Showing papers by "Stephen R. Quake published in 2018"
TL;DR: Deep single-cell RNA sequencing of microglia and related myeloid cells sorted from various regions of embryonic, postnatal, and adult mouse brains found that the majority of adult microglian signatures are remarkably similar in transcriptomes, regardless of brain region.
Abstract: Summary Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown if these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, postnatal, and adult mouse brains. We found that the majority of adult microglia with homeostatic signatures are remarkably similar in transcriptomes, regardless of brain region. By contrast, postnatal microglia represent a more heterogeneous population. We discovered that postnatal white matter-associated microglia (WAM) are strikingly different from microglia in other regions and express genes enriched in degenerative disease-associated microglia. These postnatal WAM have distinct amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease. Highlights Myeloid scRNA-seq atlas across brain regions and developmental stages Limited transcriptomic heterogeneity of homeostatic microglia in the adult brain Phase-specific gene sets of proliferating microglia along cell cycle pseudotime Phagocytic postnatal white matter-associated microglia sharing DAM gene signatures
203 citations
TL;DR: In pilot studies of pregnant women, RNA-based tests of maternal blood predicted delivery date and risk of early childbirth and hold promise for prenatal care in both the developed and developing worlds, although they require validation in larger, blinded clinical trials.
Abstract: Noninvasive blood tests that provide information about fetal development and gestational age could potentially improve prenatal care. Ultrasound, the current gold standard, is not always affordable in low-resource settings and does not predict spontaneous preterm birth, a leading cause of infant death. In a pilot study of 31 healthy pregnant women, we found that measurement of nine cell-free RNA (cfRNA) transcripts in maternal blood predicted gestational age with comparable accuracy to ultrasound but at substantially lower cost. In a related study of 38 women (23 full-term and 15 preterm deliveries), all at elevated risk of delivering preterm, we identified seven cfRNA transcripts that accurately classified women who delivered preterm up to 2 months in advance of labor. These tests hold promise for prenatal care in both the developed and developing worlds, although they require validation in larger, blinded clinical trials.
178 citations
TL;DR: It is shown that vein cells of the developing heart undergo an early cell fate switch to create a pre-artery population that subsequently builds coronary arteries, which are built by vein-derived coronary arteries that can differentiate independently of blood flow upon the release of inhibition mediated by COUP-TF2 and cell cycle factors.
Abstract: Arteries and veins are specified by antagonistic transcriptional programs. However, during development and regeneration, new arteries can arise from pre-existing veins through a poorly understood process of cell fate conversion. Here, using single-cell RNA sequencing and mouse genetics, we show that vein cells of the developing heart undergo an early cell fate switch to create a pre-artery population that subsequently builds coronary arteries. Vein cells underwent a gradual and simultaneous switch from venous to arterial fate before a subset of cells crossed a transcriptional threshold into the pre-artery state. Before the onset of coronary blood flow, pre-artery cells appeared in the immature vessel plexus, expressed mature artery markers, and decreased cell cycling. The vein-specifying transcription factor COUP-TF2 (also known as NR2F2) prevented plexus cells from overcoming the pre-artery threshold by inducing cell cycle genes. Thus, vein-derived coronary arteries are built by pre-artery cells that can differentiate independently of blood flow upon the release of inhibition mediated by COUP-TF2 and cell cycle factors.
169 citations
TL;DR: A surprising example of convergent evolution is identified in which IgE antibodies underwent identical gene rearrangements in unrelated individuals and gained high affinity and unexpected cross-reactivity to the clinically important peanut allergens Ara h 2 and Ara h 3.
Abstract: Immunoglobulin E (IgE) antibodies protect against helminth infections but can also cause life-threatening allergic reactions. Despite their role in human health, the cells that produce these antibodies are rarely observed and remain enigmatic. We isolated single IgE B cells from individuals with food allergies and used single-cell RNA sequencing to elucidate the gene expression and splicing patterns unique to these cells. We identified a surprising example of convergent evolution in which IgE antibodies underwent identical gene rearrangements in unrelated individuals. Through the acquisition of variable region mutations, these IgE antibodies gained high affinity and unexpected cross-reactivity to the clinically important peanut allergens Ara h 2 and Ara h 3. These findings provide insight into IgE B cell transcriptomics and enable biochemical dissection of this antibody class.
