RESUMO
Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have been described to have important roles in tissue regeneration, including tissue repair, control of inflammation, enhancing angiogenesis, and regulating extracellular matrix remodeling. MSC-EVs have many advantages for use in regeneration therapies such as facility for dosage, histocompatibility, and low immunogenicity, thus possessing a lower possibility of rejection. In this work, we address the potential activity of MSC-EVs isolated from adipose-derived MSCs (ADMSC-EVs) cultured on cross-linked dextran microcarriers, applied to test the scalability and reproducibility of EV production. Isolated ADMSC-EVs were added into cultured human dermal fibroblasts (NHDF-1), keratinocytes (HaCat), endothelial cells (HUVEC), and THP-1 cell-derived macrophages to evaluate cellular responses (i.e., cell proliferation, cell migration, angiogenesis induction, and macrophage phenotype-switching). ADMSC viability and phenotype were assessed during cell culture and isolated ADMSC-EVs were monitored by nanotracking particle analysis, electron microscopy, and immunophenotyping. We observed an enhancement of HaCat proliferation; NHDF-1 and HaCat migration; endothelial tube formation on HUVEC; and the expression of inflammatory cytokines in THP-1-derived macrophages. The increased expression of TGF-ß and IL-1ß was observed in M1 macrophages treated with higher doses of ADMSC-EVs. Hence, EVs from microcarrier-cultivated ADMSCs are shown to modulate cell behavior, being able to induce skin tissue related cells to migrate and proliferate as well as stimulate angiogenesis and cause balance between pro- and anti-inflammatory responses in macrophages. Based on these findings, we suggest that the isolation of EVs from ADMSC suspension cultures makes it possible to induce in vitro cellular responses of interest and obtain sufficient particle numbers for the development of in vivo concept tests for tissue regeneration studies.
Assuntos
Proliferação de Células , Vesículas Extracelulares , Macrófagos , Células-Tronco Mesenquimais , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Macrófagos/citologia , Movimento Celular , Células THP-1 , Fibroblastos/metabolismo , Fibroblastos/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Técnicas de Cultura de Células/métodos , Células Cultivadas , Queratinócitos/metabolismo , Queratinócitos/citologia , Citocinas/metabolismoRESUMO
Ischemic heart diseases are a global health problem that requires the search for alternative therapies to the current treatments. Thus, an understanding of how cardiomyogenic signals can affect cellular behavior would allow us to create strategies to improve the cell recovery in damaged tissues. In this study, we aimed to evaluate the effects of the conditioned medium (CM), collected at different time points during in vitro cardiomyogenesis of human embryonic stem cells (hESCs), to direct cell behavior. We assayed different cell types to demonstrate noncytotoxic effects from the collected CM and that the CM obtained at initial time points of cardiomyogenic differentiation could promote the cell proliferation. Otherwise, the secretome derived from cardiac committed cells during cardiomyogenesis was unable to improve angiogenesis or migration in endothelial cells, and ineffective to stimulate the differentiation of cardioblasts or increase the differentiation efficiency of hESC. Therefore, we demonstrated that the effectiveness of the CM response varies depending on the cell type and the differentiation step of hESC-derived cardiomyocytes.
Assuntos
Células-Tronco Embrionárias/fisiologia , Desenvolvimento Muscular/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Retroalimentação , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , RatosRESUMO
Myocardial infarction is a leading cause of death among all cardiovascular diseases. Cell therapies using a cell population enriched with endothelial progenitor cells (EPCs), expanded CD133+ cells, have promise as a therapeutic option for the treatment of ischemic areas after infarction. Recently, secreted membrane vesicles, including exosomes and microvesicles, have been recognized as new therapeutic candidates with important roles in intercellular and tissue communication. Expanded CD133+ cells have the ability to produce extracellular vesicles (EVs); however, their effect in the context of the heart is unknown. In the present study, we evaluated the effectiveness of the systemic application of expanded CD133+ cells and expanded CD133+ cell-derived EVs for the treatment of ischemic cardiomyopathy in a rat model of acute myocardial infarction (AMI) and examined the hypothesis that the EVs, because of their critical role in transferring regenerative signals from stem cells to the injured tissues, might elicit an equal or better therapeutic response than the expanded CD133+ cells. We demonstrate that the systemic application of expanded CD133+ cells and EVs has similar effects in infarcted rats. Few animals per group showed improvements in several heart and kidney parameters analyzed, but not significant differences were observed when comparing the groups. The systemic route may not be effective to treat ischemic cardiomyopathy; nonetheless, it may be a beneficial therapy to treat the side effects of AMI such as kidney damage.
