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1.
Anticancer Res ; 40(10): 5577-5582, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988881

RESUMO

BACKGROUND/AIM: Clusters of circulating tumor cells (CTCs) increase metastatic potential compared to single CTC. However, conventional technologies have been unable to generate an accurate analysis of single and cluster CTCs in the peripheral blood. We propose an effective strategy to detect and isolate both single and cluster CTCs using two size-selective microfilters. MATERIALS AND METHODS: Five ml of whole blood were collected from 10 patients with epidermal growth factor receptor mutation-positive non-small cell lung cancer. Single and cluster CTCs were identified using precision microfiltration membranes with two distinct pore sizes together with anti-EpCAM antibody labeling. RESULTS: Single and cluster CTCs were detected by simultaneously using two size-selective microfilters. The EGFR-L858R mutation was detected in the DNA from cells captured using both microfilters. CONCLUSION: Our method can be used to detect and isolate single and cluster CTCs in the whole blood and may facilitate the development of a liquid biopsy strategy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/sangue , DNA Tumoral Circulante/sangue , Molécula de Adesão da Célula Epitelial/sangue , Células Neoplásicas Circulantes/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/patologia , Separação Celular , Feminino , Humanos , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Mutação/genética , Células Neoplásicas Circulantes/patologia
2.
Anticancer Res ; 40(10): 5679-5685, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988893

RESUMO

BACKGROUND/AIM: The presence of circulating tumor cells (CTC) has been reported to have an impact on prognosis in different tumor entities. Little is known about CTC morphology and heterogeneity. PATIENTS AND METHODS: In a multicenter setting, pre-therapeutic peripheral blood specimens were drawn from patients with non-metastatic esophageal adenocarcinoma (EAC). CTCs were captured by size-based filtration (ScreenCell®), subsequently Giemsa-stained and evaluated by two trained readers. The isolated cells were categorized in groups based on morphologic criteria. RESULTS: Small and large single CTCs, as well as CTC-clusters, were observed in 69.2% (n=81) of the 117 specimens; small CTCs were observed most frequently (59%; n=69), followed by large CTCs (40%; n=47) and circulating cancer-associated macrophage-like cells (CAMLs; 34.2%, n=40). Clusters were rather rare (12%; n=14). CTC/CAML were heterogeneous in the cohort, but also within one specimen. Neither the presence of the CTC subtypes/CAMLs nor the exact cell count were associated with the primary clinical TNM stage. CONCLUSION: Morphologically heterogenic CTCs and CAMLs are present in patients with non-metastatic, non-pretreated EAC.


Assuntos
Adenocarcinoma/sangue , Biomarcadores Tumorais/sangue , Neoplasias Esofágicas/sangue , Células Neoplásicas Circulantes/metabolismo , Adenocarcinoma/patologia , Contagem de Células , Separação Celular , Neoplasias Esofágicas/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/patologia , Prognóstico
3.
Adv Exp Med Biol ; 1255: 7-27, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32949387

RESUMO

Within the last decade, single-cell analysis has revolutionized our understanding of cellular processes and heterogeneity across all disciplines of life science. As the transcriptome, genome, or epigenome of individual cells can nowadays be analyzed at low cost and in high-throughput within a few days by modern techniques, tremendous improvements in disease diagnosis on the one hand and the investigation of disease-relevant mechanisms on the other were achieved so far. This relies on the parallel development of reliable cell capturing and single-cell sequencing approaches that have paved the way for comprehensive single-cell studies. Apart from single-cell isolation methods in high-throughput, a variety of methods with distinct specializations were developed, allowing for correlation of transcriptomics with cellular parameters like electrophysiology or morphology.For all single-cell-based approaches, accurate and reliable isolation with proper quality controls is prerequisite, whereby different options exist dependent on sample type and tissue properties. Careful consideration of an appropriate method is required to avoid incorrect or biased data that may lead to misinterpretations.In this chapter, we will provide a broad overview of the current state of the art in matters of single-cell isolation methods mostly applied for sequencing-based downstream analysis, and their respective advantages and drawbacks. Distinct technologies will be discussed in detail addressing key parameters like sample compatibility, viability, purity, throughput, and isolation efficiency.


