RESUMO
Although mesenchymal stromal cells (MSCs) possess the capacity to modulate immune responses, little is known about the mechanisms that underpin these processes. In this study, we show that immunosupression is mediated by activation of nuclear factor kappa B (NF-κB) in human MSCs. This pathway is activated by TNF-α that is generated following TCR stimulation of T cells. Inhibition of NF-κB through silencing of IκB kinase ß or the TNF-α receptor abolishes the immunosuppressive capacity of MSCs. Our data also indicate that MSC-associated NF-κB activation primarily leads to inhibition of T-cell proliferation with little effect on expression of the activation markers CD69 and CD25. Thus, our data support the hypothesis that the TNF-α/NF-κB signalling pathway is required for the initial priming of immunosuppressive function in human MSCs. Interestingly, drugs that interfere with NF-κB activation significantly antagonise the immunoregulatory effect of MSCs, which could have important implications for immunosuppression regimens in the clinic.
Assuntos
Ativação Linfocitária/imunologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , NF-kappa B/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Proliferação de Células , Células Cultivadas , Humanos , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , NF-kappa B/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The recognition of the therapeutic potential of Multipotent Mesenchymal Stromal Cells (MSCs) is one of the most exciting recent advances in cell therapy. In just ten years, since the description of the multilineage potential of MSCs by Pittenger et al in 1999 until now, MSCs are being used in more than 150 clinical trials as therapeutic agents. The potential of these cells for cell-based therapies relies on several key properties: (1) their capacity to differentiate into several cell lineages; (2) their lack of immunogenicity and their immunomodulatory properties; (3) their ex vivo expansion potential; (4) their ability to secrete soluble factors which regulate crucial biological functions such as proliferation and differentiation over a broad spectrum of target cells; and (5) their ability to home to damaged tissues and tumor sites. Based on these properties MSCs are being exploited worldwide for a wide range of potential clinical applications including cell replacement strategies, treatment of graft-versus-host disease, autoimmune diseases and rejection after solid organ transplantation as well as their use as vehicles to deliver anti-cancer therapies. Importantly, the low inherent immunogenicity of MSCs means that they could be used not only for autologous but also for allogeneic cell therapies. In addition, increasing evidence has revealed a complex relationship between MSCs and cancer. Thus, solid evidence has placed MSCs transformed with specific mutations as the most likely cell of origin for certain sarcomas, and MSCs have been reported to both, inhibit or promote tumor growth depending on yet undefined conditions. Here we will thoroughly discuss the different potential clinical applications of MSC as well as the role of MSCs on sarcomagenesis and the control of tumor growth.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Multipotentes/fisiologia , Neoplasias/terapia , Doenças Autoimunes/terapia , Biomarcadores/metabolismo , Ensaios Clínicos como Assunto , Rejeição de Enxerto/terapia , Doença Enxerto-Hospedeiro/terapia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Neoplasias/patologia , Neoplasias/fisiopatologia , Regeneração/fisiologiaRESUMO
AAV virion biology is still lacking a complete understanding of the role that the various structural subunits (VP1, 2, and 3) play in virus assembly, infectivity, and therapeutic delivery for clinical indications. In this study, we focus on the less studied adeno-associated virus AAV3B and generate a collection of AAV plasmid substrates that assemble virion particles deficient specifically in VP1, VP2, or VP1 and 2 structural subunits. Using a collection of biological and structural assays, we observed that virions devoid of VP1, VP2, or VP1 and 2 efficiently assembled virion particles, indistinguishable by cryoelectron microscopy (cryo-EM) from that of wild type (WT), but unique in virion transduction (WT > VP2 > VP1 > VP1 and 2 mutants). We also observed that the missing structural subunit was mostly compensated by additional VP3 protomers in the formed virion particle. Using cryo-EM analysis, virions fell into three classes, namely full, empty, and partially filled, based on comparison of density values within the capsid. Further, we characterize virions described as "broken" or "disassembled" particles, and provide structural information that supports the particle dissolution occurring through the two-fold symmetry sites. Finally, we highlight the unique value of employing cryo-EM as an essential tool for release criteria with respect to AAV manufacturing.
