RESUMO
Osteoporosis is a common skeletal disorder characterized by an imbalance between bone resorption and formation, exhibiting a higher prevalence in women compared with men. While previous studies have primarily focused on genomics and genetics in osteoporosis susceptibility, there is a lack of systematic exploration of sex-specific differences in lipid levels in mouse bone marrow. Multiple reaction monitoring-based liquid chromatography-trandem mass spectrometry (LC-MS/MS) was used to quantify lipidomic profiles in bone marrow samples from three female mice and three male mice. The LC-MS/MS technique based on the multiple reaction monitoring method identified and quantified 184 lipids from 15 lipid classes. The contents of most lipids in the bone marrow cells of female mice were higher than those in male mice, including four polyunsaturated fatty acids, three phospholipids and four sphingolipids. Among all the lipid molecules, lactosylceramide (d18:0/16:0) showed the highest fold change in female mice, while its precursor lipid, glucosylceramide, was the most up-regulated in male mice. This study, focusing on bone marrow lipidomics, elucidates significant sexual dimorphism in lipid levels within bone marrow cells. It provides novel evidence supporting the higher prevalence of osteoporosis in women and enhances our understanding of the connection between sex-specific lipid levels and the risk of osteoporosis.
Assuntos
Medula Óssea , Lipidômica , Lipídeos , Espectrometria de Massas em Tandem , Animais , Feminino , Masculino , Camundongos , Lipidômica/métodos , Medula Óssea/metabolismo , Medula Óssea/química , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Lipídeos/análise , Caracteres Sexuais , Camundongos Endogâmicos C57BL , Células da Medula Óssea/metabolismo , Células da Medula Óssea/químicaRESUMO
Human biology is tightly linked to proteins, yet most measurements do not precisely determine alternatively spliced sequences or posttranslational modifications. Here, we present the primary structures of ~30,000 unique proteoforms, nearly 10 times more than in previous studies, expressed from 1690 human genes across 21 cell types and plasma from human blood and bone marrow. The results, compiled in the Blood Proteoform Atlas (BPA), indicate that proteoforms better describe protein-level biology and are more specific indicators of differentiation than their corresponding proteins, which are more broadly expressed across cell types. We demonstrate the potential for clinical application, by interrogating the BPA in the context of liver transplantation and identifying cell and proteoform signatures that distinguish normal graft function from acute rejection and other causes of graft dysfunction.
Assuntos
Células Sanguíneas/química , Proteínas Sanguíneas/química , Células da Medula Óssea/química , Bases de Dados de Proteínas , Isoformas de Proteínas/química , Proteoma/química , Processamento Alternativo , Linfócitos B/química , Proteínas Sanguíneas/genética , Linhagem da Célula , Humanos , Leucócitos Mononucleares/química , Transplante de Fígado , Plasma/química , Isoformas de Proteínas/genética , Processamento de Proteína Pós-Traducional , Proteômica , Linfócitos T/químicaRESUMO
BACKGROUND: Flow cytometry (FC) is a popular method to detect bone marrow (BM) involvement in patients with B-cell non-Hodgkin lymphoma (B-NHL). The majority of screen panels of FC still rely on finding monoclonal B-cells, e.g., B-cells with immunoglobin (Ig) light-chain restriction, which has many limitations. Therefore, exploring new markers is warranted. METHODS: A total of 52 cases of B-NHL with BM involvement were collected. The median age was 60 years. Out of these 52 cases, 34 were male, and 18 were female. A 10-color FC panel was used to detect the expression of CD54 on lymphoma cells. The expression of CD54 was calculated as the mean fluorescence index ratio (MFIR) and was described as the mean ± standard error of the mean (SEM). RESULTS: Up to 18/52 (34.62%) of BM specimens abnormally expressed an increased level of CD54, including 1/10 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), 9/13 cases of mantle cell lymphoma (MCL), 2/14 cases of follicular lymphoma (FL), 5/9 cases of marginal zone lymphoma (MZL), and 1/3 cases of high-grade B-NHL (HG B-NHL). The expression level of CD54 was significantly increased in MCL cases (53.41 ± 11.04) compared with CLL/SLL cases (11.66 ± 2.79) and FL cases (13.49 ± 2.81). The lowest percentage of CD54-positive B-cells attained 0.13%. In 5/9 cases of MZL and 1/3 cases of HG B-NHL, increased expression of CD54 was the only abnormal immunophenotype detected besides Ig light-chain restriction. No aberrant CD54 expression was identified by FC in lymphoplasmacytic lymphoma (LPL) (0/2) and Burkitt lymphoma (BL) (0/1) cases. Aberrant expression of CD54 was not related to plasma cell differentiation. CONCLUSION: Lymphoma cells, especially in MCL and MZL cases, frequently show increased expression of CD54. Such aberrant expression is not related to plasma cell differentiation. We highly recommend adding CD54 to the FC screening panel to detect BM involvement in patients with B-NHL.