139 citations
TL;DR: This work applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance, exploiting this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus.
Abstract: Dengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage and shock or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level.
124 citations
TL;DR: This model not only significantly increased predictive power by combining all datasets, but also revealed novel interactions between different biological modalities, which provides the frameworks for future studies examining deviations implicated in pregnancy‐related pathologies including preterm birth and preeclampsia.
Abstract: Motivation
Multiple biological clocks govern a healthy pregnancy. These biological mechanisms produce immunologic, metabolomic, proteomic, genomic and microbiomic adaptations during the course of pregnancy. Modeling the chronology of these adaptations during full-term pregnancy provides the frameworks for future studies examining deviations implicated in pregnancy-related pathologies including preterm birth and preeclampsia.
113 citations
TL;DR: The landscape of host transcripts and viral RNA in thousands of single blood cells from dengue patients prior to progressing to SD was profiled and cell type-specific immune activation and candidate predictive biomarkers were discovered.
Abstract: Dengue virus (DENV) infection can result in severe complications. However, the understanding of the molecular correlates of severity is limited, partly due to difficulties in defining the peripheral blood mononuclear cells (PBMCs) that contain DENV RNA in vivo. Accordingly, there are currently no biomarkers predictive of progression to severe dengue (SD). Bulk transcriptomics data are difficult to interpret because blood consists of multiple cell types that may react differently to infection. Here, we applied virus-inclusive single-cell RNA-seq approach (viscRNA-Seq) to profile transcriptomes of thousands of single PBMCs derived early in the course of disease from six dengue patients and four healthy controls and to characterize distinct leukocyte subtypes that harbor viral RNA (vRNA). Multiple IFN response genes, particularly MX2 in naive B cells and CD163 in CD14+ CD16+ monocytes, were up-regulated in a cell-specific manner before progression to SD. The majority of vRNA-containing cells in the blood of two patients who progressed to SD were naive IgM B cells expressing the CD69 and CXCR4 receptors and various antiviral genes, followed by monocytes. Bystander, non-vRNA-containing B cells also demonstrated immune activation, and IgG1 plasmablasts from two patients exhibited clonal expansions. Lastly, assembly of the DENV genome sequence revealed diversity at unexpected sites. This study presents a multifaceted molecular elucidation of natural dengue infection in humans with implications for any tissue and viral infection and proposes candidate biomarkers for prediction of SD.
111 citations
TL;DR: The authors' method detects single-nucleotide mutations of autosomal recessive diseases as early as the first trimester of pregnancy, of importance for metabolic disorders in which early diagnosis can affect management of the disease and reduce complications and anxiety related to invasive testing.
Abstract: BACKGROUND: Prenatal diagnosis in pregnancies at risk of single-gene disorders is currently performed using invasive methods such as chorionic villus sampling and amniocentesis. This is in contrast with screening for common aneuploidies, for which noninvasive methods with a single maternal blood sample have become standard clinical practice. METHODS: We developed a protocol for noninvasive prenatal diagnosis of inherited single-gene disorders using droplet digital PCR from circulating cell-free DNA (cfDNA) in maternal plasma. First, the amount of cfDNA and fetal fraction is determined using a panel of TaqMan assays targeting high-variability single-nucleotide polymorphisms. Second, the ratio of healthy and diseased alleles in maternal plasma is quantified using TaqMan assays targeting the mutations carried by the parents. Two validation approaches of the mutation assay are presented. RESULTS: We collected blood samples from 9 pregnancies at risk for different single-gene disorders, including common conditions and rare metabolic disorders. We measured cases at risk of hemophilia, ornithine transcarbamylase deficiency, cystic fibrosis, β-thalassemia, mevalonate kinase deficiency, acetylcholine receptor deficiency, and DFNB1 nonsyndromic hearing loss. We correctly differentiated affected and unaffected pregnancies (2 affected, 7 unaffected), confirmed by neonatal testing. We successfully measured an affected pregnancy as early as week 11 and with a fetal fraction as low as 3.7% (0.3). CONCLUSIONS: Our method detects single-nucleotide mutations of autosomal recessive diseases as early as the first trimester of pregnancy. This is of importance for metabolic disorders in which early diagnosis can affect management of the disease and reduce complications and anxiety related to invasive testing.