RESUMO
Pluripotent stem cells (PSC) can be used as a model to study cardiomyogenic differentiation. In vitro modeling can reproduce cardiac development through modulation of some key signaling pathways. Therefore, many studies make use of this strategy to better understand cardiomyogenesis complexity and to determine possible ways to modulate cell fate. However, challenges remain regarding efficiency of differentiation protocols, cardiomyocyte (CM) maturation and therapeutic applications. Considering that the extracellular milieu is crucial for cellular behavior control, cardiac niche studies, such as those identifying secreted molecules from adult or neonatal tissues, allow the identification of extracellular factors that may contribute to CM differentiation and maturation. This review will focus on cardiomyogenesis modeling using PSC and the elements involved in cardiac microenvironmental signaling (the secretome - extracellular vesicles, extracellular matrix and soluble factors) that may contribute to CM specification and maturation.
RESUMO
Dengue virus (DV) infection can cause either a self-limiting flu-like disease or a threatening hemorrhage that may evolve to shock and death. A variety of cell types, such as dendritic cells, monocytes, and B cells, can be infected by DV. However, despite the role of T lymphocytes in the control of DV replication, there remains a paucity of information on possible DV-T cell interactions during the disease course. In the present study, we have demonstrated that primary human naive CD4+ and CD8+ T cells are permissive for DV infection. Importantly, both T cell subtypes support viral replication and secrete viable virus particles. DV infection triggers the activation of both CD4+ and CD8+ T lymphocytes, but preactivation of T cells reduces the susceptibility of T cells to DV infection. Interestingly, the cytotoxicity-inducing protein granzyme A is highly secreted by human CD4+ but not CD8+ T cells after exposure to DV in vitro Additionally, using annexin V and polycaspase assays, we have demonstrated that T lymphocytes, in contrast to monocytes, are resistant to DV-induced apoptosis. Strikingly, both CD4+ and CD8+ T cells were found to be infected with DV in acutely infected dengue patients. Together, these results show that T cells are permissive for DV infection in vitro and in vivo, suggesting that this cell population may be a viral reservoir during the acute phase of the disease.IMPORTANCE Infection by dengue virus (DV) causes a flu-like disease that can evolve to severe hemorrhaging and death. T lymphocytes are important cells that regulate antibody secretion by B cells and trigger the death of infected cells. However, little is known about the direct interaction between DV and T lymphocytes. Here, we show that T lymphocytes from healthy donors are susceptible to infection by DV, leading to cell activation. Additionally, T cells seem to be resistant to DV-induced apoptosis, suggesting a potential role as a viral reservoir in humans. Finally, we show that both CD4+ and CD8+ T lymphocytes from acutely infected DV patients are infected by DV. Our results raise new questions about DV pathogenesis and vaccine development.