Assuntos
Capsídeo , Dependovirus , Humanos , Sorogrupo , Microscopia Crioeletrônica , Dependovirus/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/química , Vírion/genética , Células HeLaRESUMO
Interleukin (IL)-6 is a pleiotropic cytokine involved in the regulation of hematological and immune responses. IL-6 is secreted chiefly by stromal cells, but little is known about its precise role in the homeostasis of human mesenchymal stromal cells (hMSCs) and the role it may play in hMSC-mediated immunoregulation. We studied the role of IL-6 in the biology of bone marrow derived hMSC in vitro by silencing its expression using short hairpin RNA targeting. Our results show that IL-6 is involved in immunosuppression triggered by hMSCs. Cells silenced for IL-6 showed a reduced capacity to suppress activated T-cell proliferation. Moreover, silencing of IL-6 significantly blocked the capacity of hMSCs to proliferate. Notably, increasing the intracellular level of IL-6 but not recovering the extracellular level could restore the proliferative impairment observed in IL-6-silenced hMSC. Our data indicate that IL-6 signals in hMSCs by a previously undescribed intracellular mechanism.
Assuntos
Proliferação de Células , Tolerância Imunológica , Interleucina-6/imunologia , Células-Tronco Mesenquimais/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Técnicas de Cocultura , Humanos , Células-Tronco Mesenquimais/citologia , Linfócitos T/citologiaRESUMO
Mesenchymal progenitor cells (MPCs) have been hypothesized as cells of origin for sarcomas, and c-Fos transcription factor has been showed to act as an oncogene in bone tumors. In this study, we show c-Fos is present in most sarcomas with chondral phenotype, while multiple other genes are related to c-Fos expression pattern. To further define the role of c-Fos in sarcomagenesis, we expressed it in primary human MPCs (hMPCs), immortalized hMPCs and transformed murine MPCs (mMPCs). In immortalized hMPCs, c-Fos expression generated morphological changes, reduced mobility capacity and impaired adipogenic- and osteogenic-differentiation potentials. Remarkably, immortalized hMPCs or mMPCs expressing c-Fos generated tumors harboring a chondrogenic phenotype and morphology. Thus, here we show that c-Fos protein has a key role in sarcomas and that c-Fos expression in immortalized MPCs yields cell transformation and chondrogenic tumor formation.
Assuntos
Carcinogênese/genética , Transformação Celular Neoplásica/genética , Células-Tronco Mesenquimais/patologia , Proteínas Proto-Oncogênicas c-fos/genética , Sarcoma/genética , Animais , Carcinogênese/patologia , Linhagem Celular , Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Genes fos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Proto-Oncogênicas c-fos/análise , Sarcoma/patologiaRESUMO
Calnexin is a ubiquitously expressed type I membrane protein which is exclusively localized in the endoplasmic reticulum (ER). In mammalian cells, calnexin functions as a chaperone molecule and plays a key role in glycoprotein folding and quality control within the ER by interacting with folding intermediates via their monoglucosylated glycans. In order to gain more insight into the physiological roles of calnexin, we have generated calnexin gene-deficient mice. Despite its profound involvement in protein folding, calnexin is not essential for mammalian-cell viability in vivo: calnexin gene knockout mice were carried to full term, although 50% died within 48 h and the majority of the remaining mice had to be sacrificed within 4 weeks, with only a very few mice surviving to 3 months. Calnexin gene-deficient mice were smaller than their littermates and showed very obvious motor disorders, associated with a dramatic loss of large myelinated nerve fibers. Thus, the critical contribution of calnexin to mammalian physiology is tissue specific.