66 citations
TL;DR: The capacity of 6mA-Crosslinking-Exonuclease-sequencing (6mACE-seq) to detect genome-wide 6mA at single-nucleotide-resolution is established, demonstrating this by accurately mapping 6mA in synthesized DNA and bacterial genomes.
Abstract: N6-methyldeoxyadenosine (6mA) is a well-characterized DNA modification in prokaryotes but reports on its presence and function in mammals have been controversial. To address this issue, we established the capacity of 6mA-Crosslinking-Exonuclease-sequencing (6mACE-seq) to detect genome-wide 6mA at single-nucleotide-resolution, demonstrating this by accurately mapping 6mA in synthesized DNA and bacterial genomes. Using 6mACE-seq, we generated a human-genome-wide 6mA map that accurately reproduced known 6mA enrichment at active retrotransposons and revealed mitochondrial chromosome-wide 6mA clusters asymmetrically enriched on the heavy-strand. We identified a novel putative 6mA-binding protein in single-stranded DNA-binding protein 1 (SSBP1), a mitochondrial DNA (mtDNA) replication factor known to coat the heavy-strand, linking 6mA with the regulation of mtDNA replication. Finally, we characterized AlkB homologue 1 (ALKBH1) as a mitochondrial protein with 6mA demethylase activity and showed that its loss decreases mitochondrial oxidative phosphorylation. Our results show that 6mA clusters play a previously unappreciated role in regulating human mitochondrial function, despite 6mA being an uncommon DNA modification in the human genome.
64 citations
TL;DR: It is found that 5hmC sites are enriched in a disease and stage specific manner in exons, 3’UTRs and transcription termination sites with progressive disease suggesting increased transcriptional activity.
Abstract: Pancreatic cancers are typically diagnosed at late stage where disease prognosis is poor as exemplified by a 5-year survival rate of 8.2%. Earlier diagnosis would be beneficial by enabling surgical resection or earlier application of therapeutic regimens. We investigated the detection of pancreatic ductal adenocarcinoma (PDAC) in a non-invasive manner by interrogating changes in 5-hydroxymethylation cytosine status (5hmC) of circulating cell free DNA in the plasma of a PDAC cohort (n=51) in comparison with a non-cancer cohort (n=41). We found that 5hmC sites are enriched in a disease and stage specific manner in eons, 39UTRs, transcription termination sites. Our data show that the 5hmC density in H3K4me3 sites is reduced in progressive disease suggesting increased transcriptional activity. 5hmC density is differentially represented in thousands of genes, and a stringently filtered set of the most significant genes exhibited biology related to pancreas (GATA4, GATA6, PROX1, ONECUT1) and/or cancer development (YAP1, TEAD1, PROX1, ONECUT1, ONECUT2, IGF1 and IGF2). Regularized regression models were built using 5hmC densities in statistically filtered genes or a comprehensive set of highly variable gene counts and performed with an AUC = 0.94-0.96 on training data. We were able to test the ability to classify PDAC and non-cancer samples with the elastic net and lasso models on two external pancreatic cancer 5hmC data sets and found validation performance to be AUC = 0.74-0.97. The findings suggest that 5hmC changes enable classification of PDAC patients with a high fidelity and are worthy of further investigation on larger cohorts of patient samples.
56 citations
TL;DR: This work develops an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype and predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD, and refined the algorithm to handle closely related recipients and donors.
Abstract: Prediction of allograft rejection is provided based on the quantification of transplant-derived circulating cell-free DNA (dd-cfDNA levels) in the absence of a donor genotype. The technology provided herein alleviates some of the barriers to the implementation of Genome Transplant Dynamics (GTD), which will further widen its clinical application.
TL;DR: The results show that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and also suggest that haematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.
