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1.
Exp Hematol ; 80: 36-41.e3, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31812712

RESUMO

Epidemiological sequencing studies have revealed that somatic mutations characteristic of myeloid neoplasms can be detected in the blood of asymptomatic individuals decades prior to presentation of any clinical symptoms. This premalignant condition is known as clonal hematopoiesis of indeterminate potential (CHIP). Despite the fact these mutant clones become readily detectable in the blood of elderly individuals (∼10% of people over the age of 65), the overall rate of disease progression remains relatively low. Thus, in addition to genetic mutations, there are likely environmental factors that contribute to clonal evolution in people with CHIP. One environmental stress that increases with age is inflammation. Although chronic inflammation is detrimental to the long-term function of normal hematopoietic stem cells, several recent studies in animal models have indicated hematopoietic stem cells with CHIP mutations may be resistant to these deleterious effects. However, direct evidence indicating a correlation between increased inflammation and accelerated CHIP in humans is currently lacking. In this study, we sequenced the peripheral blood cells of a cohort of patients with ulcerative colitis, an autoimmune disease characterized by increased levels of pro-inflammatory cytokines. This analysis revealed that the inflammatory environment of ulcerative colitis promoted CHIP with a distinct mutational spectrum, notably positive selection of clones with DNMT3A and PPM1D mutations. We also show a specific association between elevated levels of serum interferon gamma and DNMT3A mutations. These data add to our understanding of how cell extrinsic factors select for clones with specific mutations to promote clonal hematopoiesis.


Assuntos
Colite Ulcerativa/patologia , Hematopoese/genética , Interferon gama/sangue , Mutação , Fator de Necrose Tumoral alfa/análise , Idoso , Idoso de 80 Anos ou mais , Células Clonais/citologia , Colite Ulcerativa/sangue , Colite Ulcerativa/genética , DNA (Citosina-5-)-Metiltransferases/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/sangue , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Proteína Fosfatase 2C/genética
2.
PLoS Genet ; 15(12): e1008573, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31877129

RESUMO

The ability to establish spatial organization is an essential feature of any developing tissue and is achieved through well-defined rules of cell-cell communication. Maintenance of this organization requires elimination of cells with inappropriate positional identity, a poorly understood phenomenon. Here we studied mechanisms regulating cell elimination in the context of a growing tissue, the Drosophila wing disc and its dorsal determinant Apterous. Systematic analysis of apterous mutant clones along with their twin spots shows that they are eliminated from the dorsal compartment via three different mechanisms: relocation to the ventral compartment, basal extrusion, and death, depending on the position of the clone in the wing disc. We find that basal extrusion is the main elimination mechanism in the hinge, whereas apoptosis dominates in the pouch and in the notum. In the absence of apoptosis, extrusion takes over to ensure clearance in all regions. Notably, clones in the hinge grow larger than those in the pouch, emphasizing spatial differences. Mechanistically, we find that limiting cell division within the clones does not prevent their extrusion. Indeed, even clones of one or two cells can be extruded basally, demonstrating that the clone size is not the main determinant of the elimination mechanism to be used. Overall, we revealed three elimination mechanisms and their spatial biases for preserving pattern in a growing organ.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas com Homeodomínio LIM/genética , Fatores de Transcrição/genética , Asas de Animais/crescimento & desenvolvimento , Animais , Divisão Celular , Células Clonais/citologia , Células Clonais/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/metabolismo , Mutação , Fatores de Transcrição/metabolismo , Asas de Animais/metabolismo
3.
Nat Commun ; 10(1): 4884, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31653848

RESUMO

Astrocytes play essential roles in the neural tissue where they form a continuous network, while displaying important local heterogeneity. Here, we performed multiclonal lineage tracing using combinatorial genetic markers together with a new large volume color imaging approach to study astrocyte development in the mouse cortex. We show that cortical astrocyte clones intermix with their neighbors and display extensive variability in terms of spatial organization, number and subtypes of cells generated. Clones develop through 3D spatial dispersion, while at the individual level astrocytes acquire progressively their complex morphology. Furthermore, we find that the astroglial network is supplied both before and after birth by ventricular progenitors that scatter in the neocortex and can give rise to protoplasmic as well as pial astrocyte subtypes. Altogether, these data suggest a model in which astrocyte precursors colonize the neocortex perinatally in a non-ordered manner, with local environment likely determining astrocyte clonal expansion and final morphotype.