Assuntos
Calnexina/genética , Calnexina/fisiologia , Neurônios Motores/patologia , Fibras Nervosas Mielinizadas/patologia , Alelos , Animais , Animais Recém-Nascidos , Sobrevivência Celular , Embrião de Mamíferos/citologia , Retículo Endoplasmático/metabolismo , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Modelos Genéticos , Chaperonas Moleculares , Bainha de Mielina/patologia , Fenótipo , Dobramento de Proteína , Recombinação Genética , Células-Tronco/citologia , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: Efforts are continuously made to detect and investigate the pivotal processes and interplay between the response of sentinel lymph node and malignant cells from a primary tumor. Conversely, some frequently used tumor animal models, such as human cancer xenografts, rarely feature metastasis. Therefore, lymph node alterations are seldom assessed. We consider that studying lymph node response could contribute to the understanding of host reaction to cancer. In the present study, we explored the presence of regional lymph node alterations in parallel with tumor growth using a pancreatic tumor xenograft model which does not develop metastasis. METHODS AND FINDINGS: We established an animal cancer model by the subcutaneous inoculation of PANC-1 (a metastatic human pancreatic cancer cell line) in the left upper flank of athymic nude mice. Tumor animals, along with controls (n = 7 / group) were subjected to Magnetic Resonance Imaging (MRI) in order to follow tumor growth and brachial and axillary lymph nodes alterations over several weeks. Further histological analyses were performed at the end of the study. The individual average of the different lymph nodes sizes was 15-40% larger in the tumor animals compared to control animals at week 8 to week 20. The tumor size and lymph node size were not correlated. Histological analysis of the lymph nodes showed paracortical histiocytosis. No metastasis to lymph nodes could be detected by histology. In tumor bearing animals, histiocytosis was associated with isolated apoptotic bodies and migration of human tumoral cells was confirmed by specific immunostaining of human origin markers. CONCLUSIONS: The lack of metastasis as well as the pathological manifestation of the lymph node alteration in this pre-clinical model established here parallels findings in patients with sinus histiocytosis that is correlated with improved survival.
Assuntos
Histiocitose Sinusal/diagnóstico por imagem , Linfonodos/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Neoplasias Pancreáticas/patologia , Animais , Linhagem Celular Tumoral , Humanos , Linfonodos/patologia , Camundongos , Camundongos Nus , Transplante de NeoplasiasRESUMO
Mesenchymal stem cells (MSCs), together with hematopoietic stem cells (HSCs), are the most frequently used cell type for cell-based therapeutics. As for other cell types intended for research and translational use, it is important to establish correctly typed cell lines from human tissue donations. Here, we describe methods for isolating, culturing, and identifying MSCs from various tissues obtained through human tissue donation. The methods have been used in the context of a biobank, prepared as standard operating procedures (SOPs), ensuring traceability and reproducibility of cell production.
Assuntos
Células-Tronco Mesenquimais/citologia , Bancos de Espécimes Biológicos , Técnicas de Cultura de Células/métodos , Células Cultivadas , Técnicas de Cocultura/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Reprodutibilidade dos TestesRESUMO
γδ T cells play a role in a wide range of diseases such as autoimmunity and cancer. The majority of circulating human γδ T lymphocytes express a Vγ9Vδ2+ (Vδ2+) T cell receptor (TCR) and following activation release pro-inflammatory cytokines. In this study, we show that IFNγ, produced by Vδ2+ cells, activates mesenchymal stem cell (MSC)-mediated immunosupression, which in turn exerts a negative feedback mechanism on γδ T cell function ranging from cytokine production to proliferation. Importantly, this modulatory effect is limited to a short period of time (<24 hours) post-T cell activation, after which MSCs can no longer exert their immunoregulatory capacity. Using genetically modified MSCs with the IFNγ receptor 1 constitutively silenced, we demonstrate that IFNγ is essential to this process. Activated γδ T cells induce expression of several factors by MSCs that participate in the depletion of amino acids. In particular, we show that indolamine 2,3-dioxygenase (IDO), an enzyme involved in L-tryptophan degradation, is responsible for MSC-mediated immunosuppression of Vδ2+ T cells. Thus, our data demonstrate that γδ T cell responses can be immuno-modulated by different signals derived from MSC.