Assuntos
Células da Medula Óssea , Molécula 1 de Adesão Intercelular , Linfoma de Células B , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/química , Células da Medula Óssea/metabolismo , Feminino , Citometria de Fluxo , Humanos , Molécula 1 de Adesão Intercelular/análise , Molécula 1 de Adesão Intercelular/metabolismo , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
The recent development of experimental methods for measuring chromatin state at single-cell resolution has created a need for computational tools capable of analyzing these datasets. Here we developed Signac, a comprehensive toolkit for the analysis of single-cell chromatin data. Signac enables an end-to-end analysis of single-cell chromatin data, including peak calling, quantification, quality control, dimension reduction, clustering, integration with single-cell gene expression datasets, DNA motif analysis and interactive visualization. Through its seamless compatibility with the Seurat package, Signac facilitates the analysis of diverse multimodal single-cell chromatin data, including datasets that co-assay DNA accessibility with gene expression, protein abundance and mitochondrial genotype. We demonstrate scaling of the Signac framework to analyze datasets containing over 700,000 cells.
Assuntos
Células da Medula Óssea/química , Cromatina/genética , Biologia Computacional/métodos , Leucócitos Mononucleares/química , Mitocôndrias/genética , Análise de Célula Única/métodos , Software , Células da Medula Óssea/metabolismo , Cromatina/química , Cromatina/metabolismo , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Análise de Sequência de DNARESUMO
Plasma cell leukaemia (PCL) is a rare and very aggressive plasma cell disorder. Preventing a dismal outcome of PCL requires early diagnosis with appropriate analytical tools. Therefore, the investigation of 33 patients with primary and secondary PCL was done when the quantity of circulating plasma cells (PCs) using flow cytometry (FC) and morphology assessment was evaluated. The phenotypic profile of the PCs was also analysed to determine if there is an association with clinical outcomes and to evaluate the prognostic value of analysed markers. Our results revealed that FC is an excellent method for identifying circulating PCs as a significantly higher number was identified by FC than by morphology (26·7% vs. 13·5%, P = 0·02). None of secondary PCL cases expressed CD19 or CD20. A low level of expression with similar positivity of CD27, CD28, CD81 and CD117 was found in both PCL groups. A decrease of CD44 expression was detected only in secondary PCL. Expression of CD56 was present in more than half of PCL cases as well as cytoplasmic nestin. A decreased level of platelets, Eastern Cooperative Oncology Group score of 2-3 and lack of CD20+ PC were associated with a higher risk of death. FC could be incorporated in PCL diagnostics not only to determine the number of circulating PCs, but also to assess their phenotype profile and this information should be useful in patients' diagnosis and possible prognosis.