Assuntos
Astrócitos/citologia , Diferenciação Celular , Córtex Cerebral/citologia , Animais , Astrócitos/metabolismo , Linhagem da Célula , Plasticidade Celular , Proliferação de Células , Células Clonais/citologia , Camundongos
4.
Nature ; 574(7779): 538-542, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645727

RESUMO

The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures8, which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.


Assuntos
Células Clonais/citologia , Células Clonais/patologia , Fibrose/genética , Fibrose/patologia , Fígado/citologia , Fígado/metabolismo , Mutação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Células Clonais/metabolismo , Análise Mutacional de DNA , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Filogenia , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/patologia
5.
Nature ; 574(7779): 532-537, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645730

RESUMO

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer1. However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.


Assuntos
Colo/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Mutação , Sintomas Prodrômicos , Reto/citologia , Adenoma/genética , Adenoma/patologia , Idoso , Proteína Axina/genética , Carcinoma/genética , Carcinoma/patologia , Transformação Celular Neoplásica , Células Clonais/citologia , Células Clonais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Feminino , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Células-Tronco/citologia , Células-Tronco/metabolismo
6.
Nature ; 574(7776): 122-126, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554970

RESUMO

B cells are important in the pathogenesis of many, and perhaps all, immune-mediated diseases. Each B cell expresses a single B cell receptor (BCR)1, and the diverse range of BCRs expressed by the total B cell population of an individual is termed the 'BCR repertoire'. Our understanding of the BCR repertoire in the context of immune-mediated diseases is incomplete, and defining this could provide new insights into pathogenesis and therapy. Here, we compared the BCR repertoire in systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Crohn's disease, Behçet's disease, eosinophilic granulomatosis with polyangiitis, and immunoglobulin A (IgA) vasculitis by analysing BCR clonality, use of immunoglobulin heavy-chain variable region (IGHV) genes and-in particular-isotype use. An increase in clonality in systemic lupus erythematosus and Crohn's disease that was dominated by the IgA isotype, together with skewed use of the IGHV genes in these and other diseases, suggested a microbial contribution to pathogenesis. Different immunosuppressive treatments had specific and distinct effects on the repertoire; B cells that persisted after treatment with rituximab were predominately isotype-switched and clonally expanded, whereas the inverse was true for B cells that persisted after treatment with mycophenolate mofetil. Our comparative analysis of the BCR repertoire in immune-mediated disease reveals a complex B cell architecture, providing a platform for understanding pathological mechanisms and designing treatment strategies.


Assuntos
Doenças do Sistema Imunitário/imunologia , Isotipos de Imunoglobulinas/análise , Isotipos de Imunoglobulinas/imunologia , Receptores de Antígenos de Linfócitos B/análise , Receptores de Antígenos de Linfócitos B/imunologia , Adulto , Idoso , Células Clonais/citologia , Células Clonais/imunologia , Humanos , Imunoglobulina A/análise , Imunoglobulina A/imunologia , Switching de Imunoglobulina/imunologia , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Pessoa de Meia-Idade , Adulto Jovem
7.
Nature ; 571(7764): 205-210, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270459

RESUMO

The mammalian brain contains neurogenic niches that comprise neural stem cells and other cell types. Neurogenic niches become less functional with age, but how they change during ageing remains unclear. Here we perform single-cell RNA sequencing of young and old neurogenic niches in mice. The analysis of 14,685 single-cell transcriptomes reveals a decrease in activated neural stem cells, changes in endothelial cells and microglia, and an infiltration of T cells in old neurogenic niches. T cells in old brains are clonally expanded and are generally distinct from those in old blood, which suggests that they may experience specific antigens. T cells in old brains also express interferon-γ, and the subset of neural stem cells that has a high interferon response shows decreased proliferation in vivo. We find that T cells can inhibit the proliferation of neural stem cells in co-cultures and in vivo, in part by secreting interferon-γ. Our study reveals an interaction between T cells and neural stem cells in old brains, opening potential avenues through which to counteract age-related decline in brain function.


