Showing papers on "Cellular differentiation published in 2018"

Microglia and macrophages in brain homeostasis and disease

Abstract: Microglia and non-parenchymal macrophages in the brain are mononuclear phagocytes that are increasingly recognized to be essential players in the development, homeostasis and diseases of the central nervous system. With the availability of new genetic, molecular and pharmacological tools, considerable advances have been made towards our understanding of the embryonic origins, developmental programmes and functions of these cells. These exciting discoveries, some of which are still controversial, also raise many new questions, which makes brain macrophage biology a fast-growing field at the intersection of neuroscience and immunology. Here, we review the current knowledge of how and where brain macrophages are generated, with a focus on parenchymal microglia. We also discuss their normal functions during development and homeostasis, the disturbance of which may lead to various neurodegenerative and neuropsychiatric diseases.

Advanced maturation of human cardiac tissue grown from pluripotent stem cells

Abstract: Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) can serve as platforms for patient-specific studies of physiology and disease1–6. However, the predictive power of these models is presently limited by the immature state of the cells1,2,5,6. Here we show that this fundamental limitation can be overcome if cardiac tissues are formed from early-stage iPSC-derived cardiomyocytes soon after the initiation of spontaneous contractions and are subjected to physical conditioning with increasing intensity over time. After only four weeks of culture, for all iPSC lines studied, such tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiological sarcomere length (2.2 µm) and density of mitochondria (30%), the presence of transverse tubules, oxidative metabolism, a positive force–frequency relationship and functional calcium handling. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiological responses to isoproterenol and recapitulating pathological hypertrophy, supporting the utility of this tissue model for studies of cardiac development and disease. A tissue culture system that provides an increasing intensity of electromechanical stimulation over time enables an in vitro model of cardiac tissue derived from human induced pluripotent stem cells to develop many of the characteristics of adult cardiac tissue.

Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease

Abstract: Our understanding of kidney disease pathogenesis is limited by an incomplete molecular characterization of the cell types responsible for the organ’s multiple homeostatic functions. To help fill this knowledge gap, we characterized 57,979 cells from healthy mouse kidneys by using unbiased single-cell RNA sequencing. On the basis of gene expression patterns, we infer that inherited kidney diseases that arise from distinct genetic mutations but share the same phenotypic manifestation originate from the same differentiated cell type. We also found that the collecting duct in kidneys of adult mice generates a spectrum of cell types through a newly identified transitional cell. Computational cell trajectory analysis and in vivo lineage tracing revealed that intercalated cells and principal cells undergo transitions mediated by the Notch signaling pathway. In mouse and human kidney disease, these transitions were shifted toward a principal cell fate and were associated with metabolic acidosis.

A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte

Abstract: The functions of epithelial tissues are dictated by the types, abundance and distribution of the differentiated cells they contain. Attempts to restore tissue function after damage require knowledge of how physiological tasks are distributed among cell types, and how cell states vary between homeostasis, injury–repair and disease. In the conducting airway, a heterogeneous basal cell population gives rise to specialized luminal cells that perform mucociliary clearance1. Here we perform single-cell profiling of human bronchial epithelial cells and mouse tracheal epithelial cells to obtain a comprehensive census of cell types in the conducting airway and their behaviour in homeostasis and regeneration. Our analysis reveals cell states that represent known and novel cell populations, delineates their heterogeneity and identifies distinct differentiation trajectories during homeostasis and tissue repair. Finally, we identified a novel, rare cell type that we call the ‘pulmonary ionocyte’, which co-expresses FOXI1, multiple subunits of the vacuolar-type H+-ATPase (V-ATPase) and CFTR, the gene that is mutated in cystic fibrosis. Using immunofluorescence, modulation of signalling pathways and electrophysiology, we show that Notch signalling is necessary and FOXI1 expression is sufficient to drive the production of the pulmonary ionocyte, and that the pulmonary ionocyte is a major source of CFTR activity in the conducting airway epithelium. Single-cell RNA sequencing analysis is used to identify cell types in the tracheal epithelium, including previously unidentified ionocytes, which express high levels of the cystic fibrosis transmembrane conductance regulator, CFTR.

Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis

Abstract: The quantity of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is a robust prognostic factor for improved patient survival, particularly in triple-negative and HER2-overexpressing BC subtypes1. Although T cells are the predominant TIL population2, the relationship between quantitative and qualitative differences in T cell subpopulations and patient prognosis remains unknown. We performed single-cell RNA sequencing (scRNA-seq) of 6,311 T cells isolated from human BCs and show that significant heterogeneity exists in the infiltrating T cell population. We demonstrate that BCs with a high number of TILs contained CD8+ T cells with features of tissue-resident memory T (TRM) cell differentiation and that these CD8+ TRM cells expressed high levels of immune checkpoint molecules and effector proteins. A CD8+ TRM gene signature developed from the scRNA-seq data was significantly associated with improved patient survival in early-stage triple-negative breast cancer (TNBC) and provided better prognostication than CD8 expression alone. Our data suggest that CD8+ TRM cells contribute to BC immunosurveillance and are the key targets of modulation by immune checkpoint inhibition. Further understanding of the development, maintenance and regulation of TRM cells will be crucial for successful immunotherapeutic development in BC.

Single-cell mapping of gene expression landscapes and lineage in the zebrafish embryo

Abstract: High-throughput mapping of cellular differentiation hierarchies from single-cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single-cell RNA sequencing to >92,000 cells from zebrafish embryos during the first day of development. Using a graph-based approach, we mapped a cell-state landscape that describes axis patterning, germ layer formation, and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon-based barcoding approach (TracerSeq) for reconstructing single-cell lineage histories. Clonally related cells were often restricted by the state landscape, including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in embryos lacking the chordin gene. We provide web-based resources for further analysis of the single-cell data.

Natural Killer Cells: Development, Maturation, and Clinical Utilization

Abstract: Natural killer cells are the predominant innate lymphocyte subsets that mediate anti-tumor and anti-viral responses, and therefore possess promising clinical utilization. NK cells do not express polymorphic clonotypic receptors and utilize inhibitory receptors (KIR and Ly49) to develop, mature, and recognize ‘self’ from ‘non-self’. The essential roles of common gamma cytokines such as IL-2, IL-7, and IL-15 in the commitment and development of NK cells are well-established. However, the critical functions of proinflammatory cytokines IL-12, IL-18, IL-27, and IL-35 in the transcriptional-priming of NK cells are only starting to emerge. Recent studies have highlighted multiple shared characteristics between NK cells the adaptive immune lymphocytes. NK cells utilize unique signaling pathways that offer exclusive ways to genetically manipulate to improve their effector functions. Here, we summarize the recent advances made in the understanding of how NK cells develop, mature, and their potential translational use in the clinic.

Breast cancer development and progression: Risk factors, cancer stem cells, signaling pathways, genomics, and molecular pathogenesis.

Abstract: As the most commonly occurring cancer in women worldwide, breast cancer poses a formidable public health challenge on a global scale. Breast cancer consists of a group of biologically and molecularly heterogeneous diseases originated from the breast. While the risk factors associated with this cancer varies with respect to other cancers, genetic predisposition, most notably mutations in BRCA1 or BRCA2 gene, is an important causative factor for this malignancy. Breast cancers can begin in different areas of the breast, such as the ducts, the lobules, or the tissue in between. Within the large group of diverse breast carcinomas, there are various denoted types of breast cancer based on their invasiveness relative to the primary tumor sites. It is important to distinguish between the various subtypes because they have different prognoses and treatment implications. As there are remarkable parallels between normal development and breast cancer progression at the molecular level, it has been postulated that breast cancer may be derived from mammary cancer stem cells. Normal breast development and mammary stem cells are regulated by several signaling pathways, such as estrogen receptors (ERs), HER2, and Wnt/β-catenin signaling pathways, which control stem cell proliferation, cell death, cell differentiation, and cell motility. Furthermore, emerging evidence indicates that epigenetic regulations and noncoding RNAs may play important roles in breast cancer development and may contribute to the heterogeneity and metastatic aspects of breast cancer, especially for triple-negative breast cancer. This review provides a comprehensive survey of the molecular, cellular and genetic aspects of breast cancer.