Abstract: Haematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are haematopoietic stem cells (HSCs), which are multipotent and self-renewing, and generate the entire repertoire of blood and immune cells throughout an animal's life1. Although there have been comprehensive studies on self-renewal, differentiation, physiological regulation and niche occupation in vertebrate HSCs, relatively little is known about the evolutionary origin and niches of these cells. Here we describe the haematopoietic system of Botryllus schlosseri, a colonial tunicate that has a vasculature and circulating blood cells, and interesting stem-cell biology and immunity characteristics2-8. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other3,4,7. Using flow cytometry, whole-transcriptome sequencing of defined cell populations and diverse functional assays, we identify HSCs, progenitors, immune effector cells and an HSC niche, and demonstrate that self-recognition inhibits allospecific cytotoxic reactions. Our results show that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and also suggest that haematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.
TL;DR: A synthetic microbial community is created using heritable genetic barcodes and tracked community composition over repeated rounds of subculture with immigration to show that there is a clear transition between neutral and selective regimes that depends on the rate of immigration as well as the fitness differences.
Abstract: Ecologists debate the relative importance of selective vs. neutral processes in understanding biodiversity. This debate is especially pertinent to microbial communities, which play crucial roles in areas such as health, disease, industry, and the environment. Here, we created a synthetic microbial community using heritable genetic barcodes and tracked community composition over repeated rounds of subculture with immigration. Consistent with theory, we find a transition exists between neutral and selective regimes, and the crossover point depends on the fraction of immigrants and the magnitude of fitness differences. Neutral models predict an increase in diversity with increased carrying capacity, while our selective model predicts a decrease in diversity. The community here lost diversity with an increase in carrying capacity, highlighting that using the correct model is essential for predicting community response to change. Together, these results emphasize the importance of including selection to obtain realistic models of even simple systems.
TL;DR: Using a simple, in vitro comparative RNA-seq approach, novel genes pertinent to fast bony antler regeneration were identified and their proliferative/osteogenic function was verified via gene overexpression, knockdown, and immunostaining.
Abstract: Deer antlers are bony structures that re-grow at very high rates, making them an attractive model for studying rapid bone regeneration. To identify the genes that are involved in this fast pace of bone growth, an in vitro RNA-seq model that paralleled the sharp differences in bone growth between deer antlers and humans was established. Subsequently, RNA-seq (> 60 million reads per library) was used to compare transcriptomic profiles. Uniquely expressed deer antler proliferation as well as mineralization genes were identified via a combination of differential gene expression and subtraction analysis. Thereafter, the physiological relevance as well as contributions of these identified genes were determined by immunofluorescence, gene overexpression, and gene knockdown studies. Cell characterization studies showed that in vitro-cultured deer antler-derived reserve mesenchyme (RM) cells exhibited high osteogenic capabilities and cell surface markers similar to in vivo counterparts. Under identical culture conditions, deer antler RM cells proliferated faster (8.6–11.7-fold increase in cell numbers) and exhibited increased osteogenic differentiation (17.4-fold increase in calcium mineralization) compared to human mesenchymal stem cells (hMSCs), paralleling in vivo conditions. Comparative RNA-seq identified 40 and 91 previously unknown and uniquely expressed fallow deer (FD) proliferation and mineralization genes, respectively, including uhrf1 and s100a10. Immunofluorescence studies showed that uhrf1 and s100a10 were expressed in regenerating deer antlers while gene overexpression and gene knockdown studies demonstrated the proliferation contributions of uhrf1 and mineralization capabilities of s100a10. Using a simple, in vitro comparative RNA-seq approach, novel genes pertinent to fast bony antler regeneration were identified and their proliferative/osteogenic function was verified via gene overexpression, knockdown, and immunostaining. This combinatorial approach may be applicable to discover unique gene contributions between any two organisms for a given phenomenon-of-interest.
TL;DR: Hypothermic storage of intact primary tissues in organ transplant preservative maintains the quality and stability of the transcriptome of cells for single cell RNAseq analysis.