Assuntos
Envelhecimento/fisiologia , Encéfalo/citologia , Movimento Celular , Células-Tronco Neurais/citologia , Neurogênese , Análise de Célula Única , Nicho de Células-Tronco/fisiologia , Linfócitos T/citologia , Animais , Sangue , Proliferação de Células , Células Clonais/citologia , Técnicas de Cocultura , Células Endoteliais/citologia , Interferon gama/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Análise de Sequência de RNA , Transdução de Sinais , Linfócitos T/metabolismo , Transcriptoma/genética
8.
Eur J Pharmacol ; 856: 172400, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31103630

RESUMO

Eosinophils and their granular proteins are crucial for combating allergic airway diseases. Eosinophils derived from HL-60 clone 15 (HC15) cells have been established as a feasible alternative cell model for human primary eosinophils. Simvastatin, a cholesterol-lowering agent, has been shown to exhibit anti-inflammatory and anti-allergic effects. Among the granular eosinophil proteins, eosinophil cationic protein (ECP) is the one best recognised in allergic airway diseases. The aim of our study is to investigate the effect and regulatory mechanisms of simvastatin on ECP levels derived from eosinophils. Both HC15 cell counts and ECP levels decreased after simvastatin treatment in the animal and cell models; however, after a cell count adjustment, simvastatin was not observed to exert a significantly inhibitory effect on ECP expression. Real-time polymerase chain reaction and Western blotting analyses demonstrated that simvastatin did not inhibit the intracellular formation or release of ECP. Cell cycle analysis showed that the percentage of HC15 cells in the G1 and S phases significantly increased and decreased, respectively, after simvastatin treatment. Simvastatin inhibited the proliferation of HC15-derived eosinophils by inducing G1/S cell cycle arrest in a dose-dependent manner. Its effect on the cell cycle involved the downregulation of cyclin A but without the presence of mevalonate; therefore, total ECP expression from eosinophils decreased, not by suppressing the actual formation or release of ECP but by arresting the G1/S cell cycle phase and inhibiting subsequent cell proliferation through the mevalonate pathway.


Assuntos
Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Sinvastatina/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Clonais/citologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Masculino , Ácido Mevalônico/farmacologia , Ratos , Ratos Sprague-Dawley , Sinvastatina/antagonistas & inibidores
9.
Nature ; 571(7763): 117-121, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31142833

RESUMO

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1 month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50 cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.


Assuntos
Técnicas de Cultura de Células/métodos , Autorrenovação Celular/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Meios de Cultura/química , Meios de Cultura/farmacologia , Feminino , Fibronectinas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Masculino , Camundongos , Álcool de Polivinil/farmacologia , Albumina Sérica , Fator de Células-Tronco/farmacologia , Trombopoetina/farmacologia , Fatores de Tempo , Condicionamento Pré-Transplante
10.
Nature ; 569(7757): 497-502, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31092920

RESUMO

Cell competition-the sensing and elimination of less fit 'loser' cells by neighbouring 'winner' cells-was first described in Drosophila. Although cell competition has been proposed as a selection mechanism to optimize tissue and organ development, its evolutionary generality remains unclear. Here, by using live imaging, lineage tracing, single-cell transcriptomics and genetics, we identify two cell competition mechanisms that sequentially shape and maintain the architecture of stratified tissue during skin development in mice. In the single-layered epithelium of the early embryonic epidermis, winner progenitors kill and subsequently clear neighbouring loser cells by engulfment. Later, as the tissue begins to stratify, the basal layer instead expels losers through upward flux of differentiating progeny. This cell competition switch is physiologically relevant: when it is perturbed, so too is barrier formation. Our findings show that cell competition is a selective force that optimizes vertebrate tissue function, and illuminate how a tissue dynamically adjusts cell competition strategies to preserve fitness as its architectural complexity increases during morphogenesis.