Cancer stem cells as key drivers of tumour progression

Abstract: Cancer stem cells (CSCs) are subpopulations of cancer cells sharing similar characteristics as normal stem or progenitor cells such as self-renewal ability and multi-lineage differentiation to drive tumour growth and heterogeneity. Throughout the cancer progression, CSC can further be induced from differentiated cancer cells via the adaptation and cross-talks with the tumour microenvironment as well as a response from therapeutic pressures, therefore contributes to their heterogeneous phenotypes. Challengingly, conventional cancer treatments target the bulk of the tumour and are unable to target CSCs due to their highly resistance nature, leading to metastasis and tumour recurrence. This review highlights the roles of CSCs in tumour initiation, progression and metastasis with a focus on the cellular and molecular regulators that influence their phenotypical changes and behaviours in the different stages of cancer progression. We delineate the cross-talks between CSCs with the tumour microenvironment that support their intrinsic properties including survival, stemness, quiescence and their cellular and molecular adaptation in response to therapeutic pressure. An insight into the distinct roles of CSCs in promoting angiogenesis and metastasis has been captured based on in vitro and in vivo evidences. Given dynamic cellular events along the cancer progression and contributions of resistance nature by CSCs, understanding their molecular and cellular regulatory mechanism in a heterogeneous nature, provides significant cornerstone for the development of CSC-specific therapeutics.

SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation.

Abstract: Some exciting biological questions require quantifying thousands of proteins in single cells. To achieve this goal, we develop Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) and validate its ability to identify distinct human cancer cell types based on their proteomes. We use SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem cells. The single-cell proteomes enable us to deconstruct cell populations and infer protein abundance relationships. Comparison between single-cell proteomes and transcriptomes indicates coordinated mRNA and protein covariation, yet many genes exhibit functionally concerted and distinct regulatory patterns at the mRNA and the protein level.

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells

Abstract: Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

m 6 A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer

Abstract: N6-methyladenosine (m6A) messenger RNA methylation is a gene regulatory mechanism affecting cell differentiation and proliferation in development and cancer. To study the roles of m6A mRNA methylation in cell proliferation and tumorigenicity, we investigated human endometrial cancer in which a hotspot R298P mutation is present in a key component of the methyltransferase complex (METTL14). We found that about 70% of endometrial tumours exhibit reductions in m6A methylation that are probably due to either this METTL14 mutation or reduced expression of METTL3, another component of the methyltransferase complex. These changes lead to increased proliferation and tumorigenicity of endometrial cancer cells, likely through activation of the AKT pathway. Reductions in m6A methylation lead to decreased expression of the negative AKT regulator PHLPP2 and increased expression of the positive AKT regulator mTORC2. Together, these results reveal reduced m6A mRNA methylation as an oncogenic mechanism in endometrial cancer and identify m6A methylation as a regulator of AKT signalling.

scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells.

Abstract: Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

Wnt signaling in development and tissue homeostasis.

Abstract: The Wnt-β-catenin signaling pathway is an evolutionarily conserved cell-cell communication system that is important for stem cell renewal, cell proliferation and cell differentiation both during embryogenesis and during adult tissue homeostasis. Genetic or epigenetic events leading to hypo- or hyper-activation of the Wnt-β-catenin signaling cascade have also been associated with human diseases such as cancer. Understanding how this pathway functions is thus integral for developing therapies to treat diseases or for regenerative medicine approaches. Here, and in the accompanying poster, we provide an overview of Wnt-β-catenin signaling and briefly highlight its key functions during development and adult tissue homeostasis.

The dynamics of gene expression in vertebrate embryogenesis at single-cell resolution

Abstract: Time series of single-cell transcriptome measurements can reveal dynamic features of cell differentiation pathways. From measurements of whole frog embryos spanning zygotic genome activation through early organogenesis, we derived a detailed catalog of cell states in vertebrate development and a map of differentiation across all lineages over time. The inferred map recapitulates most if not all developmental relationships and associates new regulators and marker genes with each cell state. We find that many embryonic cell states appear earlier than previously appreciated. We also assess conflicting models of neural crest development. Incorporating a matched time series of zebrafish development from a companion paper, we reveal conserved and divergent features of vertebrate early developmental gene expression programs.

The Balance of Th17 versus Treg Cells in Autoimmunity.

Abstract: T helper type 17 (Th17) cells and pTreg cells, which share a common precursor cell (the naive CD4 T cell), require a common tumor growth factor (TGF)-β signal for initial differentiation. However, terminally differentiated cells fulfill opposite functions: Th17 cells cause autoimmunity and inflammation, whereas Treg cells inhibit these phenomena and maintain immune homeostasis. Thus, unraveling the mechanisms that affect the Th17/Treg cell balance is critical if we are to better understand autoimmunity and tolerance. Recent studies have identified many factors that influence this balance; these factors range from signaling pathways triggered by T cell receptors, costimulatory receptors, and cytokines, to various metabolic pathways and the intestinal microbiota. This review article summarizes recent advances in our understanding of the Th17/Treg balance and its implications with respect to autoimmune disease.

Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart

Abstract: Fibroblasts are a dynamic cell type that achieve selective differentiated states to mediate acute wound healing and long-term tissue remodeling with scarring. With myocardial infarction injury, cardiomyocytes are replaced by secreted extracellular matrix proteins produced by proliferating and differentiating fibroblasts. Here, we employed 3 different mouse lineage-tracing models and stage-specific gene profiling to phenotypically analyze and classify resident cardiac fibroblast dynamics during myocardial infarction injury and stable scar formation. Fibroblasts were activated and highly proliferative, reaching a maximum rate within 2 to 4 days after infarction injury, at which point they expanded 3.5-fold and were maintained long term. By 3 to 7 days, these cells differentiated into myofibroblasts that secreted abundant extracellular matrix proteins and expressed smooth muscle α-actin to structurally support the necrotic area. By 7 to 10 days, myofibroblasts lost proliferative ability and smooth muscle α-actin expression as the collagen-containing extracellular matrix and scar fully matured. However, these same lineage-traced initial fibroblasts persisted within the scar, achieving a new molecular and stable differentiated state referred to as a matrifibrocyte, which was also observed in the scars of human hearts. These cells express common and unique extracellular matrix and tendon genes that are more specialized to support the mature scar.

Stem cell fate in cancer growth, progression and therapy resistance

Abstract: Although we have come a long way in our understanding of the signals that drive cancer growth, and how these signals can be targeted, effective control of this disease remains a key scientific and medical challenge. The therapy resistance and relapse that are commonly seen are driven in large part by the inherent heterogeneity within cancers that allows drugs to effectively eliminate some, but not all, malignant cells. Here, we focus on the fundamental drivers of this heterogeneity by examining emerging evidence that shows that these traits are often controlled by the disruption of normal cell fate and aberrant adoption of stem cell signals. We discuss how undifferentiated cells are preferentially primed for transformation and often serve as the cell of origin for cancers. We also consider evidence showing that activation of stem cell programmes in cancers can lead to progression, therapy resistance and metastatic growth and that targeting these attributes may enable better control over a difficult disease.

Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics

Abstract: INTRODUCTION Understanding the differentiation of stem cells into the vast amount of cell types that form the human body is a central problem of basic and medical science. The recent advances in single-cell sequencing techniques now make it possible to capture the transcriptomes of thousands of cells in a fast and cost-effective manner, opening a new way to study the cell composition of organs, tissues, and developmental stages. Yet, single-cell transcriptomics per se just provides a snapshot of cellular dynamics and transient cell populations. Computational algorithms have emerged that infer a pseudotemporal ordering of cells based on comparison of their transcriptomic profiles, allowing new insights into stem cell biology and tissue differentiation. However, these algorithms were designed for relatively simple scenarios, such as the differentiation of cells belonging to a specific lineage or the lineage relationships among cells from a particular tissue, and cannot evaluate all possible cellular differentiation trajectories in complex animals. To this end, we use single-cell transcriptomic approaches to improve our molecular understanding of how stem cells differentiate into the set of cell types that make an entire complex adult animal. RATIONALE Freshwater planarians such as Schmidtea mediterranea offer a unique opportunity to approach this question. These animals are immortal and constantly renew and regenerate all tissues owing to the presence of a large pool of pluripotent stem cells that continuously differentiate into all mature cell types. Therefore, we reasoned that an unbiased single-cell transcriptomic approach should allow us to capture not only terminally differentiated cell types but also intermediate cellular states, possibly enabling cell lineage reconstruction of the whole animal from transcriptomic data. RESULTS We performed massively parallel single-cell transcriptomics profiling of thousands of cells from adult planarians. At the molecular level, we identified and characterized dozens of cell types, including stem cells, progenitors, and terminally differentiated cells. We then applied a new computational algorithm, partition-based graph abstraction (PAGA), which can predict a lineage tree for the whole animal in an unbiased way. By combining the predictions from PAGA with several independent lines of evidence, including single-cell transcriptome data from purified stem cells and stem cell–depleted animals, analysis of gene expression dynamics, and a method called velocyto that predicts future gene expression from mRNA metabolism, we produced a consolidated lineage tree that included all identified cell types rooted to a single stem cell group. We used this information to identify gene sets co-regulated during the differentiation of many specific cell types. To show the power of our approach, we applied single-cell transcriptomics to regenerating planarians and characterized how each cell type in the adult planarian body dynamically responds to regenerative body remodeling at the transcriptomic and cellular levels. Our results highlight that some cell types that had been previously overlooked in molecular studies quickly decrease their abundance, indicating that they may serve as an energy reservoir that fuels the regeneration process. CONCLUSION We have shown that it is possible to use single-cell transcriptomics to (i) build a cell atlas of an adult animal, (ii) reconstruct the lineage relationships of its cells in an unbiased way, and (iii) identify gene sets which likely contain genes that are involved in programming the lineage tree. Moreover, we demonstrated how single-cell transcriptomics can be used to study complex and dynamic cellular processes such as regeneration. Notably, our approach is applicable to other model and non–model organisms, assuming that their differentiation processes are sampled with sufficient time resolution. To foster future studies, we provide a detailed tutorial on the application of our approach, and we make our data available through an interactive web interface. This study opens the door to powerful approaches for understanding molecular mechanisms of development and regeneration in animals.