Assuntos
Interferon gama/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Retroalimentação Fisiológica/efeitos dos fármacos , Humanos , Tolerância Imunológica/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
We demonstrate a straightforward method to encapsulate siRNA into naturally available and unmodified human apoferritin. The encapsulation into apoferritin is independent of the sequence of the siRNA and provides superior protection for those sensitive molecules. High efficiency in transfection can be achieved in human tumorigenic cells, human primary mesenchymal stem cells (hMSC) and peripheral blood mononuclear cells (PBMCs). In contrast to Lipofectamine, highly effective gene silencing can be achieved with ferritin as the delivery agent in both tumor cells and PBMCs at low siRNA concentrations (10 nM). As an endogenous delivery agent, apoferritin does not induce immune activation of T- and B-cells in human PBMCs. Apoferritin shows intrinsic anti-inflammatory effects and apoferritin-mediated delivery shows a preference for immune-activated T- and B-cells, a natural selectivity which may turn useful for drug delivery in case of infections or inflammatory diseases.
Assuntos
Ferritinas/metabolismo , Inativação Gênica , Técnicas de Transferência de Genes , Células-Tronco Mesenquimais/metabolismo , RNA Interferente Pequeno/administração & dosagem , Linfócitos T/metabolismo , Células CACO-2 , Células Hep G2 , Humanos , Lisossomos/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Cardiac progenitor cells (CPCs) from adult myocardium offer an alternative cell therapy approach for ischaemic heart disease. Improved clinical performance of CPCs in clinical trials requires a comprehensive definition of their biology and specific interactions with the environment. In this work we characterize specific human CPC surface markers and study some of their related functions. c-kit(pos) human CPCs (hCPCs) were characterized for cell surface marker expression, pluripotency, early and late cardiac differentiation markers and therapeutic activity in a rat model of acute myocardial infarction. The results indicate that hCPCs are a mesenchymal stem cell (MSC)-like population, with a similar immunoregulatory capacity. A partial hCPC membrane proteome was analysed by liquid chromatography-mass spectrometry/mass spectrometry and 36 proteins were identified. Several, including CD26, myoferlin and podocalyxin-like protein 1 (PODXL), have been previously described in other stem-cell systems. Suppression and overexpression analysis demonstrated that PODXL regulates hCPC activation, migration and differentiation; it also modulates their local immunoregulatory capacity. Therefore, hCPCs are a resident cardiac population that shares many features with hMSCs, including their capacity for local immunoregulation. Expression of PODXL appears to favour the immature state of hCPCs, while its downregulation facilitates their differentiation. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Antígenos de Diferenciação/biossíntese , Células-Tronco Mesenquimais/metabolismo , Miocárdio/metabolismo , Proteínas Proto-Oncogênicas c-kit/biossíntese , Sialoglicoproteínas/biossíntese , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Miocárdio/citologiaRESUMO
Mesenchymal stem cells (MSC) are effective in treating myocardial infarction (MI) and previous reports demonstrated that hypoxia improves MSC self-renewal and therapeutics. Considering that hypoxia-inducible factor-1 alpha (HIF-1α) is a master regulator of the adaptative response to hypoxia, we hypothesized that HIF-1α overexpression in MSC could mimic some of the mechanisms triggered by hypoxia and increase their therapeutic potential without hypoxia stimulation. Transduction of MSC with HIF-1α lentivirus vectors (MSC-HIF) resulted in increased cell adhesion and migration, and activation of target genes coding for paracrine factors. When MSC-HIF were intramyocardially injected in infarcted nude rats, significant improvement was found (after treatment of infarcted rats with MSC-HIF) in terms of cardiac function, angiogenesis, cardiomyocyte proliferation, and reduction of fibrotic tissue with no induction of cardiac hypertrophy. This finding provides evidences for a crucial role of HIF-1α on MSC biology and suggests the stabilization of HIF-1α as a novel strategy for cellular therapies.