Assuntos
Comunicação Celular , Células Epidérmicas/citologia , Epiderme/embriologia , Morfogênese , Animais , Apoptose , Células Clonais/citologia , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Células Epidérmicas/metabolismo , Feminino , Masculino , Camundongos , Fagocitose , Análise de Célula Única
11.
Nature ; 568(7752): 344-350, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30944469

RESUMO

Stem cells underlie tissue homeostasis, but their dynamics during ageing-and the relevance of these dynamics to organ ageing-remain unknown. Here we report that the expression of the hemidesmosome component collagen XVII (COL17A1) by epidermal stem cells fluctuates physiologically through genomic/oxidative stress-induced proteolysis, and that the resulting differential expression of COL17A1 in individual stem cells generates a driving force for cell competition. In vivo clonal analysis in mice and in vitro 3D modelling show that clones that express high levels of COL17A1, which divide symmetrically, outcompete and eliminate adjacent stressed clones that express low levels of COL17A1, which divide asymmetrically. Stem cells with higher potential or quality are thus selected for homeostasis, but their eventual loss of COL17A1 limits their competition, thereby causing ageing. The resultant hemidesmosome fragility and stem cell delamination deplete adjacent melanocytes and fibroblasts to promote skin ageing. Conversely, the forced maintenance of COL17A1 rescues skin organ ageing, thereby indicating potential angles for anti-ageing therapeutic intervention.


Assuntos
Homeostase , Envelhecimento da Pele/patologia , Envelhecimento da Pele/fisiologia , Pele/citologia , Pele/patologia , Células-Tronco/citologia , Células-Tronco/patologia , Animais , Atrofia , Autoantígenos/química , Autoantígenos/metabolismo , Divisão Celular , Proliferação de Células , Células Clonais/citologia , Células Epidérmicas/citologia , Células Epidérmicas/patologia , Feminino , Genoma , Hemidesmossomos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Colágenos não Fibrilares/química , Colágenos não Fibrilares/metabolismo , Estresse Oxidativo , Proteólise
12.
Nature ; 567(7747): 234-238, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30814736

RESUMO

Longitudinal bone growth in children is sustained by growth plates, narrow discs of cartilage that provide a continuous supply of chondrocytes for endochondral ossification1. However, it remains unknown how this supply is maintained throughout childhood growth. Chondroprogenitors in the resting zone are thought to be gradually consumed as they supply cells for longitudinal growth1,2, but this model has never been proved. Here, using clonal genetic tracing with multicolour reporters and functional perturbations, we demonstrate that longitudinal growth during the fetal and neonatal periods involves depletion of chondroprogenitors, whereas later in life, coinciding with the formation of the secondary ossification centre, chondroprogenitors acquire the capacity for self-renewal, resulting in the formation of large, stable monoclonal columns of chondrocytes. Simultaneously, chondroprogenitors begin to express stem cell markers and undergo symmetric cell division. Regulation of the pool of self-renewing progenitors involves the hedgehog and mammalian target of rapamycin complex 1 (mTORC1) signalling pathways. Our findings indicate that a stem cell niche develops postnatally in the epiphyseal growth plate, which provides a continuous supply of chondrocytes over a prolonged period.