Assuntos
Contagem de Células Sanguíneas , Citometria de Fluxo/métodos , Leucemia Plasmocitária/sangue , Células Neoplásicas Circulantes , Plasmócitos , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/análise , Antígenos de Neoplasias/análise , Contagem de Células Sanguíneas/métodos , Medula Óssea/patologia , Células da Medula Óssea/química , Detecção Precoce de Câncer , Reações Falso-Negativas , Feminino , Humanos , Imunofenotipagem , Estimativa de Kaplan-Meier , Leucemia Plasmocitária/mortalidade , Masculino , Pessoa de Meia-Idade , Plasmócitos/química , Plasmócitos/ultraestrutura , Intervalo Livre de ProgressãoRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia that is most frequent in children and is characterized by the presence of few chromosomal rearrangements and 10 to 20 somatic mutations in protein-coding regions at diagnosis. The majority of T-ALL cases harbor activating mutations in NOTCH1 together with mutations in genes implicated in kinase signaling, transcriptional regulation, or protein translation. To obtain more insight in the level of clonal heterogeneity at diagnosis and during treatment, we used single-cell targeted DNA sequencing with the Tapestri platform. We designed a custom ALL panel and obtained accurate single-nucleotide variant and small insertion-deletion mutation calling for 305 amplicons covering 110 genes in about 4400 cells per sample and time point. A total of 108 188 cells were analyzed for 25 samples of 8 T-ALL patients. We typically observed a major clone at diagnosis (>35% of the cells) accompanied by several minor clones of which some were less than 1% of the total number of cells. Four patients had >2 NOTCH1 mutations, some of which present in minor clones, indicating a strong pressure to acquire NOTCH1 mutations in developing T-ALL cells. By analyzing longitudinal samples, we detected the presence and clonal nature of residual leukemic cells and clones with a minor presence at diagnosis that evolved to clinically relevant major clones at later disease stages. Therefore, single-cell DNA amplicon sequencing is a sensitive assay to detect clonal architecture and evolution in T-ALL.
Assuntos
Evolução Clonal , DNA de Neoplasias/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Análise de Célula Única/métodos , Células Sanguíneas/química , Células da Medula Óssea/química , Criança , Humanos , Mutação INDEL , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Neoplasia Residual/diagnóstico , PTEN Fosfo-Hidrolase/genética , Filogenia , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Notch1/genética , Receptor Notch1/fisiologia , Recidiva , Terapia de Salvação , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
Deletion of the long arm of chromosome 6 (del6q) is the most frequent cytogenetic abnormality in Waldenström macroglobulinaemia (WM), occurring in approximately 50% of patients. Its effect on patient outcome has not been completely established. We used fluorescence in situ hybridisation to analyse the prevalence of del6q in selected CD19+ bone marrow cells of 225 patients with newly diagnosed immunoglobulin M (IgM) monoclonal gammopathies. Del6q was identified in one of 27 (4%) cases of IgM-monoclonal gammopathy of undetermined significance, nine of 105 (9%) of asymptomatic WM (aWM), and 28/93 (30%) of symptomatic WM (sWM), and was associated with adverse prognostic features and higher International Prognostic Scoring System for WM (IPSSWM) score. Asymptomatic patients with del6q ultimately required therapy more often and had a shorter time to transformation (TT) to symptomatic disease (median TT, 30 months vs. 199 months, respectively, P < 0·001). When treatment was required, 6q-deleted patients had shorter progression-free survival (median 20 vs. 47 months, P < 0·001). The presence of del6q translated into shorter overall survival (OS), irrespective of the initial diagnosis, with a median OS of 90 compared with 131 months in non-del6q patients (P = 0·01). In summary, our study shows that del6q in IgM gammopathy is associated with symptomatic disease, need for treatment and poorer clinical outcomes.
Assuntos
Transformação Celular Neoplásica/genética , Macroglobulinemia de Waldenstrom/genética , Idoso , Doenças Assintomáticas , Células da Medula Óssea/química , Células da Medula Óssea/ultraestrutura , Deleção Cromossômica , Cromossomos Humanos Par 6/genética , Feminino , Humanos , Imunoglobulina M/sangue , Imunofenotipagem , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Gamopatia Monoclonal de Significância Indeterminada/genética , Paraproteínas/análise , Prognóstico , Intervalo Livre de Progressão , Medição de Risco , Análise de Sobrevida , Fatores de Tempo , Resultado do Tratamento , Macroglobulinemia de Waldenstrom/patologiaRESUMO
Mammalian hematopoietic stem cells (HSCs) maintain life-long hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after bone marrow injury or in vitro culture. We have recently demonstrated the ability to maintain HSC quiescence in vitro by mimicking the bone marrow microenvironment. Here, we provide a detailed protocol for keeping functional HSCs in the quiescent state in vitro. For complete details on the use and execution of this protocol, please refer to Kobayashi et al. (2019).