Assuntos
Coração/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Infarto do Miocárdio/terapia , Animais , Células da Medula Óssea/fisiologia , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Vasos Coronários/fisiopatologia , Humanos , Masculino , Infarto do Miocárdio/patologia , Neovascularização Fisiológica , Ratos , Ratos Nus , Regeneração , Transdução de Sinais , Transcriptoma , Regulação para Cima , CicatrizaçãoRESUMO
Mesenchymal stromal cells (MSCs) are multipotent cells found in connective tissues that can differentiate into bone, cartilage, and adipose tissue. Interestingly, they can regulate immune responses in a paracrine way and allogeneic MSCs do not elicit immune response. These properties have encouraged a number of clinical trials in a broad range of regenerative therapies. Although these trials were first focused on their differentiation properties, in the last years, the immunosuppressive features have gained most of the attention. In this review, we will summarize the up-to-date knowledge about the immunosuppressive mechanisms of MSCs in vivo and in vitro and the most promising approaches in clinical investigation.
RESUMO
Epigenetic changes are regarded as emerging major players for hematopoietic stem cell (HSC) biology. Although some histone deacetylase (HDAC) inhibitors, such as valproic acid (VA), induce differentiation and apoptosis in a variety of leukemic cells in vitro, they produce a favorable effect on the expansion of normal HSCs. In this study, we have identified the VA target HDAC3 as a negative regulator of umbilical cord blood HSC expansion. We demonstrate that knockdown of the transcript dramatically improves CD34+ cell expansion, which correlates with a higher potential to generate colony-forming units in functional assays. We show that this effect is mediated at the level of primitive hematopoietic cells and that it is not due to negative effects on specific cell commitment or alterations in the cell cycle. HDAC3 inhibition does not block commitment to the monocytic lineage and the maturation of monocyte precursors, which are the main inhibited pathways in the presence of VA. Therefore, our results identify HDAC3 as a promising target for therapies aiming to expand HSCs.
Assuntos
Proliferação de Células , Células-Tronco Hematopoéticas/enzimologia , Histona Desacetilases/metabolismo , Antígenos CD34/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem da Célula , Proliferação de Células/efeitos dos fármacos , Sangue Fetal/citologia , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Humanos , Lentivirus/genética , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , RNA Interferente Pequeno/genética , Ácido Valproico/farmacologiaRESUMO
Mesenchymal stem cells are often transplanted into inflammatory environments where they are able to survive and modulate host immune responses through a poorly understood mechanism. In this paper we analyzed the responses of MSC to IL-1ß: a representative inflammatory mediator. Microarray analysis of MSC treated with IL-1ß revealed that this cytokine activateds a set of genes related to biological processes such as cell survival, cell migration, cell adhesion, chemokine production, induction of angiogenesis and modulation of the immune response. Further more detailed analysis by real-time PCR and functional assays revealed that IL-1ß mainly increaseds the production of chemokines such as CCL5, CCL20, CXCL1, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11 and CX(3)CL1, interleukins IL-6, IL-8, IL23A, IL32, Toll-like receptors TLR2, TLR4, CLDN1, metalloproteins MMP1 and MMP3, growth factors CSF2 and TNF-α, together with adhesion molecules ICAM1 and ICAM4. Functional analysis of MSC proliferation, migration and adhesion to extracellular matrix components revealed that IL-1ß did not affect proliferation but also served to induce the secretion of trophic factors and adhesion to ECM components such as collagen and laminin. IL-1ß treatment enhanced the ability of MSC to recruit monocytes and granulocytes in vitro. Blockade of NF-κß transcription factor activation with IκB kinase beta (IKKß) shRNA impaired MSC migration, adhesion and leucocyte recruitment, induced by IL-1ß demonstrating that NF-κB pathway is an important downstream regulator of these responses. These findings are relevant to understanding the biological responses of MSC to inflammatory environments.