Assuntos
Condrócitos/citologia , Células Clonais/citologia , Lâmina de Crescimento/citologia , Nicho de Células-Tronco/fisiologia , Envelhecimento , Animais , Cartilagem/citologia , Autorrenovação Celular , Células Clonais/metabolismo , Feminino , Lâmina de Crescimento/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos
13.
Science ; 364(6438)2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30898844

RESUMO

The ability to generate induced pluripotent stem cells from differentiated cell types has enabled researchers to engineer cell states. Although studies have identified molecular networks that reprogram cells to pluripotency, the cellular dynamics of these processes remain poorly understood. Here, by combining cellular barcoding, mathematical modeling, and lineage tracing approaches, we demonstrate that reprogramming dynamics in heterogeneous populations are driven by dominant "elite" clones. Clones arise a priori from a population of poised mouse embryonic fibroblasts derived from Wnt1-expressing cells that may represent a neural crest-derived population. This work highlights the importance of cellular dynamics in fate programming outcomes and uncovers cell competition as a mechanism by which cells with eliteness emerge to occupy and dominate the reprogramming niche.


Assuntos
Reprogramação Celular/fisiologia , Evolução Clonal , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Reprogramação Celular/genética , Técnicas de Reprogramação Celular , Células Clonais/citologia , DNA/genética , Fibroblastos/citologia , Camundongos , Modelos Teóricos
14.
Cell Biol Int ; 43(5): 456-465, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30729622

RESUMO

Urothelial cell populations which differ in morphology and proliferation capacities can be isolated from the urinary bladder. The goal of this study was to analyze a clonal, proliferative, and self-renewing potential of porcine urothelial cells and to compare expression of selected adhesion and tight junction molecules, urothelial and stem cell markers for the urothelial clone types. Urothelial cells were isolated from 10 porcine urinary bladders. Three different clone types: holoclone-, meroclone-and paraclone-like colonies were identified based on their morphology. To characterize and compare the urothelial clones the immunofluorescent stains were performed. Expression of pancytokeratin (PanCK), Ki-67 and p63 was higher for holoclone- like cells compared to meroclone-and paraclone-like cells (P < 0.05). Meroclone-like cells expressed higher levels of p63 compared to paraclone- like cells (P < 0.05). The level of Ki-67 and PanCK for meroclone- and paraclone- like cells was comparable (P > 0.05). ß1 and ß4 integrins were not expressed. Expression of zonula occludens-1 (ZO-1) in cell-cell junctions for paraclone-, meroclone-and holoclone-like cells was 17.6 ± 0.6, 14.7 ± 0.5, and 16.1 ± 0.4, respectively. The results of actin filaments (F-actin) expression were 253,634 ± 6,920 for meroclone-like cells, 198,512 ± 7,977 for paraclone-like cells and 133,544 ± 3,169 for holoclone-like cells. Three urothelial cell types with differing features can be isolated from the bladder. Holoclone-like cells are the richest in stem cells and should be used in further studies for construction of neo-bladder or neo-conduit using tissue engineering methods.


Assuntos
Células Clonais/citologia , Bexiga Urinária/citologia , Urotélio/citologia , Animais , Biomarcadores/metabolismo , Adesão Celular/fisiologia , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Células Clonais/fisiologia , Antígeno Ki-67/análise , Masculino , Células-Tronco Neoplásicas/metabolismo , Cultura Primária de Células/métodos , Suínos/metabolismo , Junções Íntimas/fisiologia , Bexiga Urinária/metabolismo
15.
Blood ; 133(18): 1927-1942, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30782612

RESUMO

Although many recent studies describe the emergence and prevalence of "clonal hematopoiesis of indeterminate potential" in aged human populations, a systematic analysis of the numbers of clones supporting steady-state hematopoiesis throughout mammalian life is lacking. Previous efforts relied on transplantation of "barcoded" hematopoietic stem cells (HSCs) to track the contribution of HSC clones to reconstituted blood. However, ex vivo manipulation and transplantation alter HSC function and thus may not reflect the biology of steady-state hematopoiesis. Using a noninvasive in vivo color-labeling system, we report the first comprehensive analysis of the changing global clonal complexity of steady-state hematopoiesis during the natural murine lifespan. We observed that the number of clones (ie, clonal complexity) supporting the major blood and bone marrow hematopoietic compartments decline with age by ∼30% and ∼60%, respectively. Aging dramatically reduced HSC in vivo-repopulating activity and lymphoid potential while increasing functional heterogeneity. Continuous challenge of the hematopoietic system by serial transplantation provoked the clonal collapse of both young and aged hematopoietic systems. Whole-exome sequencing of serially transplanted aged and young hematopoietic clones confirmed oligoclonal hematopoiesis and revealed mutations in at least 27 genes, including nonsense, missense, and deletion mutations in Bcl11b, Hist1h2ac, Npy2r, Notch3, Ptprr, and Top2b.