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Células da Medula Óssea/química , Células da Medula Óssea/classificação , Células da Medula Óssea/citologia , Proliferação de Células , Células Cultivadas , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/classificação , CamundongosRESUMO
Stroke is one of the most common causes of disability and death, and currently, ideal clinical treatment is lacking. Stem cell transplantation is a widely-used treatment approach for stroke. When compared with other types of stem cells, bone marrow mesenchymal stem cells (BMSCs) have been widely studied because of their many advantages. The paracrine effect is the primary mechanism for stem cells to play their role, and exosomes play an essential role in the paracrine effect. When compared with cell therapy, cell-free exosome therapy can prevent many risks and difficulties, and therefore, represents a promising and novel approach for treatment. In this study, we reviewed the research progress in the application of BMSCs-derived exosomes (BMSCs-exos) and BMSCs in the treatment of stroke. In addition, the advantages and disadvantages of cell therapy and cell-free exosome therapy were described, and the possible factors that hinder the introduction of these two treatments into the clinic were analyzed. Furthermore, we reviewed the current optimization methods of cell therapy and cell-free exosome therapy. Taken together, we hypothesize that cell-free exosome therapy will have excellent research prospects in the future, and therefore, it is worth further exploring. There are still some issues that need to be further addressed. For example, differences between the in vivo microenvironment and in vitro culture conditions will affect the paracrine effect of stem cells. Most importantly, we believe that more preclinical and clinical design studies are required to compare the efficacy of stem cells and exosomes.
Assuntos
Exossomos , Células-Tronco , Acidente Vascular Cerebral/terapia , Animais , Células da Medula Óssea/química , Humanos , Transplante de Células-Tronco , Células-Tronco/químicaRESUMO
Mast cells (MCs) are secretory cells that are central players in human allergic disease and immune responses. With the exception of a few pathological situations, MCs are usually present at relatively low frequencies in most tissues. Since their first description, MCs in tissues were identified mostly using their morphological characteristics and their typical coloration when stained with aniline dyes. However, increasing availability of highly specific antibodies now permits the use of fluorescence-based flow cytometry as the method of choice for the quantification, characterization, and purification of cells in suspension. This technique allows for a rapid analysis of thousands of events and for the identification of cells present at frequencies as low as one event in 106 unwanted cells. This method also permits for simultaneous characterization of multiple antigens at a single cell level, which is ideal in order to study rare populations of cells like MCs. Here we describe the basis of flow cytometry-based immunophenotyping applied to the study of MC. The protocol focuses on the study of human MCs present in body fluids (mainly bone marrow) but can easily be adapted to studying MCs from other tissues and species.