Assuntos
Quimiotaxia de Leucócito , Interleucina-1beta/fisiologia , Células-Tronco Mesenquimais/fisiologia , NF-kappa B/metabolismo , Adesão Celular , Movimento Celular , Proliferação de Células , Quimiocinas/genética , Quimiocinas/metabolismo , Colágeno/metabolismo , Fibronectinas/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Mediadores da Inflamação/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Laminina/metabolismo , Leucócitos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , Transdução de SinaisRESUMO
Although marrow adipocytes and osteoblasts derive from a common bone marrow stromal cells (BMSCs), the mechanisms that underlie osteoporosis-associated bone loss and marrow adipogenesis during prolonged steroid treatment are unclear. We show in human BMSCs (hBMSCs) that glucocorticoid receptor (GR) signaling in response to high concentrations of glucocorticoid (GC) supports adipogenesis but inhibits osteogenesis by reducing c-Jun expression and hBMSC proliferation. Conversely, significantly lower concentrations of GC, which permit hBMSC proliferation, are necessary for normal bone mineralization. In contrast, platelet-derived growth factor (PDGF) signaling increases both JNK/c-Jun activity and hBMSC expansion, favoring osteogenic differentiation instead of adipogenesis. Indeed, PDGF antagonizes the proadipogenic qualities of GC/GR signaling. Thus our results reveal a novel c-Jun-centered regulatory network of signaling pathways in differentiating hBMSCs that controls the proliferation-dependent balance between osteogenesis and adipogenesis.
Assuntos
Células da Medula Óssea/citologia , Receptores de Glucocorticoides/fisiologia , Células Estromais/citologia , Fator de Transcrição AP-1/fisiologia , Adipogenia/fisiologia , Diferenciação Celular , Proliferação de Células , Citometria de Fluxo , Humanos , Osteogênese/fisiologiaRESUMO
OBJECTIVES: The purpose of this study was to compare the ability of human CD34(+) hematopoietic stem cells and bone marrow mesenchymal stem cells (MSC) to treat myocardial infarction (MI) in a model of permanent left descendent coronary artery (LDA) ligation in nude rats. BACKGROUND: Transplantation of human CD34(+) cells and MSC has been proved to be effective in treating MI, but no comparative studies have been performed to elucidate which treatment prevents left ventricular (LV) remodelling more efficiently. METHODS: Human bone marrow MSC or freshly isolated CD34(+) cells from umbilical cord blood were injected intramyocardially in infarcted nude rats. Cardiac function was analyzed by echocardiography. Ventricular remodelling was evaluated by tissue histology and electron microscopy, and neo-formed vessels were quantified by immunohistochemistry. Chronic local inflammatory infiltrates were evaluated in LV wall by hematoxylin-eosin staining. Apoptosis of infarcted tissue was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. RESULTS: Both cell types induced an improvement in LV cardiac function and increased tissue cell proliferation in myocardial tissue and neoangiogenesis. However, MSC were more effective for the reduction of infarct size and prevention of ventricular remodelling. Scar tissue was 17.48 +/- 1.29% in the CD34 group and 10.36 +/- 1.07% in the MSC group (p < 0.001 in MSC vs. CD34). Moreover, unlike MSC, CD34(+)-treated animals showed local inflammatory infiltrates in LV wall that persisted 4 weeks after transplantation. CONCLUSIONS: Mesenchymal stem cells might be more effective than CD34(+) cells for the healing of the infarct. This study contributes to elucidate the mechanisms by which these cell types operate in the course of MI treatment.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Infarto do Miocárdio/terapia , Animais , Antígenos CD34/metabolismo , Antígenos CD34/uso terapêutico , Proliferação de Células , Imuno-Histoquímica , Transplante de Células-Tronco Mesenquimais , Comunicação Parácrina/fisiologia , Ratos , Ratos Nus , Função Ventricular EsquerdaRESUMO