Subepithelial telocytes are an important source of Wnts that supports intestinal crypts

Abstract: Tissues that undergo rapid cellular turnover, such as the mammalian haematopoietic system or the intestinal epithelium, are dependent on stem and progenitor cells that proliferate to provide differentiated cells to maintain organismal health. Stem and progenitor cells, in turn, are thought to rely on signals and growth factors provided by local niche cells to support their function and self-renewal. Several cell types have been hypothesized to provide the signals required for the proliferation and differentiation of the intestinal stem cells in intestinal crypts1-6. Here we identify subepithelial telocytes as an important source of Wnt proteins, without which intestinal stem cells cannot proliferate and support epithelial renewal. Telocytes are large but rare mesenchymal cells that are marked by expression of FOXL1 and form a subepithelial plexus that extends from the stomach to the colon. While supporting the entire epithelium, FOXL1+ telocytes compartmentalize the production of Wnt ligands and inhibitors to enable localized pathway activation. Conditional genetic ablation of porcupine (Porcn), which is required for functional maturation of all Wnt proteins, in mouse FOXL1+ telocytes causes rapid cessation of Wnt signalling to intestinal crypts, followed by loss of proliferation of stem and transit amplifying cells and impaired epithelial renewal. Thus, FOXL1+ telocytes are an important source of niche signals to intestinal stem cells.

The adult human testis transcriptional cell atlas

Abstract: Human adult spermatogenesis balances spermatogonial stem cell (SSC) self-renewal and differentiation, alongside complex germ cell-niche interactions, to ensure long-term fertility and faithful genome propagation. Here, we performed single-cell RNA sequencing of ~6500 testicular cells from young adults. We found five niche/somatic cell types (Leydig, myoid, Sertoli, endothelial, macrophage), and observed germline-niche interactions and key human-mouse differences. Spermatogenesis, including meiosis, was reconstructed computationally, revealing sequential coding, non-coding, and repeat-element transcriptional signatures. Interestingly, we identified five discrete transcriptional/developmental spermatogonial states, including a novel early SSC state, termed State 0. Epigenetic features and nascent transcription analyses suggested developmental plasticity within spermatogonial States. To understand the origin of State 0, we profiled testicular cells from infants, and identified distinct similarities between adult State 0 and infant SSCs. Overall, our datasets describe key transcriptional and epigenetic signatures of the normal adult human testis, and provide new insights into germ cell developmental transitions and plasticity.

Cell type transcriptome atlas for the planarian Schmidtea mediterranea.