Assuntos
Apoptose/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Vírus da Dengue/imunologia , Dengue/imunologia , Ativação Linfocitária/imunologia , Adolescente , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Dengue/virologia , Vírus da Dengue/fisiologia , Feminino , Granzimas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Replicação Viral/imunologia , Adulto JovemRESUMO
Adult stem cells have beneficial effects when exposed to damaged tissue due, at least in part, to their paracrine activity, which includes soluble factors and extracellular vesicles (EVs). Given the multiplicity of signals carried by these vesicles through the horizontal transfer of functional molecules, human mesenchymal stem cell (hMSCs) and CD133+ cell-derived EVs have been tested in various disease models and shown to recover damaged tissues. In this study, we profiled the protein content of EVs derived from expanded human CD133+ cells and bone marrow-derived hMSCs with the intention of better understanding the functions performed by these vesicles/cells and delineating the most appropriate use of each EV in future therapeutic procedures. Using LC-MS/MS analysis, we identified 623 proteins for expanded CD133+-EVs and 797 proteins for hMSCs-EVs. Although the EVs from both origins were qualitatively similar, when protein abundance was considered, hMSCs-EVs and CD133+-EVs were different. Gene Ontology (GO) enrichment analysis in CD133+-EVs revealed proteins involved in a variety of angiogenesis-related functions as well proteins related to the cytoskeleton and highly implicated in cell motility and cellular activation. In contrast, when overrepresented proteins in hMSCs-EVs were analyzed, a GO cluster of immune response-related genes involved with immune response-regulating factors acting on phagocytosis and innate immunity was identified. Together our data demonstrate that from the point of view of protein content, expanded CD133+-EVs and hMSCs-EVs are in part similar but also sufficiently different to reflect the main beneficial paracrine effects widely reported in pre-clinical studies using expanded CD133+ cells and/or hBM-MSCs.
Assuntos
Vesículas Extracelulares/metabolismo , Sangue Fetal/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteoma/metabolismo , Medicina Regenerativa/métodos , Antígeno AC133/sangue , Apoptose , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proliferação de Células , Células Cultivadas , Cromatografia Líquida , Exossomos/metabolismo , Exossomos/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Sangue Fetal/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Microscopia Eletrônica de Transmissão , Necrose , Proteômica/métodos , Espectrometria de Massas em TandemRESUMO
Although fibroblasts and multipotent stromal/stem cells, including adipose-derived stromal cells (ADSCs), have been extensively studied, they cannot be clearly distinguished from each other. We, therefore, investigated the cellular and molecular characteristics of ADSCs and fibroblasts. ADSCs and fibroblasts share several morphological similarities and surface markers, but were clearly found to be different types of cells. Contrary to previous reports, fibroblasts were not able to differentiate into adipocytes, osteoblasts, or chondrocytes. Polysome-bound mRNA profiling revealed that â¼ 1,547 genes were differentially expressed (DE) in the two cell types; the genes were related to cell adhesion, the extracellular matrix, differentiation, and proliferation. These findings were confirmed by functional analyses showing that ADSCs had a greater adhesion capacity than fibroblasts; the proliferation rate of fibroblasts was also higher than that of ADSCs. Importantly, 185 DE genes were integral to the plasma membrane and, thus, candidate markers for ADSC isolation and manipulation. We also observed that an established marker of fibroblasts and ADSCs, CD105, was overexpressed in ADSCs at both mRNA and protein levels. CD105 expression seemed to be related to differentiation capacity, at least for adipogenesis. This study shows that ADSCs and fibroblasts are distinct cell types. These findings should be taken into account when using these two cell types in basic and therapeutic studies.
Assuntos
Adipócitos/fisiologia , Tecido Adiposo/fisiologia , Fibroblastos/fisiologia , Polirribossomos/metabolismo , Células Estromais/fisiologia , Adipócitos/metabolismo , Adipogenia/genética , Adipogenia/fisiologia , Tecido Adiposo/metabolismo , Adesão Celular/genética , Adesão Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/fisiologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Fibroblastos/metabolismo , Humanos , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Polirribossomos/genética , RNA Mensageiro/genética , Células Estromais/metabolismoRESUMO
Adipocyte stem cells (hASCs) can proliferate and self-renew and, due to their multipotent nature, they can differentiate into several tissue-specific lineages, making them ideal candidates for use in cell therapy. Most attempts to determine the mRNA profile of self-renewing or differentiating stem cells have made use of total RNA for gene expression analysis. Several lines of evidence suggest that self-renewal and differentiation are also dependent on the control of protein synthesis by posttranscriptional mechanisms. We used adipogenic differentiation as a model, to investigate the extent to which posttranscriptional regulation controlled gene expression in hASCs. We focused on the initial steps of differentiation and isolated both the total mRNA fraction and the subpopulation of mRNAs associated with translating ribosomes. We observed that adipogenesis is committed in the first days of induction and three days appears as the minimum time of induction necessary for efficient differentiation. RNA-seq analysis showed that a significant percentage of regulated mRNAs were posttranscriptionally controlled. Part of this regulation involves massive changes in transcript untranslated regions (UTR) length, with differential extension/reduction of the 3'UTR after induction. A slight correlation can be observed between the expression levels of differentially expressed genes and the 3'UTR length. When we considered association to polysomes, this correlation values increased. Changes in the half lives were related to the extension of the 3'UTR, with longer UTRs mainly stabilizing the transcripts. Thus, changes in the length of these extensions may be associated with changes in the ability to associate with polysomes or in half-life.