Human adipose-derived mesenchymal stem cells (hASCs) are mesenchymal stem cells (MSCs) with reduced immunogenicity and capability to modulate immune responses. Whereas the immunosuppressive activity of bone marrow-MSCs has received considerable attention during the last few years, the specific mechanisms underlying hASC-mediated immunosuppression have been poorly studied. Recent studies comparing both cell types have reported differences at transcriptional and proteomic levels, suggesting that hASCs and bone marrow-MSCs, while having similarities, are quite different. This suggests that different mechanisms of immunosuppression may apply. Here, we report that hASCs inhibit peripheral blood mononuclear cells (PBMCs), and CD4(+) and CD8(+) T cell proliferation in both cell-cell contact and transwell conditions, which is accompanied by a reduction of proinflammatory cytokines. We demonstrate that hASCs do not constitutively express immunomodulatory factors. Conditioned supernatants from hASCs stimulated by IFN-gamma, PBMCs, or activated PBMCs highly inhibited PBMC proliferation, indicating that inhibitory factors are released upon hASC activation. Many factors have been involved in MSC-mediated immunosuppression, including IFN-gamma, IL-10, hepatocyte growth factor, prostaglandin E2, transforming growth factor-beta1, indoleamine 2,3-dioxygenase (IDO), nitric oxide, and IL-10. Using pharmacological inhibitors, neutralizing antibodies, and genetically modified hASCs that constitutively express or silence IDO enzyme, we demonstrate that, in the case of hASCs, the IFN-gamma/IDO axis is essential. Taken together, our data support the key role of IDO in the therapeutic use of hASC on immunomediated diseases.
Assuntos
Adipócitos/citologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama/metabolismo , Linfócitos/citologia , Células-Tronco Mesenquimais/citologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Proliferação de Células , Dinoprostona/metabolismo , Citometria de Fluxo , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Interleucina-10/metabolismo , Leucócitos Mononucleares/citologia , Linfócitos/imunologia , Óxido Nítrico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Engenharia Tecidual , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Human Mesenchymal Stem Cells (hMSC), derived mainly from adult bone marrow, are valuable models for the study of processes involved in stem cell self-renewal and differentiation. As the Extracellular signal-Regulated Kinase (ERK) signalling pathway is a major contributor to cellular growth, differentiation and survival, we have studied the functions of this kinase in hMSC activity. Ablation of ERK2 gene expression (but not ERK1) by RNA interference significantly reduced proliferation of hMSC. This reduction was due to a defect in Cyclin D1 expression and subsequent arrest in the G0/G1 phase of the cell cycle. hMSC growth is enhanced through culture medium supplementation with growth factors (GFs) such as Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF). However, these supplements could not rescue the defect observed after ERK2 knockdown, suggesting a common signalling pathway used by these GFs for proliferation. In contrast, ERK1/2 may be dissociated from chemotactic signalling induced by the same GFs. Additionally, hMSCs were capable of differentiating into adipocytes even in the absence of either ERK1 or ERK2 proteins. Our data show that hMSCs do not require cell division to enter the adipogenic differentiation process, indicating that clonal amplification of these cells is not a critical step. However, cell-cell contact seems to be an essential requirement to be able to differentiate into mature adipocytes.
Assuntos
Células-Tronco Mesenquimais/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Adipogenia , Diferenciação Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Interferência de RNARESUMO
Vav1 is a guanine nucleotide exchange factor (GEF) for Rho-family GTPases, which is activated by tyrosine phosphorylation following TCR stimulation. Vav1-deficient mice have defects in positive and negative selection of thymocytes as well as TCR-induced proliferation in mature T cells, demonstrating a critical role for Vav1 in transducing TCR signals. Binding of phospholipids to the PH domain of Vav1 has been proposed to regulate its GEF activity in vitro. To test this model in vivo, we have generated mice carrying a point mutation in the PH domain of Vav1, and we show that they have defects in T cell development and activation. We demonstrate that the mutation affects the function of Vav1 as a GEF and perturbs PI3K-dependent pathways downstream of Vav1. Unexpectedly, the mutation selectively affects TCR-induced proliferation of CD4(+) but not CD8(+) T cells, demonstrating differences in the wiring of TCR signaling pathways between the two lineages.