Abstract: INTRODUCTION The complete sequence of animal genomes has had a transformative impact on biological research. Whereas the genome sequence of an organism contains the information for its development and physiology, the transcriptomes (the sets of actively transcribed genes) of the cell types in an organism define how the genome is used for the unique functions of its cells. Cell number and complexity have historically made the identification of all cell types, much less their transcriptomes, an extreme challenge for most multicellular organisms. Recent advances in single-cell RNA sequencing (SCS) have greatly enhanced the ability to determine cell type transcriptomes, with SCS of thousands of cells readily achievable. RATIONALE We reasoned that it might be possible, given these advances, to determine the transcriptomes of essentially every cell type of a complete organism possessing an unknown number of cell types. The planarian Schmidtea mediterranea , famous for its regeneration ability, is an attractive case study for such an undertaking. Planarians possess a complex anatomy with diverse differentiated cell types, including many found across animals. Furthermore, planarians contain a proliferating cell population called neoblasts that includes pluripotent stem cells. Neoblasts mediate regeneration and constitutive tissue turnover. Consequently, lineage precursors for essentially all differentiated cells types are also present in adults. Finally, planarians constitutively express positional information guiding tissue turnover. Therefore, comprehensive SCS at a single time point (the adult) could allow transcriptome determination for all differentiated cell types and for lineage precursors, and could identify patterning information that guides new cell production and organization. Capturing this information in most organisms would require sampling adults and many transient embryonic stages. RESULTS We used the SCS method Drop-seq to determine the transcriptomes for 66,783 individual cells from adult planarians. We locally saturated cell type coverage by iteratively sequencing distinct body regions and assessing the frequency of known rare cell types in the data. Clustering the cells by shared gene expression grouped cells into broad tissue classes. Subclustering of each broad tissue type in isolation enabled separation of cells into the cell populations constituting each tissue. These analyses enabled the identification of a previously unidentified tissue group and the classification of poorly characterized tissues into their constituent cell types, including numerous previously unknown cell types. Transcriptomes were identified for many rare cell types, including those that exist as rarely as ~10 cells in an animal that has 10 5 to 10 6 cells, which suggests that near-to-complete cellular saturation was reached. In addition, transcriptomes for known and novel lineage precursors, from pluripotent stem cell to differentiated cell types, were generated. Precursor transcriptomes identified transcription factors required for maintenance of associated differentiated cells during homeostatic cell turnover. Finally, the data were used to identify genes regionally expressed in muscle, which is the site of planarian patterning gene expression. CONCLUSION We successfully used SCS to generate transcriptomes for most to all cells of a complete organism. This resource provides a wealth of data regarding the cellular site of expression of thousands of conserved genes and the transcriptomes for cell types widely used in animals. These data will inform studies of these genes and cell types broadly, and will provide a resource for the fields of planarian biology and comparative evolutionary biology. This work also provides a template for the generation of cell type transcriptome atlases, which can be applied to a large array of organisms.

Oxidative stress preconditioning of mouse perivascular myogenic progenitors selects a subpopulation of cells with a distinct survival advantage in vitro and in vivo.

Abstract: Cell engraftment, survival and integration during transplantation procedures represent the crux of cell-based therapies. Thus, there have been many studies focused on improving cell viability upon implantation. We used severe oxidative stress to select for a mouse mesoangioblast subpopulation in vitro and found that this subpopulation retained self-renewal and myogenic differentiation capacities while notably enhancing cell survival, proliferation and migration relative to unselected cells. Additionally, this subpopulation of cells presented different resistance and recovery properties upon oxidative stress treatment, demonstrating select advantages over parental mesoangioblasts in our experimental analysis. Specifically, the cells were resistant to oxidative environments, demonstrating survival, continuous self-renewal and improved migration capability. The primary outcome of the selected cells was determined in in vivo experiments in which immunocompromised dystrophic mice were injected intramuscularly in the tibialis anterior with selected or non-selected mesoangioblasts. Resistant mesoangioblasts exhibited markedly enhanced survival and integration into the host skeletal muscle, accounting for a more than 70% increase in engraftment compared with that of the unselected mesoangioblast cell population and leading to remarkable muscle recovery. Thus, the positive effects of sorting on mesoangioblast cell behaviour in vitro and in vivo suggest that a selection step involving oxidative stress preconditioning may provide a novel methodology to select for resistant cells for use in regenerative tissue applications to prevent high mortality rates upon transplantation.

T H 2 cell development and function

Abstract: T helper 2 (TH2) cells orchestrate protective type 2 immune responses, such as those that target helminths and facilitate tissue repair, but also contribute to chronic inflammatory diseases, such as asthma and allergy. Here, we review recent insights into how diverse molecular signals from cellular sources, including dendritic cells, innate lymphoid cells and the epithelium, are integrated by T cells to guide the transcriptional and epigenetic changes necessary for TH2 cell differentiation. Our improved understanding of these pathways has opened new avenues for therapeutically targeting TH2 cells in asthma and allergy. The advent of comprehensive single-cell transcriptomics along with improvements in single-cell proteomics and the generation of novel in vivo cell fate mapping techniques promise to expand our understanding of T cell diversity and offer new insight into disease-related heterogeneity and plasticity of TH cell responses.