Abstract: High-fidelity preservation strategies for primary tissues are in great demand in the single cell RNAseq community. A reliable method would greatly expand the scope of feasible multi-site collaborations and maximize the utilization of technical expertise. When choosing a method, standardizability and fidelity are important factors to consider due to the susceptibility of single-cell RNAseq analysis to technical noise. Existing approaches such as cryopreservation and chemical fixation are less than ideal for failing to satisfy either or both of these standards. Here we propose a new strategy that leverages preservation schemes developed for organ transplantation. We evaluated the strategy by storing intact mouse kidneys in organ transplant preservative solution at hypothermic temperature for up to 4 days (6 h, 1, 2, 3, and 4 days), and comparing the quality of preserved and fresh samples using FACS and single cell RNAseq. We demonstrate that the strategy effectively maintained cell viability, transcriptome integrity, cell population heterogeneity, and transcriptome landscape stability for samples after up to 3 days of preservation. The strategy also facilitated the definition of the diverse spectrum of kidney resident immune cells, to our knowledge the first time at single cell resolution. Hypothermic storage of intact primary tissues in organ transplant preservative maintains the quality and stability of the transcriptome of cells for single cell RNAseq analysis. The strategy is readily generalizable to primary specimens from other tissue types for single cell RNAseq analysis.
01 Oct 2018
TL;DR: In this paper, the authors present a case report describing the unique pathological and genomic characteristics of a tailgut cyst that metastasized to liver, and the histologic and immunohistochemical findings were consistent with a well-differentiated NET.
Abstract: Neuroendocrine tumors (NETs) arising from tailgut cysts are a rare but increasingly reported entity with gene expression profiles that may be indicative of the gastrointestinal cell of origin. We present a case report describing the unique pathological and genomic characteristics of a tailgut cyst NET that metastasized to liver. The histologic and immunohistochemical findings were consistent with a well-differentiated NET. Genomic testing indicates a germline frameshift in BRCA1 and a few somatic mutations of unknown significance. Transcriptomic analysis suggests an enteroendocrine L cell in the tailgut as a putative cell of origin. Genomic profiling of a rare NET and metastasis provides insight into its origin, development, and potential therapeutic options.
TL;DR: The self-assembling manifolds (SAM) algorithm, which dynamically rescales gene expression to amplify differences between cells, is presented and its advantage over other methods is demonstrated by analyzing stem cells from Schistosoma, a parasite that infects >250 million people.
Abstract: Analysis of single-cell transcriptomes remains a challenge in that subtle differences of cell types are difficult to resolve. Here we present the self-assembling manifolds (SAM) algorithm, which dynamically rescales gene expression to amplify differences between cells. We demonstrate its advantage over other methods by analyzing stem cells from Schistosoma, a parasite that infects >250 million people. Benchmarking on another 47 datasets, SAM consistently improves cell clustering and marker gene identification.
TL;DR: The transcriptomic transformation of human endometrium is characterized at single cell resolution, dissecting multidimensional cellular heterogeneity of the tissue across the entire natural menstrual cycle and revealing signatures in the luminal and glandular epithelium during epithelial gland reconstruction, and suggesting a mechanism for adult gland formation.
Abstract: In a human menstrual cycle, the endometrium undergoes remodeling, shedding, and regeneration which are driven by substantial gene expression changes in the underlying cellular hierarchy. Despite its importance in human fertility and regenerative biology, mechanistic understanding of this unique type of tissue homeostasis remains rudimentary. Here, we characterized the transcriptomic transformation of human endometrium at single cell resolution, dissecting multidimensional cellular heterogeneity of the tissue across the entire natural menstrual cycle. We analyzed 6 endometrial cell types, including a previously uncharacterized ciliated epithelial cell type, during four major phases of endometrial transformation, and found characteristic signatures for each cell type and phase. We discovered that human window of implantation opens up with an abrupt and discontinuous transcriptomic activation in the epithelium, accompanied with widespread decidualized feature in the stroma. These data reveal signatures in the luminal and glandular epithelium during epithelial gland reconstruction, and suggest a mechanism for adult gland formation.
TL;DR: In this paper, the authors characterized the transcriptomic transformation of human endometrium at single cell resolution, dissecting multidimensional cellular heterogeneity of the tissue across the entire natural menstrual cycle.
TL;DR: The discovery of a complete and divergent hydrogenotrophic methanogenesis pathway in a novel, thermophilic order of the Verstraetearchaeota is reported, as well as the presence of the Wood-Ljungdahl pathway in the crenarchaeal order Desulfurococcales.