Assuntos
Envelhecimento/fisiologia , Células Clonais/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Animais , Transplante de Células-Tronco Hematopoéticas , Camundongos
16.
Nature ; 566(7744): 398-402, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30760926

RESUMO

The human genome contains approximately 20 thousand protein-coding genes1, but the size of the collection of antigen receptors of the adaptive immune system that is generated by the recombination of gene segments with non-templated junctional additions (on B cells) is unknown-although it is certainly orders of magnitude larger. It has not been established whether individuals possess unique (or private) repertoires or substantial components of shared (or public) repertoires. Here we sequence recombined and expressed B cell receptor genes in several individuals to determine the size of their B cell receptor repertoires, and the extent to which these are shared between individuals. Our experiments revealed that the circulating repertoire of each individual contained between 9 and 17 million B cell clonotypes. The three individuals that we studied shared many clonotypes, including between 1 and 6% of B cell heavy-chain clonotypes shared between two subjects (0.3% of clonotypes shared by all three) and 20 to 34% of λ or κ light chains shared between two subjects (16 or 22% of λ or κ light chains, respectively, were shared by all three). Some of the B cell clonotypes had thousands of clones, or somatic variants, within the clonotype lineage. Although some of these shared lineages might be driven by exposure to common antigens, previous exposure to foreign antigens was not the only force that shaped the shared repertoires, as we also identified shared clonotypes in umbilical cord blood samples and all adult repertoires. The unexpectedly high prevalence of shared clonotypes in B cell repertoires, and identification of the sequences of these shared clonotypes, should enable better understanding of the role of B cell immune repertoires in health and disease.


Assuntos
Anticorpos/genética , Anticorpos/imunologia , Linfócitos B/imunologia , Células Clonais/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Adulto , Sequência de Aminoácidos , Anticorpos/química , Antígenos/imunologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Sequência de Bases , Células Clonais/citologia , Células Clonais/metabolismo , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Voluntários Saudáveis , Humanos , Recém-Nascido , Masculino , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Análise de Sequência de DNA
17.
PLoS One ; 14(1): e0211213, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30682127

RESUMO

Tumors are heterogeneous in the sense that they consist of multiple subpopulations of cells, referred to as subclones, each of which is characterized by a distinct profile of genomic variations such as somatic mutations. Inferring the underlying clonal landscape has become an important topic in that it can help in understanding cancer development and progression, and thereby help in improving treatment. We describe a novel state-space model, based on the feature allocation framework and an efficient sequential Monte Carlo (SMC) algorithm, using the somatic mutation data obtained from tumor samples to estimate the number of subclones, as well as their characterization. Our approach, by design, is capable of handling any number of mutations. Via extensive simulations, our method exhibits high accuracy, in most cases, and compares favorably with existing methods. Moreover, we demonstrated the validity of our method through analyzing real tumor samples from patients from multiple cancer types (breast, prostate, and lung). Our results reveal driver mutation events specific to cancer types, and indicate clonal expansion by manual phylogenetic analysis. MATLAB code and datasets are available to download at: https://github.com/moyanre/tumor_clones.