Assuntos
Separação Celular/métodos , Análise de Célula Única/métodos , Animais , Genoma , Humanos , Transcriptoma
4.
PLoS One ; 15(8): e0237308, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790691

RESUMO

The isolation and analysis of circulating tumor cells (CTC) has the potential to provide minimally invasive diagnostic, prognostic and predictive information. Widespread clinical implementation of CTC analysis has been hampered by a lack of comparative investigation between different analytic methodologies in clinically relevant settings. The objective of this study was to evaluate four different CTC isolation techniques-those that rely on surface antigen expression (EpCAM or CD45 using DynaBeads® or EasySep™ systems) or the biophysical properties (RosetteSep™ or ScreenCell®) of CTCs. These were evaluated using cultured cells in order to calculate isolation efficiency at various levels including; inter-assay and inter-operator variability, protocol complexity and turn-around time. All four techniques were adequate at levels above 100 cells/mL which is commonly used for the evaluation of new isolation techniques. Only the RosetteSep™ and ScreenCell® techniques were found to provide adequate sensitivity at a level of 10 cells/mL. These techniques were then applied to the isolation and analysis of circulating tumor cells blood drawn from metastatic breast cancer patients where CTCs were detected in 54% (15/28) of MBC patients using the RosetteSep™ and 75% (6/8) with ScreenCell®. Overall, the ScreenCell® method had better sensitivity.


Assuntos
Neoplasias da Mama/secundário , Separação Celular/métodos , Células Neoplásicas Circulantes/patologia , Adulto , Idoso , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/análise , Feminino , Humanos , Antígenos Comuns de Leucócito/análise , Pessoa de Meia-Idade
5.
Science ; 369(6503): 530-537, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732419

RESUMO

Microglia, immune cells of the central nervous system (CNS), are important for tissue development and maintenance and are implicated in CNS disease, but we lack understanding of human fetal microglia development. Single-cell gene expression and bulk chromatin profiles of microglia at 9 to 18 gestational weeks (GWs) of human fetal development were generated. Microglia were heterogeneous at all studied GWs. Microglia start to mature during this developmental period and increasingly resemble adult microglia with CNS-surveilling properties. Chromatin accessibility increases during development with associated transcriptional networks reflective of adult microglia. Thus, during early fetal development, microglia progress toward a more mature, immune-sensing competent phenotype, and this might render the developing human CNS vulnerable to environmental perturbations during early pregnancy.


Assuntos
Encéfalo/embriologia , Desenvolvimento Embrionário/imunologia , Feto/imunologia , Microglia/imunologia , Fagocitose/imunologia , Encéfalo/citologia , Separação Celular , Células Cultivadas , Desenvolvimento Embrionário/genética , Redes Reguladoras de Genes , Humanos , Fagocitose/genética , Transcriptoma
6.
Nat Commun ; 11(1): 3953, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769974

RESUMO

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder. We reveal gene expression signatures that demarcate fibroblasts from mural cells and provide molecular signatures for cell subtype identification. We observe striking inter- and intra-organ heterogeneity amongst the fibroblasts, primarily reflecting differences in the expression of extracellular matrix components. Fibroblast subtypes localize to discrete anatomical positions offering novel predictions about physiological function(s) and regulatory signaling circuits. Our data shed new light on the diversity of poorly defined classes of cells and provide a foundation for improved understanding of their roles in physiological and pathological processes.


Assuntos
Diferenciação Celular , Fibroblastos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Miócitos de Músculo Liso/fisiologia , Pericitos/fisiologia , Animais , Separação Celular , Vasos Coronários/citologia , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Citometria de Fluxo , Intestinos/irrigação sanguínea , Intestinos/citologia , Masculino , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/citologia , Músculo Liso Vascular/citologia , Miocárdio/citologia , Miócitos de Músculo Liso/citologia , Pericitos/citologia , RNA-Seq , Análise de Célula Única , Bexiga Urinária/irrigação sanguínea , Bexiga Urinária/citologia
7.
Nat Commun ; 11(1): 3452, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651381

RESUMO

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.


