However, cells isolated by spontaneous migration are in a mature state, and CD141 cross-presenting DCs (cDC1) are more difficult to define. Different pathogen-binding receptors are susceptible to cleavage by blends of collagenase, demonstrating that great care must be taken in choosing the correct enzyme blend to digest tissue if carrying out pathogen-interaction assays. Finally, we have optimized mononuclear phagocyte culture conditions to enhance their survival after liberation from the tissue.

Langerhans cells and sexual transmission of HIV and HSV.

Langerhans cells (LCs) situated in stratified squamous epithelium of the skin and mucosal tissue are amongst the first cells that sexually transmitted pathogens encounter during transmission. They are potent antigen presenting cells and play a key role in the host mounting an appropriate immune response. As such, viruses have evolved complex strategies to manipulate these cells to facilitate successful transmission. One of best studied examples is HIV, which manipulates the natural function of these cells to interact with CD4 T cells, which are the main target cell for HIV in which rapid replication occurs. However, there is controversy in the literature as to the role that LCs play in this process. Langerhans cells also play a key role in the way the body mounts an immune response to HSV, and there is also a complex interplay between the transmission of HSV and HIV that involves LCs. In this article, we review both past and present literatures with a particular focus on a few very recent studies that shed new light on the role that LCs play in the transmission and immune response to these 2 pathogens.