Assuntos
Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Mastócitos/citologia , Antígenos/análise , Antígenos/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Líquidos Corporais/química , Líquidos Corporais/imunologia , Células da Medula Óssea/química , Células da Medula Óssea/imunologia , Fluorescência , Humanos , Mastócitos/química , Mastócitos/imunologia , Mastócitos/patologia , Mastocitose/diagnóstico , Mastocitose/imunologia , Coloração e Rotulagem/métodosRESUMO
BACKGROUND: The oncolytic viruses have shown promising results for the treatment of multiple myeloma. However, the use of human viruses is limited by the patients' antiviral immune response. In this study, we investigated an alternative oncolytic strategy using non-human pathogen viruses as the bovine viral diarrhea virus (BVDV) that were able to interact with CD46. METHODS: We treated several human myeloma cell lines and non-myeloma cell lines with BVDV to evaluate the expression of CD46 and to study the effect on cell viability by flow cytometry. The possible synergistic effect of bortezomib in combination with BVDV was also tested. Moreover, we infected the bone marrow mononuclear cells obtained from myeloma patients and we checked the BVDV effect on different cell populations, defined by CD138, CD14, CD3, CD19, and CD56 expression evaluated by flow cytometry. Finally, the in vivo BVDV effect was tested in NOD-SCID mice injected subcutaneously with myeloma cell lines. RESULTS: Human myeloma cells were selectively sensitive to BVDV treatment with an increase of cell death and, consequently, of apoptotic markers. Consistently, bone marrow mononuclear cells isolated from myeloma patients treated with BVDV, showed a significant selective decrease of the percentage of viable CD138+ cells. Interestingly, bortezomib pre-treatment significantly increased the cytotoxic effect of BVDV in myeloma cell lines with a synergistic effect. Finally, the in vitro data were confirmed in an in vivo myeloma mouse model showing that BVDV treatment significantly reduced the tumoral burden compared to the vehicle. CONCLUSIONS: Overall, our data indicate, for the first time, a direct oncolytic effect of the BVDV in human myeloma cells suggesting its possible use as novel alternative anti-myeloma virotherapy strategy.
Assuntos
Vírus da Diarreia Viral Bovina , Mieloma Múltiplo/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos CD/análise , Apoptose , Células da Medula Óssea/química , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/virologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Vírus da Diarreia Viral Bovina/fisiologia , Feminino , Herpesvirus Bovino 4 , Humanos , Masculino , Proteína Cofatora de Membrana/biossíntese , Proteína Cofatora de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Vírus Oncolíticos/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Organismos Livres de Patógenos EspecíficosRESUMO
Bone marrow mesenchymal stem cells (BM-MSC) have recently been predicted to have a major therapeutic potential due to secretion of soluble factors and the release of cytokines and growth factors, which could mediate the cellular communication to induce cell differentiation/maturity. The aim of the present study was to determine the effect of mBM condition medium on morphine-induced cell death in PC12, U87, AGS and MCF-7 cell lines. The condition media were harvested as mBM soup (mBM soup 24 and mBM soup 48h, respectively). To investigate the effect of mBM soup on cell lines, morphological changes were studied with an inverted microscope, the viability of cells was determined with trypan blue staining and MTT assay, the type of cell death was determined using Hoescht / PI staining, and NO secretion analysis. Viability assay showed that mBM soup (24 and 48 h) in time-dependent manner increased cell viability (pndings suggest that mBM soup can enhance the proliferation and growth of cell lines and can suppress cell death induced by morphine (Fig. 8, Ref. 59). Text in PDF www.elis.sk. Keywords: morphine, BM-MSC soup, cell viability, cell death, NO.
Assuntos
Células da Medula Óssea/química , Meios de Cultivo Condicionados/farmacologia , Células-Tronco Mesenquimais/química , Morfina/farmacologia , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Humanos , Células MCF-7 , Células PC12 , RatosRESUMO
BACKGROUND: The current study aims to analyze the expression of miR-34a in bone marrow mononuclear cells (BMMCs) of acute myeloid leukemia (AML) patients and its possible role in the development of leukemia. METHODS: Real-time fluorescent quantitative PCR was used to detect the expression of miR-34a in bone marrow of 40 patients with initial treatment of AML, 22 patients with complete remission AML, 13 patients with refractory recurrence, 20 patients with non-hematological tumors and healthy persons. Pearson's correlation was performed to analyze the correlation between the expression level of miR-34a and clinical indicators in patients. RESULTS: The expression of miR-34a in the initial treatment group, complete remission group and refractory relapse group of AML patients was higher than that in the control group. In addition, the expression of miR-34a in the complete remission group was higher than that in the initial treatment group. Moreover, the expression of miR-34a in the refractory relapse group was lower than that in the initial treatment group. The expression level of miR-34a in initial treatment AML group was negatively correlated with the number of bone marrow primary immature cells, but not with the number of peripheral blood leukocytes, platelet count, hemoglobin content, age, gender, and chromosomal abnormalities. CONCLUSIONS: The expression of miR-34a in bone marrow mononuclear cells of patients with AML was down-regulated and related to tumor load. It may play an important role in the occurrence and development of leukemia and may become a new molecular marker for the diagnosis of AML.