Abstract: Methanogenic archaea are major contributors to the global carbon cycle and were long thought to belong exclusively to the euryarchaeotal phylum. Discovery of the methanogenesis gene cluster methyl-coenzyme M reductase (Mcr) in the Bathyarchaeota and thereafter the Verstraetearchaeota led to a paradigm shift, pushing back the evolutionary origin of methanogenesis to pre-date that of the Euryarchaeota. The methylotrophic methanogenesis found in the non-Euryarchaota distinguished itself from the predominantly hydrogenotrophic methanogens found in euryarchaeal orders as the former do not couple methanogenesis to carbon fixation through the reductive acetyl-coenzyme A (Wood-Ljungdahl) pathway, which was interpreted as evidence for independent evolution of the two methanogenesis pathways. Here, we report the discovery of a complete and divergent hydrogenotrophic methanogenesis pathway in a novel, thermophilic order of the Verstraetearchaeota which we have named Candidatus Methanohydrogenales, as well as the presence of the Wood-Ljungdahl pathway in the crenarchaeal order Desulfurococcales. Our findings support the ancient origin of hydrogenotrophic methanogenesis, suggest that methylotrophic methanogenesis might be a later adaptation of specific orders, and provide insight into how transition from hydrogenotrophic to methylotrophic methanogenesis might occur.
TL;DR: The authors' method detects single nucleotide mutations of autosomal recessive diseases as early as the first trimester of pregnancy, of importance for metabolic disorders where early diagnosis can affect management of the disease and reduce complications and anxiety related to invasive testing.
Abstract: Methods for detection of single nucleotide mutations of autosomal recessive diseases as early as the first trimester of pregnancy are provided. This is of importance for metabolic disorders where early diagnosis can affect management of the disease and reduce complications and anxiety related to invasive testing.
TL;DR: In some aspects, the methods include quantitating one or more placental or fetal-tissue specific genes in a biological sample from the woman as mentioned in this paper, and identifying woman is risk for preterm delivery.
Abstract: The invention is directed to methods of predicting gestational age of a fetus. The invention is also directed to methods of identifying woman is risk for preterm delivery. In some aspects, the methods include quantitating one or more placental or fetal-tissue specific genes in a biological sample from the woman.
TL;DR: Single cell RNA sequencing of freshly isolated human foetal and healthy adult liver identified hepatocyte, hepatoblast and liver progenitor cell (hLPC) populations and support the idea of a true progenitors existing within healthy adult Liver that can be activated upon injury.
Abstract: The liver is largely composed of hepatocytes and bile duct epithelial cells (BECs). Controversy exists as to whether a liver stem/progenitor cell capable of renewing both hepatocytes and BECs exists. Single cell RNA sequencing of freshly isolated human foetal and healthy adult liver identified hepatocyte, hepatoblast and liver progenitor cell (hLPC) populations. hLPCs, found at the interface between hepatocytes and bile ducts in both foetal and adult tissue, were distinguishable from BECs by their negative expression of TROP-2. Prospective isolation followed by in vitro culture demonstrated their potential for expansion and bi-lineage differentiation. The hLPC expression signature was also conserved within expanded cell populations specific to certain cases of liver injury and cancer. These data support the idea of a true progenitor existing within healthy adult liver that can be activated upon injury. Further work to define the mechanisms regulating hLPC behaviour could advance understanding of human development and disease.
TL;DR: This work systematically profiled the expression of four sRNA classes across eleven mouse tissues by RNA-seq, yielding the most comprehensive catalog of specific and ubiquitous small RNAs in individual tissues to date.
Abstract: Small non-coding RNAs (sRNAs) play a vital role in a broad range of biological processes both in health and disease. A comprehensive quantitative reference of sRNA expression would significantly advance our understanding of sRNA roles in shaping tissue functions. Here, we systematically profiled the expression of four sRNA classes across eleven mouse tissues by RNA-seq. Using fourteen biological replicates spanning both genders, we identified 3,962 various sRNAs; 473 of these sRNAs are novel and identified for the first time by this work. We found that 40% of these transcripts were distributed across the body in a tissue-specific manner, and this tissue-specificity extends through multiple sRNA classes; furthermore, some sRNAs are also sexually dimorphic. By combining these findings with machine learning, we were able to accurately classify tissue types from sRNA data generated by other studies. These results yield the most comprehensive catalog of specific and ubiquitous small RNAs in individual tissues to date, and we expect that this catalog will be a resource for the further identification of sRNAs involved in tissue-function in health and dysfunction in disease.