Assuntos
Células Clonais/citologia , Mutação , Neoplasias/genética , Algoritmos , Contagem de Células , Células Clonais/química , Progressão da Doença , Genótipo , Humanos , Modelos Teóricos , Método de Monte Carlo , Filogenia
18.
Nature ; 566(7744): 393-397, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30664748

RESUMO

In principle, humans can produce an antibody response to any non-self-antigen molecule in the appropriate context. This flexibility is achieved by the presence of a large repertoire of naive antibodies, the diversity of which is expanded by somatic hypermutation following antigen exposure1. The diversity of the naive antibody repertoire in humans is estimated to be at least 1012 unique antibodies2. Because the number of peripheral blood B cells in a healthy adult human is on the order of 5 × 109, the circulating B cell population samples only a small fraction of this diversity. Full-scale analyses of human antibody repertoires have been prohibitively difficult, primarily owing to their massive size. The amount of information encoded by all of the rearranged antibody and T cell receptor genes in one person-the 'genome' of the adaptive immune system-exceeds the size of the human genome by more than four orders of magnitude. Furthermore, because much of the B lymphocyte population is localized in organs or tissues that cannot be comprehensively sampled from living subjects, human repertoire studies have focused on circulating B cells3. Here we examine the circulating B cell populations of ten human subjects and present what is, to our knowledge, the largest single collection of adaptive immune receptor sequences described to date, comprising almost 3 billion antibody heavy-chain sequences. This dataset enables genetic study of the baseline human antibody repertoire at an unprecedented depth and granularity, which reveals largely unique repertoires for each individual studied, a subpopulation of universally shared antibody clonotypes, and an exceptional overall diversity of the antibody repertoire.


Assuntos
Anticorpos/genética , Anticorpos/imunologia , Variação Genética/genética , Anticorpos/química , Antígenos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sequência de Bases , Células Clonais/citologia , Células Clonais/imunologia , Células Clonais/metabolismo , Humanos , Análise de Sequência de DNA
19.
Biol Blood Marrow Transplant ; 25(3): 417-423, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30359734

RESUMO

Owing to the difficulty in isolating T cells from human biopsy samples, the characteristics of T cells that are infiltratinghuman acute graft-versus-host disease (GVHD) tissues remain largely uninvestigated. In the present study, TCR-ß deep sequencing of various GVHD tissue samples and concurrent peripheral blood obtained from transplant recipients was performed in combination with functional assays of tissue-infiltrating T cell clones. The T cell repertoire was more skewed in GVHD tissues than in the peripheral blood. The frequent clonotypes differed from tissue to tissue in the same patient, and the frequent clonotypes in the same tissue differed from patient to patient. Two T cell clones were successfully isolated from GVHD skin of a patient. In a cytotoxicity assay, both Tcell clones lysed patient peripheral blood mononuclear cells, but not donor-derived Epstein-Barr virus-transformed lymphoblastoid cells. Their clonotypes were identical to the most and second most frequent T cell clonotypes in the original GVHD skin and accounted for almost all of the skin-infiltrating T cells. These results suggest that human acute GVHD may result from only a few different alloreactive cytotoxic T cell clones, which differ from tissue to tissue and from patient to patient. The characterization of T cells infiltrating human GVHD tissues should be further investigated.


Assuntos
Doença Enxerto-Hospedeiro/patologia , Linfócitos T Citotóxicos/citologia , Movimento Celular , Células Clonais/citologia , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Pele/imunologia , Pele/patologia , Transplante Homólogo
20.
Methods Mol Biol ; 1905: 19-27, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30536087

RESUMO

Cholangiocytes are proliferative and are one of the sources for liver progenitor cells. Clonogenic cholangiocytes are defined as cells capable of clonally proliferating and differentiating cholangiocytes both in vitro and in vivo. In this protocol, we describe the method for isolation of primary cholangiocytes from mouse. To study the heterogeneity of cholangiocytes, we used flow cytometry-based cell sorting to isolate different subsets of cholangiocytes. Organoid-forming efficiencies from sorted single cells are compared within different cholangiocyte populations to identify clonogenic cholangiocytes.


Assuntos
Ductos Biliares/citologia , Separação Celular/métodos , Organoides/citologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Clonais/citologia , Células Epiteliais/citologia , Citometria de Fluxo , Camundongos
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