Assuntos
Células da Medula Óssea/química , Leucemia Mieloide Aguda/diagnóstico , MicroRNAs/análise , Adulto , Idoso , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e EspecificidadeRESUMO
A simple, sensitive, and rapid liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method was developed for the simultaneous determination of arginine and its pathway-related metabolites (ornithine, proline, citrulline, glutamate, agmatine, spermidine, and spermine) in cellular extracts. Cells were lysed and cellular proteins precipitated by the addition of acetonitrile followed by ultra-sonication. Supernatants were analyzed using a Chromolith High Resolution RP-18 endcapped column (100 × 4.6 mm, 1.15 µm, 150 Å), with mobile phases of 0.1% formic acid solution and 0.1% formic acid in acetonitrile. Detection was carried out in multiple reaction monitoring (MRM) mode. Calibration curves showed linearity (r2 > 0.99) for all metabolites over the calibration ranges used. The intra- and inter-day precision was less than 13.5%, and the accuracy was between 91.3 and 114.7%. The method developed in this study was successfully applied to measure arginine and its pathway-related metabolites, which are related to nitric oxide synthase/arginase pathways in mouse bone marrow-derived dendritic cells (BMDCs). The ability to simultaneously measure arginine and its pathway-related metabolites is valuable for better understanding local and systemic inflammatory processes.
Assuntos
Aminoácidos/análise , Arginina/análise , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Agmatina/análise , Agmatina/metabolismo , Aminoácidos/metabolismo , Animais , Arginina/metabolismo , Células da Medula Óssea/química , Células da Medula Óssea/metabolismo , Células Cultivadas , Células Dendríticas/química , Células Dendríticas/metabolismo , Limite de Detecção , Modelos Lineares , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Espermidina/análise , Espermidina/metabolismoRESUMO
RNA quality (purity and integrity) and quantity are of critical importance to ensure reliable gene expression analysis, reproducibility of RNA sequencing and microarray data and validation by RT-PCR. Currently available methods for isolating RNA either are labor intensive (requiring the use of toxic organic solvents and separate DNase treatment) or require automation (with extensive setup and startup costs). To optimize both the quality and quantity of RNA from bone marrow, we recommend stabilization and storage of bone marrow mononuclear cells in RNAprotect® Cell Reagent, followed by extraction using the RNeasy® Protect Cell Mini Kit (Qiagen, Hilden, Germany). This method achieves optimal quantity and high-quality RNA for sequencing and RT-PCR while remaining efficient and cost effective.
Assuntos
Células da Medula Óssea/química , Fracionamento Químico/métodos , RNA/isolamento & purificação , Células da Medula Óssea/citologia , DNA/química , Humanos , Reação em Cadeia da Polimerase , RNA/análise , RNA/química , Análise EspectralRESUMO
Traditional tendon-to-bone repair where the tendon is reattached to bone via suture anchors often results in disorganized scar production rather than the formation of a zonal insertion. In contrast, ligament reconstructions where tendon grafts are passed through bone tunnels can yield zonal tendon-to-bone attachments between the graft and adjacent bone. Therefore, ligament reconstructions can be used to study mechanisms that regulate zonal tendon-to-bone repair in the adult. Anterior cruciate ligament (ACL) reconstructions are one of the most common reconstruction procedures and while we know that cells from outside the graft produce the attachments, we have not yet established specific cell populations that give rise to this tissue. To address this knowledge gap, we performed ACL reconstructions in lineage tracing mice where α-smooth muscle actin (αSMACreERT2) was used to label αSMA-expressing progenitors within the bone marrow that produced zonal attachments. Expression of αSMA was increased during early stages of the repair process such that the contribution of SMA-labeled cells to the tunnel integration was highest when tamoxifen was delivered in the first week post-surgery. The zonal attachments shared features with normal entheses, including tidemarks oriented perpendicularly to collagen fibers, Col1a1-expressing cells, alkaline phosphatase activity, and proteoglycan-rich staining. Finally, the integration strength increased with time, requiring 112% greater force to remove the graft from the tunnel at 28 days compared with 14 days post-surgery. Future studies will target these progenitor cells to define the pathways that regulate zonal tendon-to-bone repair in the adult. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:105-116, 2020.