Assuntos
Separação Celular/métodos , Análise Espectral Raman/métodos , Animais , Humanos
8.
Cytometry A ; 97(9): 887-890, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32654350

RESUMO

In patients with severe SARS-CoV-2 infection, the development of cytokine storm induces extensive lung damage, and monocytes play a role in this pathological process. Non-classical (NC) and intermediate (INT) monocytes are known to be involved during viral and bacterial infections. In this study, 30 patients with different manifestations of acute SARS-CoV-2 infection were investigated with a flow cytometric study of NC, INT, and classical (CL) monocytes. Significantly reduced NC and INT monocytes and a downregulated HLA-DR were found in acute patients with severe SARS-CoV-2 symptoms. Conversely in patients with moderate symptoms NC and INT monocytes and CD11b expression were increased. © 2020 International Society for Advancement of Cytometry.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Monócitos/imunologia , Pneumonia Viral/imunologia , Idoso , Betacoronavirus/patogenicidade , Biomarcadores/análise , Antígeno CD11b/análise , Separação Celular , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Feminino , Citometria de Fluxo , Interações entre Hospedeiro e Microrganismos , Humanos , Leucócitos , Masculino , Pessoa de Meia-Idade , Monócitos/virologia , Pandemias , Fenótipo , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Índice de Gravidade de Doença
9.
Proc Natl Acad Sci U S A ; 117(30): 17832-17841, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661178

RESUMO

Spermatogonial stem cells (SSCs) are essential for the generation of sperm and have potential therapeutic value for treating male infertility, which afflicts >100 million men world-wide. While much has been learned about rodent SSCs, human SSCs remain poorly understood. Here, we molecularly characterize human SSCs and define conditions favoring their culture. To achieve this, we first identified a cell-surface protein, PLPPR3, that allowed purification of human primitive undifferentiated spermatogonia (uSPG) highly enriched for SSCs. Comparative RNA-sequencing analysis of these enriched SSCs with differentiating SPG (KIT+ cells) revealed the full complement of genes that shift expression during this developmental transition, including genes encoding key components in the TGF-ß, GDNF, AKT, and JAK-STAT signaling pathways. We examined the effect of manipulating these signaling pathways on cultured human SPG using both conventional approaches and single-cell RNA-sequencing analysis. This revealed that GDNF and BMP8B broadly support human SPG culture, while activin A selectively supports more advanced human SPG. One condition-AKT pathway inhibition-had the unique ability to selectively support the culture of primitive human uSPG. This raises the possibility that supplementation with an AKT inhibitor could be used to culture human SSCs in vitro for therapeutic applications.


Assuntos
Transdução de Sinais , Espermatogônias/citologia , Espermatogônias/metabolismo , Transcriptoma , Biomarcadores , Separação Celular , Células Cultivadas , Biologia Computacional , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imunofenotipagem , Masculino , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo
10.
PLoS One ; 15(7): e0234986, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32634135

RESUMO

Glioblastoma is a common, malignant brain tumor whose disease incidence increases with age. Glioblastoma stem-like cells (GSCs) are thought to contribute to cancer therapy resistance and to be responsible for tumor initiation, maintenance, and recurrence. This study utilizes both SNP array and gene expression profiling to better understand GSCs and their relation to malignant disease. Peripheral blood and primary glioblastoma tumor tissue were obtained from patients, the latter of which was used to generate GSCs as well as a CD133pos./CD15pos. subpopulation. The stem cell features of GSCs were confirmed via the immunofluorescent expression of Nestin, SOX2, and CD133. Both tumor tissue and the isolated primary cells shared unique abnormal genomic characteristics, including a gain of chromosome 7 as well as either a partial or complete loss of chromosome 10. Individual genomic differences were also observed, including the loss of chromosome 4 and segmental uniparental disomy of 9p24.3→p21.3 in GSCs. Gene expression profiling revealed 418 genes upregulated in tumor tissue vs. CD133pos./CD15pos. cells and 44 genes upregulated in CD133pos./CD15pos. cells vs. tumor tissue. Pathway analyses demonstrated that upregulated genes in CD133pos./CD15pos. cells are relevant to cell cycle processes and cancerogenesis. In summary, we detected previously undescribed genomic and gene expression differences when comparing tumor tissue and isolated stem-like subpopulations.


Assuntos
Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Antígeno AC133/análise , Separação Celular/métodos , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Antígenos CD15/análise , Polimorfismo de Nucleotídeo Único/genética , Manejo de Espécimes , Regulação para Cima
11.
Proc Natl Acad Sci U S A ; 117(29): 16839-16847, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641515

RESUMO

Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.