TL;DR: The isolation of IgE producing B cells from the blood of individuals with food allergies is described, followed by a detailed study of their properties by single cell RNA sequencing (scRNA-seq), and splicing within B cells of all isotypes reveals polarized germline transcription of the IgE, but not IgG4, isotype.
Abstract: IgE antibodies provide defense against helminth infections, but can also cause life-threatening allergic reactions. Despite their importance to human health, these antibodies and the cells that produce them remain enigmatic due to their scarcity in humans; much of our knowledge of their properties is derived from model organisms. Here we describe the isolation of IgE producing B cells from the blood of individuals with food allergies, followed by a detailed study of their properties by single cell RNA sequencing (scRNA-seq). We discovered that IgE B cells are deficient in membrane immunoglobulin expression and that the IgE plasmablast state is more immature than that of other antibody producing cells. Through recombinant expression of monoclonal antibodies derived from single cells, we identified IgE antibodies which had unexpected cross-reactive specificity for major peanut allergens Ara h 2 and Ara h 3; not only are these among the highest affinity native human antibodies discovered to date, they represent a surprising example of convergent evolution in unrelated individuals who independently evolved nearly identical antibodies. Finally, we discovered that splicing within B cells of all isotypes reveals polarized germline transcription of the IgE, but not IgG4, isotype as well as several examples of biallelic expression of germline transcripts. Our results offer insights into IgE B cell transcriptomics, clonality and regulation, provide a striking example of adaptive immune convergence, and offer an approach for accelerating mechanistic disease understanding by characterizing a rare B cell population underlying IgE-mediated disease at single cell resolution.
TL;DR: Virus-inclusive single-cell RNA-Seq is a powerful approach to quantitatively assess the complex interplay between virus and host at the single cell level and at a genome-wide scale, and to elucidate the cellular pathways involved in viral infection.
Abstract: Dengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level.
TL;DR: A principled model which depends only on the genome sequence is described, which captures many interesting relationships between viral families, and which creates clusters which correlate well with both the Baltimore and ICTV classifications.
Abstract: We describe a new genome alignment-based model for classification of viruses based on evolutionary genetic relationships. This approach uses information theory and a physical model to determine the information shared by the genes in two genomes. Pairwise comparisons of genes from the viruses are created from alignments using NCBI BLAST, and their match scores are combined to produce a metric between genomes, which is in turn used to determine a global classification using the 5,817 viruses on RefSeq. In cases where there is no measurable alignment between any genes, the method falls back to a coarser measure of genome relationship: the mutual information of k-mer frequency. This results in a principled model which depends only on the genome sequence, which captures many interesting relationships between viral families, and which creates clusters which correlate well with both the Baltimore and ICTV classifications. The incremental computational cost of classifying a novel virus is low and therefore newly discovered viruses can be quickly identified and classified.
TL;DR: This work develops a general analysis tool to quantitatively study the spatial correlations of gene expression in fixed tissue sections and builds a statistical model to characterize correlations in the spatial distribution of the expression level among different genes.
Abstract: Recently high-throughput image-based transcriptomic methods were developed and enabled researchers to spatially resolve gene expression variation at the molecular level for the first time. In this work, we develop a general analysis tool to quantitatively study the spatial correlations of gene expression in fixed tissue sections. As an illustration, we analyze the spatial distribution of single mRNA molecules measured by in situ sequencing on human fetal pancreas at three developmental time points 80, 87 and 117 days post-fertilization. We develop a density profile-based method to capture the spatial relationship between gene expression and other morphological features of the tissue sample such as position of nuclei and endocrine cells of the pancreas. In addition, we build a statistical model to characterize correlations in the spatial distribution of the expression level among different genes. This model enables us to infer the inhibitory and clustering effects throughout different time points. Our analysis framework is applicable to a wide variety of spatially-resolved transcriptomic data to derive biological insights.