Assuntos
Actinas/análise , Reconstrução do Ligamento Cruzado Anterior/métodos , Células da Medula Óssea/metabolismo , Osso e Ossos/cirurgia , Células-Tronco/metabolismo , Tendões/cirurgia , Actinas/fisiologia , Animais , Células da Medula Óssea/química , Camundongos , CicatrizaçãoRESUMO
Mesenchymal stem or stromal cells are currently under clinical investigation for multiple diseases. While their mechanism of action is still not fully elucidated, vesicles secreted by MSCs are believed to recapitulate their therapeutic potentials to some extent. Microvesicles (MVs), also called as microparticles or ectosome, are among secreted vesicles that could transfer cytoplasmic cargo, including RNA and proteins, from emitting (source) cells to recipient cells. Given the importance of MVs, we here attempted to establish a method to isolate and characterize MVs secreted from unmodified human bone marrow derived MSCs (referred to as native MSCs, and their microvesicles as Native-MVs) and IFNγ stimulated MSCs (referred to as IFNγ-MSCs, and their microvesicles as IFNγ-MVs). We first describe an ultracentrifugation technique to isolate MVs from the conditioned cell culture media of MSCs. Next, we describe characterization and quality control steps to analyze the protein and RNA content of MVs. Finally, we examined the potential of MVs to exert immunomodulatory effects through induction of regulatory T cells (Tregs). Secretory vesicles from MSCs are promising alternatives for cell therapy with applications in drug delivery, regenerative medicine, and immunotherapy.
Assuntos
Micropartículas Derivadas de Células/química , Sistemas de Liberação de Medicamentos/métodos , Células-Tronco Mesenquimais/química , Proteômica/métodos , Medicina Regenerativa/métodos , Animais , Células da Medula Óssea/química , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Separação Celular/métodos , Micropartículas Derivadas de Células/imunologia , Meios de Cultivo Condicionados/química , Humanos , Imunoterapia/métodos , Interferon gama/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/imunologia , Proteínas/classificação , Proteínas/isolamento & purificação , RNA/classificação , RNA/isolamento & purificação , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologiaRESUMO
Cell-free DNA (cfDNA) analysis to detect circulating tumor DNA has been focused on monitoring malignant lymphomas. However, clonal hematopoiesis of indeterminate potential (CHIP)-associated mutations can also be detected by cfDNA analysis. Our aim is to investigate the origin of mutations detected in cfDNA among B-cell lymphoma patients. MYD88/CD79B, DNMT3A, and TP53 were chosen as genes of interest, representing each of the following categories: lymphoma driver genes, CHIP-related genes, and genes shared between lymphoma and CHIP. Seventy-five B-cell lymphoma patients were included in this retrospective study. Serum cfDNAs at time of complete metabolic response (CMR) were sequenced for TP53 (N = 75) and DNMT3A (N = 49). MYD88 p.L265P and CD79B p.Y196C/H mutations were analyzed in diffuse large B-cell lymphoma (DLBCL) patients whose tumor samples were available (N = 29). Two and seven mutations in TP53 and DNMT3A, respectively, were detected in cfDNA at CMR. These mutations were detected in either bone marrow mononuclear cells (BMMC) or PBMC. Although four DNMT3A mutations were also detected in tumors, median variant allele frequencies in the tumors (<1.0%) were significantly lower than those in both BMMC (6.1%) and serum (5.2%) obtained before the therapy. Conversely, five MYD88 and three CD79B mutations detected in tumors were confirmed in cfDNA before therapy, but not in BMMC nor in cfDNA at CMR. Thus, all TP53 and DNMT3A mutations detected in cfDNA at remission seemed to originate from CHIP rather than from residual disease. Results of liquid biopsy should be carefully interpreted, especially in genes shared between lymphomas and CHIP.