Assuntos
Separação Celular/métodos , Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Células Neoplásicas Circulantes/classificação , Linhagem Celular Tumoral , Separação Celular/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Leucaférese/métodos , Campos Magnéticos , Microfluídica/instrumentação
12.
PLoS One ; 15(7): e0229967, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645012

RESUMO

Phthalic acid esters (phthalates) are male reproductive toxicants, which exert their most potent toxicity during fetal development. In the fetal rat, exposure to phthalates reduces testosterone biosynthesis, alters the development of seminiferous cords and other male reproductive tissues, and induces the formation of abnormal multinucleated germ cells (MNGs). Identification of MNGs is a time-intensive process, and it requires specialized training to identify MNGs in histological sections. As a result, MNGs are not routinely quantified in phthalate toxicity experiments. In order to speed up and standardize this process, we have developed an improved method for automated detection of MNGs. Using hand-labeled histological section images with human-identified MNGs, we trained a convolutional neural network with a U-Net architecture to identify MNGs on unlabeled images. With unseen hand-labeled images not used in model training, we assessed the performance of the model, using five different configurations of the data. On average, the model reached near human accuracy, and in the best model, it exceeded it. The use of automated image analysis will allow data on this histopathological endpoint to be more readily collected for analysis of phthalate toxicity. Our trained model application code is available for download at github.com/brown-ccv/mngcount.


Assuntos
Automação Laboratorial/métodos , Núcleo Celular , Separação Celular/métodos , Células Germinativas/patologia , Processamento de Imagem Assistida por Computador , Animais , Feminino , Masculino , Redes Neurais de Computação , Ácidos Ftálicos/toxicidade , Ratos Sprague-Dawley , Testículo/patologia
13.
J Vis Exp ; (159)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32510487

RESUMO

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were >90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.


Assuntos
Separação Celular/métodos , Fibroblastos/citologia , Neurônios Motores/citologia , Células de Schwann/citologia , Células Receptoras Sensoriais/citologia , Animais , Regeneração Nervosa , Fenótipo , Ratos
15.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510488

RESUMO

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1-3-day-old (P1-3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6-12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células-Tronco Neurais/citologia , Neurogênese , Animais , Astrócitos/citologia , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Hipocampo/citologia , Ventrículos Laterais/citologia , Camundongos , Neurônios/citologia , Oligodendroglia/citologia
16.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510511

RESUMO

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague-Dawley rats. In this method, the neurons and glia were incubated in a 37 °C, 5% CO2 incubator for 5-7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by ß-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Neuroglia/citologia , Neurônios/citologia , Bexiga Urinária/citologia , Animais , Neuroglia/metabolismo , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley
17.
Nat Commun ; 11(1): 2898, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518258

RESUMO

The sequential generation of layer-specific cortical neurons requires radial glia cells (RGCs) to precisely balance self-renewal and lineage commitment. While specific cell-cycle phases have been associated with these decisions, the mechanisms linking the cell-cycle machinery to cell-fate commitment remain obscure. Using single-cell RNA-sequencing, we find that the strongest transcriptional signature defining multipotent RGCs is that of G2/M-phase, and particularly CYCLIN-B1/2, while lineage-committed progenitors are enriched in G1/S-phase genes, including CYCLIN-D1. These data also reveal cell-surface markers that allow us to isolate RGCs and lineage-committed progenitors, and functionally confirm the relationship between cell-cycle phase enrichment and cell fate competence. Finally, we use cortical electroporation to demonstrate that CYCLIN-B1/2 cooperate with CDK1 to maintain uncommitted RGCs by activating the NOTCH pathway, and that CYCLIN-D1 promotes differentiation. Thus, this work establishes that cell-cycle phase-specific regulators act in opposition to coordinate the self-renewal and lineage commitment of RGCs via core stem cell regulatory pathways.