Assuntos
Adipócitos/fisiologia , Polirribossomos/fisiologia , Células-Tronco/fisiologia , Regiões 3' não Traduzidas , Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia , Adulto , Diferenciação Celular/fisiologia , Feminino , Regulação da Expressão Gênica , Glutationa/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Polirribossomos/genética , Polirribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Transcrição Gênica , Adulto JovemRESUMO
The use of conditioned medium (CM) from human cardiac explants (HCEs) as a potential source of paracrine factors for adult stem cell signaling has never been evaluated. We hypothesized that HCEs might provide a source of soluble factors triggering the differentiation of mesenchymal stem cells (MSCs) into cardiomyocyte-like cells. By using two-dimensional electrophoresis (2-DE) gels/mass spectrometry and antibody macroarray assays, we found that HCEs release macromolecules, including cytokines, growth factors and myocardial and metabolism-related proteins into the culture medium. We identified a total of 20 proteins in the HCE-CM. However, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-DE, these 20 proteins account for only a fraction of the total number of proteins present in the HCE-CM. We also found that CM increased the proliferation of bone marrow-derived-MSCs (BM-MSCs) in vitro. Unlike the other effects, this effect was most evident after 48 h of culture. Moreover, we examined the effect of HCE-CM on levels of mRNA and protein for specific cardiac markers. We showed that a surprisingly big fraction of BM-MSCs (3.4-5.0%) treated in vitro with HCE-CM became elongated and began to express cardiac markers, consistent with their possible differentiation into cardiomyocyte-like cells. Our in vitro model may be useful not only per se, but also for studies of the mechanisms of action of soluble factors involved in cell differentiation, paving the way for possible new protein-based treatments in the future.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Técnicas de Cultura , Citocinas/química , Citocinas/farmacologia , Eletroforese em Gel Bidimensional , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Espectrometria de Massas , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Análise em Microsséries , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas/química , Proteínas/farmacologia , RNA Mensageiro/metabolismo , Solubilidade , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
The ephrin-Eph ligand receptor pair is known to control the repulsion/adhesion process in different tissues, including the immune system. Herein, we evaluated the role of EphB2 receptors in T cell progenitor migration during in vitro thymus colonization and to ECM or chemokine stimuli. EphB2 and their ligands, ephrin-B1 and ephrin-B2, are expressed in BM-derived progenitors, and EphB2(-/-) cells had diminished thymus colonization capacity. Conversely, EphB2(LacZ) cells, which maintain a preserved ephrin-binding domain, were capable of colonizing WT thymuses similarly to WT progenitors, highlighting the importance of reverse signals transmitted to normal fetal thymus. However, the EphB2 receptor expressed by microenvironmental cells also drives progenitor immigration, as recolonization of EphB2-deficient fetal thymuses was compromised profoundly. Additionally, we observed lower depositions of ECM and chemokines on EphB2-deficient thymuses but no changes in their receptor expression on BM-derived progenitors and developing thymocytes. Migration of EphB2-deficient progenitors and thymocytes was also reduced through ECM or chemokine stimuli. Furthermore, ephrin-B1 costimulation also inhibited haptotaxis and chemotaxis of WT but not EphB2(LacZ) cells, demonstrating the specific involvement of EphB2 signaling on T cell progenitor migration. Our data suggest the relevance of a nonactivated EphB2 for regulating T cell progenitor migration and its modulation upon ephrin-B engagement.