Assuntos
Células Clonais/química , DNA (Citosina-5-)-Metiltransferases/genética , Hematopoese , Linfoma de Células B/genética , Mutação , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/química , Ácidos Nucleicos Livres/genética , DNA Metiltransferase 3A , Feminino , Frequência do Gene , Humanos , Leucócitos Mononucleares/química , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Monócitos/química , Indução de Remissão , Estudos Retrospectivos , Análise de Sequência de DNA/métodosRESUMO
Multiple myeloma (MM) is malignant tumor with abnormal proliferation of bone marrow plasma cells. The existing clinical tools used to determine treatment response and tumor relapse are limited in sensitivity. We investigated the CD138+ microparticles (MPs) of MM patients to find out whether MPs could provide a novel means to monitor the malignant cells in MM patients. Our study showed that the levels of MPs were significantly elevated in MM patients. The MP counts in peripheral blood from new diagnosed MM patients were significantly higher than patients in CR and HD. Consist with the total MPs, the number of the PC-derived MPs (CD138+) increased in BM from MM patients compared with CR and HD. The ratio of the PC-derived MPs (CD138+) in BM increased in MM patients compared with CR and HD. The correlation test revealed that the CD138+ MPs in BM and PB were all positively correlated with the plasmacyte ratio in bone marrow (BMPC) and the ß2 -MG. New diagnosed MM patients and controls were compared, and ROC curves were used to identify cutoff points with optimal sensitivity and specificity concerning the ratios and counts of CD138+ MPs in BM and PB. The AUC of the CD138+ MP counts in BM was 0.767, and in PB was 0.680. The AUC of the CD138+ MP ratios in BM was 0.714, and in PB was 0.666. According to this, the counts of CD138+ MPs in BM showed to be a powerful marker of diagnosis. We demonstrated that CD138+ MPs from the plasma provide support for a potential monitoring biomarker of MM.
Assuntos
Células da Medula Óssea/química , Medula Óssea/patologia , Micropartículas Derivadas de Células/química , Mieloma Múltiplo/sangue , Proteínas de Neoplasias/sangue , Sindecana-1/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Biomarcadores Tumorais/sangue , Separação Celular/métodos , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/patologia , Plasmócitos/patologia , Curva ROC , Sensibilidade e Especificidade , Sindecana-1/análise , Microglobulina beta-2/análiseRESUMO
Systemic Sclerosis (SSc) is a disease with limited therapeutic possibilities. Mesenchymal stem cells (MSCs)-therapy could be a promising therapeutic option, however the ideal MSCs source has not yet been found. To address this problem, we perform comparison between bone marrow (BM)-MSCs and adipose (A)-MSCs, by the miRs expression profile, to identify the gene modulation in these two MSCs source. MicroRNAs (miRs) are RNAs sequences, regulating gene expression and MSCs, derived from different tissues, may differently respond to the SSc microenvironment. The miRs array was used for the miRs profiling and by DIANA-mirPath tool we identified the biological functions of the dysregulated miRs. In SSc-BM-MSCs, 6 miRs were significantly down-regulated and 4 miRs up-regulated. In SSc-A-MSCs, 11 miRs were significantly down-regulated and 3 miRs up-regulated. Interestingly, in both the sources, the involved pathways included the senescence mechanisms and the pro-fibrotic behaviour. Furthermore, both the MSCs sources showed potential compensatory ability. A deeper knowledge of this miRs signature might give more information about some pathogenic steps of the disease and in the same time clarify the possible therapeutic role of autologous MSCs in the regenerative therapy in SSc.