Assuntos
Ciclina B1/fisiologia , Ciclina B2/fisiologia , Ciclina D1/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Proteína Quinase CDC2/fisiologia , Ciclo Celular , Diferenciação Celular , Linhagem da Célula , Separação Celular , Córtex Cerebral/embriologia , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Células-Tronco/citologia
18.
Proc Natl Acad Sci U S A ; 117(25): 14342-14353, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513716

RESUMO

Immature T cells undergo a process of positive selection in the thymus when their new T cell receptor (TCR) engages and signals in response to self-peptides. As the T cell matures, a slew of negative regulatory molecules, including the inhibitory surface glycoprotein CD5, are up-regulated in proportion to the strength of the self-peptide signal. Together these regulators dampen TCR-proximal signaling and help avoid any subsequent peripheral activation of T cells by self-peptides. Paradoxically, antigen-specific T cells initially expressing more CD5 (CD5hi) have been found to better persist as effector/memory cells after a peripheral challenge. The molecular mechanisms underlying such a duality in CD5 function is not clear. We found that CD5 alters the basal activity of the NF-κB signaling in resting peripheral T cells. When CD5 was conditionally ablated, T cells were unable to maintain higher expression of the cytoplasmic NF-κB inhibitor IκBα. Consistent with this, resting CD5hi T cells expressed more of the NF-κB p65 protein than CD5lo cells, without significant increases in transcript levels, in the absence of TCR signals. This posttranslationally stabilized cellular NF-κB depot potentially confers a survival advantage to CD5hi T cells over CD5lo ones. Taken together, these data suggest a two-step model whereby the strength of self-peptide-induced TCR signal lead to the up-regulation of CD5, which subsequently maintains a proportional reserve of NF-κB in peripheral T cells poised for responding to agonistic antigen-driven T cell activation.


Assuntos
Antígenos CD5/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Inibidor de NF-kappaB alfa/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Linfócitos T/imunologia , Transferência Adotiva , Animais , Apresentação do Antígeno/imunologia , Antígenos CD5/genética , Linhagem Celular Tumoral , Separação Celular , Sobrevivência Celular/imunologia , Feminino , Citometria de Fluxo , Lipopolissacarídeos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , Modelos Animais , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Timo/citologia , Timo/crescimento & desenvolvimento , Timo/imunologia , Fator de Transcrição RelA/metabolismo , Regulação para Cima
19.
Nat Protoc ; 15(7): 2163-2185, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572244

RESUMO

Circulating tumor cells (CTCs) enable noninvasive liquid biopsy and identification of cancer. Various approaches exist for the capture and release of CTCs, including microfluidic methods and those involving magnetic beads or nanostructured solid interfaces. However, the concomitant cell damage and fragmentation that often occur during capture make it difficult to extensively characterize and analyze living CTCs. Here, we describe an aptamer-trigger-clamped hybridization chain reaction (atcHCR) method for the capture of CTCs by porous 3D DNA hydrogels. The 3D environment of the DNA networks minimizes cell damage, and the CTCs can subsequently be released for live-cell analysis. In this protocol, initiator DNAs with aptamer-toehold biblocks specifically bind to the epithelial cell adhesion molecule (EpCAM) on the surface of CTCs, which triggers the atcHCR and the formation of a DNA hydrogel. The DNA hydrogel cloaks the CTCs, facilitating quantification with minimal cell damage. This method can be used to quantitively identify as few as 10 MCF-7 cells in a 2-µL blood sample. Decloaking of tumor cells via gentle chemical stimulus (ATP) is used to release living tumor cells for subsequent cell culture and live-cell analysis. We also describe how to use the protocol to encapsulate and release cells of cancer cell lines, which can be used in preliminary experiments to model CTCs. The whole protocol takes ~2.5 d to complete, including downstream cell culture and analysis.


Assuntos
Separação Celular/métodos , DNA/química , Hidrogéis/química , Células Neoplásicas Circulantes/patologia , Cápsulas , Sobrevivência Celular , Humanos , Células MCF-7 , Hibridização de Ácido Nucleico
20.
J Vis Exp ; (159)2020 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-32449745

RESUMO

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 µm thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 ± 11.9 vs. 7.89 ± 3.6%, p < 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 ± 15.0 vs. 6.87 ± 5.23 cells/mg, p < 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9-200 cells/mg) from minimal amounts of tissue (<50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.


Assuntos
Separação Celular/métodos , Ventrículos do Coração/citologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Potenciais de Ação , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Sobrevivência Celular , Feminino , Humanos , Perfusão , Ratos Wistar , Reprodutibilidade dos Testes
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