Assuntos
Comunicação Celular/imunologia , Movimento Celular/imunologia , Efrina-B2/metabolismo , Feto/citologia , Células-Tronco/citologia , Linfócitos T/citologia , Timo/embriologia , Animais , Células da Medula Óssea/citologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Membrana Celular/metabolismo , Quimiocinas/metabolismo , Ensaio de Unidades Formadoras de Colônias , Efrina-B1/metabolismo , Efrina-B2/deficiência , Proteínas da Matriz Extracelular/metabolismo , Ligantes , Camundongos , Receptor EphB2/metabolismo , Receptores Fc/metabolismo , Células-Tronco/imunologia , Linfócitos T/imunologia , Timo/citologia , Timo/imunologiaRESUMO
Las Eph son la mayor familia de receptores tirosina quinasa presentesen la mayoría de tipos celulares. Junto con sus ligandos, las ephrinas, lasEph participan en la organogénesis de muchos tejidos regulando numerosos procesos, como el posicionamiento y la migración celular, los cualesson claves para el correcto funcionamiento del timo, un órgano linfoide primario implicado en la maduración de las células T. En el presente trabajo,revisamos diferentes resultados sobre el papel que estas moléculas juegan en la biología del timo. La mayoría de las Eph y ephrinas se expresanen el timo adulto y fetal, tanto en los timocitos como en las células del estroma. Estas moléculas tienen un papel esencial regulando el tamaño del timo,a través del control de la supervivencia de los timocitos y de las células epiteliales tímicas (TEC). Además, estudios in vivo e in vitro demuestran quemodificaciones en la señalización de Eph y ephrinas resultan en fenotipostímicos específicos, concluyendo que dicha señalización determina finalmente tanto el patrón de maduración y diferenciación de los timocitos comoel de las TEC. El papel de Eph y ephrinas en la función del timo aparecepronto en la ontogenia. En este sentido, varios resultados apoyan su relevancia en procesos claves para la organización del órgano, tales como elreclutamiento de los progenitores linfoides al primordio tímico, el patrónde ramificación del epitelio tímico y el posicionamiento de los timocitos ylas TEC en el timo en crecimiento. Algunas de las alteraciones fenotípicasobservadas en el timo de ratones deficientes en Eph y ephrinas se observantambién en los órganos linfoides periféricos, pero no hay evidencias de unaalteración funcional en sus sistemas inmunes (AU)
Eph are the largest family of protein tyrosine kinases, which are described in most cell types. Together with their ligands, ephrins, Eph participate in the organogenesis of many tissues mediating numerous processes, such as cell positioning and cell migration, which are key for thefunctioning of the thymus: a primary lymphoid organ involved in T-cellmaturation. In the present study, we review available data on the role played by these families of molecules in the biology of the thymus gland.Most Eph and ephrin are expressed in adult and fetal thymus, frequentlyin both thymocytes and thymic stromal cells. They appear to play an essential role in governing thymus size through the control of survival of thymocytes and thymic epithelial cells (TEC). Furthermore, studies in vivoand in vitro demonstrate that altered Eph/ephrin signalling results in specific thymus phenotypes and conclude that the balance of Eph/ephrinsignals finally determine the pattern of maturation/differentiation of thymocytes and TEC. Indeed, the role of Eph/ephrin in thymus functionappears early in ontogeny. In this regard, several results emphasize theirrelevance in key processes for thymus organization, such as the recruitment of lymphoid progenitors to the thymic primordium, the branchingpattern of thymus epithelium and the positioning of thymocytes and TECin the growing gland. Some of the phenotypic alterations observed in thethymus of Eph/ephrin deficient mice are reflected in the peripheral lymphoid organs, but there is no evidence for alterations in the function of theirimmune systems (AU)