RESUMO
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).
Assuntos
Células da Medula Óssea/citologia , Medula Óssea , Síndrome de Down/sangue , Síndrome de Down/imunologia , Feto/citologia , Hematopoese , Sistema Imunitário/citologia , Linfócitos B/citologia , Células Dendríticas/citologia , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Células Endoteliais/patologia , Eosinófilos/citologia , Células Eritroides/citologia , Granulócitos/citologia , Humanos , Imunidade , Células Mieloides/citologia , Células Estromais/citologiaRESUMO
BACKGROUND: Human genome sequencing has transformed our understanding of genomic variation and its relevance to health and disease, and is now starting to enter clinical practice for the diagnosis of rare diseases. The question of whether and how some categories of genomic findings should be shared with individual research participants is currently a topic of international debate, and development of robust analytical workflows to identify and communicate clinically relevant variants is paramount. METHODS: The Deciphering Developmental Disorders (DDD) study has developed a UK-wide patient recruitment network involving over 180 clinicians across all 24 regional genetics services, and has performed genome-wide microarray and whole exome sequencing on children with undiagnosed developmental disorders and their parents. After data analysis, pertinent genomic variants were returned to individual research participants via their local clinical genetics team. FINDINGS: Around 80,000 genomic variants were identified from exome sequencing and microarray analysis in each individual, of which on average 400 were rare and predicted to be protein altering. By focusing only on de novo and segregating variants in known developmental disorder genes, we achieved a diagnostic yield of 27% among 1133 previously investigated yet undiagnosed children with developmental disorders, whilst minimising incidental findings. In families with developmentally normal parents, whole exome sequencing of the child and both parents resulted in a 10-fold reduction in the number of potential causal variants that needed clinical evaluation compared to sequencing only the child. Most diagnostic variants identified in known genes were novel and not present in current databases of known disease variation. INTERPRETATION: Implementation of a robust translational genomics workflow is achievable within a large-scale rare disease research study to allow feedback of potentially diagnostic findings to clinicians and research participants. Systematic recording of relevant clinical data, curation of a gene-phenotype knowledge base, and development of clinical decision support software are needed in addition to automated exclusion of almost all variants, which is crucial for scalable prioritisation and review of possible diagnostic variants. However, the resource requirements of development and maintenance of a clinical reporting system within a research setting are substantial. FUNDING: Health Innovation Challenge Fund, a parallel funding partnership between the Wellcome Trust and the UK Department of Health.
Assuntos
Deficiências do Desenvolvimento/diagnóstico , Genoma Humano/genética , Adolescente , Criança , Pré-Escolar , Deficiências do Desenvolvimento/genética , Feminino , Variação Genética/genética , Estudo de Associação Genômica Ampla/métodos , Heterozigoto , Humanos , Achados Incidentais , Lactente , Recém-Nascido , Disseminação de Informação , Masculino , Fenótipo , Manejo de EspécimesRESUMO
Schistosoma mansoni is a parasitic flatworm that causes the major neglected tropical disease schistosomiasis. The miracidium is the first larval stage of the life cycle. It swims and infects a freshwater snail, transforms into a mother sporocyst, where its stem cells generate daughter sporocysts that give rise to human-infective cercariae larvae. To understand the miracidium at cellular and molecular levels, we created a whole-body atlas of its ~365 cells. Single-cell RNA sequencing identified 19 transcriptionally distinct cell clusters. In situ hybridisation of tissue-specific genes revealed that 93% of the cells in the larva are somatic (57% neural, 19% muscle, 13% epidermal or tegument, 2% parenchyma, and 2% protonephridia) and 7% are stem. Whereas neurons represent the most diverse somatic cell types, trajectory analysis of the two main stem cell populations indicates that one of them is the origin of the tegument lineage and the other likely contains pluripotent cells. Furthermore, unlike the somatic cells, each of these stem populations shows sex-biased transcriptional signatures suggesting a cell-type-specific gene dosage compensation for sex chromosome-linked loci. The miracidium represents a simple developmental stage with which to gain a fundamental understanding of the molecular biology and spatial architecture of schistosome cells.
Assuntos
Larva , Schistosoma mansoni , Análise de Célula Única , Animais , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/genética , Estágios do Ciclo de Vida/genéticaRESUMO
BACKGROUND: As normal cells transform into cancers, their cell state changes, which may drive cancer cells into a stem-like or more primordial, foetal, or embryonic cell state. The transcriptomic profile of this final state may encode information about cancer's origin and how cancers relate to their normal cell counterparts. METHODS: Here, we used single-cell atlases to study cancer transformation in transcriptional terms. We utilised bulk transcriptomes across a wide spectrum of adult and childhood cancers, using a previously established method to interrogate their relationship to normal cell states. We extend and validate these findings using single-cell cancer transcriptomes and organ-specific atlases of colorectal and liver cancer. RESULTS: Our bulk transcriptomic data reveals that adult cancers rarely return to an embryonic state, but that a foetal state is a near-universal feature of childhood cancers. This finding was confirmed with single-cell cancer transcriptomes. CONCLUSIONS: Our findings provide a nuanced picture of transformation in human cancer, indicating cancer-specific rather than universal patterns of transformation pervade adult epithelial cancers.
Assuntos
Neoplasias Hepáticas , Adulto , Humanos , Neoplasias Hepáticas/genética , Desenvolvimento Embrionário , Feto , Perfilação da Expressão Gênica , TranscriptomaRESUMO
A fundamental step of tumour single cell mRNA analysis is separating cancer and non-cancer cells. We show that the common approach to separation, using shifts in average expression, can lead to erroneous biological conclusions. By contrast, allelic imbalances representing copy number changes directly detect the cancer genotype and accurately separate cancer from non-cancer cells. Our findings provide a definitive approach to identifying cancer cells from single cell mRNA sequencing data.
Assuntos
Neoplasias , Transcriptoma , Desequilíbrio Alélico/genética , Genótipo , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genéticaRESUMO
During activation, T cells undergo extensive gene expression changes that shape the properties of cells to exert their effector function. Understanding the regulation of this process could help explain how genetic variants predispose to immune diseases. Here, we mapped genetic effects on gene expression (expression quantitative trait loci (eQTLs)) using single-cell transcriptomics. We profiled 655,349 CD4+ T cells, capturing transcriptional states of unstimulated cells and three time points of cell activation in 119 healthy individuals. This identified 38 cell clusters, including transient clusters that were only present at individual time points of activation. We found 6,407 genes whose expression was correlated with genetic variation, of which 2,265 (35%) were dynamically regulated during activation. Furthermore, 127 genes were regulated by variants associated with immune-mediated diseases, with significant enrichment for dynamic effects. Our results emphasize the importance of studying context-specific gene expression regulation and provide insights into the mechanisms underlying genetic susceptibility to immune-mediated diseases.
Assuntos
Doenças do Sistema Imunitário , Locos de Características Quantitativas , Linfócitos T CD4-Positivos , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Doenças do Sistema Imunitário/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas/genética , TranscriptomaRESUMO
Tumor cells may share some patterns of gene expression with their cell of origin, providing clues into the differentiation state and origin of cancer. Here, we study the differentiation state and cellular origin of 1300 childhood and adult kidney tumors. Using single cell mRNA reference maps of normal tissues, we quantify reference "cellular signals" in each tumor. Quantifying global differentiation, we find that childhood tumors exhibit fetal cellular signals, replacing the presumption of "fetalness" with a quantitative measure of immaturity. By contrast, in adult cancers our assessment refutes the suggestion of dedifferentiation towards a fetal state in most cases. We find an intimate connection between developmental mesenchymal populations and childhood renal tumors. We demonstrate the diagnostic potential of our approach with a case study of a cryptic renal tumor. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer.
Assuntos
Neoplasias Renais/genética , Rim/metabolismo , RNA Mensageiro/genética , RNA-Seq/métodos , Análise de Célula Única/métodos , Transcriptoma , Adulto , Algoritmos , Criança , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Rim/embriologia , Neoplasias Renais/embriologia , Neoplasias Renais/metabolismo , Modelos Genéticos , Transdução de Sinais/genéticaRESUMO
We compared ChromID VRE and Brilliance VRE media for the detection of vancomycin-resistant enterococci (VRE). Using a panel of 28 enterococcal isolates, 10 vanA Enterococcus faecium and three vanA Enterococcus faecalis isolates grew as per manufacturers' instructions whilst growth of two vanC and eight vancomycin-susceptible enterococci was inhibited on both media. Important differences were noted in the selectivity and chromogenic properties of the two media for vanA Enterococcus raffinosus and vanB E. faecium. The two media were further evaluated using 295 stool samples from nursing home residents, 34 of which grew VRE (11.5%). ChromID and Brilliance had comparable sensitivity, which was increased markedly by prolonging incubation to 48 hours (from 29% to 82%, and from 41% to 85%, respectively) and by a pre-enrichment step (to 97% and 100%, respectively). Brilliance VRE agar had higher selectivity at 48 hours, and after pre-enrichment.
Assuntos
Técnicas Bacteriológicas/métodos , Portador Sadio/diagnóstico , Meios de Cultura/química , Infecções por Bactérias Gram-Positivas/diagnóstico , Casas de Saúde , Enterococos Resistentes à Vancomicina/isolamento & purificação , Portador Sadio/microbiologia , Compostos Cromogênicos , Enterococcus faecalis/isolamento & purificação , Enterococcus faecium/isolamento & purificação , Fezes/microbiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
ChromID ESBL agar and Brilliance ESBL agar were compared for the isolation of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae from 298 stools. These had comparable sensitivity and selectivity for the 116 positive samples. Pre-enrichment with cefpodoxime and extending incubation to 48 hours after direct plating both significantly increased sensitivity but reduced selectivity of both agars.