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The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we establish compensating amino acid replacements that fully rescue the integration defect of these mutants, suggesting epistasis between these amino acid residues. We further show that the transposons excised by the exc+/int- transposase mutants form extrachromosomal circles that cannot undergo a further round of transposition, thereby representing dead-end products of the excision reaction. Finally, we demonstrate the utility of the exc+/int- transposase in cassette removal for the generation of reprogramming factor-free induced pluripotent stem cells. Lack of genomic integration and formation of transposon circles following excision is reminiscent of signal sequence removal during V(D)J recombination, and implies that cut-and-paste DNA transposition can be converted to a unidirectional process by a single amino acid change.
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Reprogramação Celular , Elementos de DNA Transponíveis , Células-Tronco Pluripotentes Induzidas/metabolismo , Transposases/genética , Substituição de Aminoácidos , Animais , Epistasia Genética , Engenharia Genética/métodos , Células HeLa , Células Hep G2 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Mutação , Transposases/metabolismoRESUMO
Adoptive Tcell therapies are emerging tools to combat various human diseases. CART cells are approved and marketed as last line therapeutics in advanced Bcell lymphomas and leukemias. TCR-engineered T cells are being evaluated in clinical trials for a variety of hematological and solid tumors. Genetically modified regulatory T cells, however, are still in the initial stages of clinical development for the induction of immune tolerance in various indications.Here we outline the general role of regulatory T cells in establishing self-tolerance and the mechanisms by which these suppress the effector immune cells. Further, the role of regulatory T cells in the pathomechanism of certain immune diseases is presented, and the current status of clinical developments of genetically modified Treg cells is discussed. We also present the regulatory framework for genetically modified regulatory T cells as advanced therapy medicinal products, including aspects of manufacture and quality control, as well as nonclinical and clinical development requirements.
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Imunoterapia Adotiva , Linfócitos T Reguladores , Alemanha , HumanosRESUMO
Recent evidence implicates the myocardin-related transcription factors (MRTFs) as key mediators of the phenotypic plasticity leading to the conversion of various cell types into myofibroblasts. This review highlights the function of MRTFs during development, fibrosis and cancer, and the role of MRTFs during epithelial-mesenchymal transitions (EMTs) underlying these processes. EMT is a sequentially orchestrated process where cells undergo a rearrangement of their cell contacts and activate a fibrogenic and myogenic expression program. MRTFs interact with and regulate the major signaling pathways and the expression of key markers and transcription factors involved in EMT. These functions indicate a central role for MRTFs in controlling the process of EMT. Developmental Dynamics 247:396-404, 2018. © 2017 Wiley Periodicals, Inc.
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Transição Epitelial-Mesenquimal , Proteínas Nucleares , Transativadores , Fatores de Transcrição/fisiologia , Animais , Fibrose/patologia , Humanos , Neoplasias/patologia , Transdução de SinaisRESUMO
OBJECTIVES: The purpose of this study was to determine the level of heterogeneity in high grade serous ovarian cancer (HGSOC) by analyzing RNA expression in single epithelial and cancer associated stromal cells. In addition, we explored the possibility of identifying subgroups based on pathway activation and pre-defined signatures from cancer stem cells and chemo-resistant cells. METHODS: A fresh, HGSOC tumor specimen derived from ovary was enzymatically digested and depleted of immune infiltrating cells. RNA sequencing was performed on 92 single cells and 66 of these single cell datasets passed quality control checks. Sequences were analyzed using multiple bioinformatics tools, including clustering, principle components analysis, and geneset enrichment analysis to identify subgroups and activated pathways. Immunohistochemistry for ovarian cancer, stem cell and stromal markers was performed on adjacent tumor sections. RESULTS: Analysis of the gene expression patterns identified two major subsets of cells characterized by epithelial and stromal gene expression patterns. The epithelial group was characterized by proliferative genes including genes associated with oxidative phosphorylation and MYC activity, while the stromal group was characterized by increased expression of extracellular matrix (ECM) genes and genes associated with epithelial-to-mesenchymal transition (EMT). Neither group expressed a signature correlating with published chemo-resistant gene signatures, but many cells, predominantly in the stromal subgroup, expressed markers associated with cancer stem cells. CONCLUSIONS: Single cell sequencing provides a means of identifying subpopulations of cancer cells within a single patient. Single cell sequence analysis may prove to be critical for understanding the etiology, progression and drug resistance in ovarian cancer.
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Células Epiteliais/patologia , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Células Estromais/patologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Epiteliais e Glandulares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Análise de Célula Única/métodos , Células Estromais/metabolismoRESUMO
Endothelial-mesenchymal transition (EndMT) is an important mechanism during organ development and in certain pathological conditions. For example, EndMT contributes to myofibroblast formation during organ fibrosis, and it has been identified as an important source of cancer-associated fibroblasts, facilitating tumor progression. Recently, EndMT was proposed to modulate endothelial function during intravasation and extravasation of metastatic tumor cells. Evidence suggests that endothelial cells are not passive actors during transendothelial migration (TEM) of cancer cells, as there are profound changes in endothelial junctional protein expression, signaling, permeability, and contractility. This review describes these alterations in endothelial characteristics during TEM of metastatic tumor cells and discusses them in the context of EndMT. EndMT could play an important role during metastatic intravasation and extravasation, a novel hypothesis that may lead to new therapeutic approaches to tackle metastatic disease.
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Células Endoteliais/patologia , Transição Epitelial-Mesenquimal , Neoplasias/patologia , Migração Transendotelial e Transepitelial , Animais , Comunicação Celular , Células Endoteliais/metabolismo , Humanos , Metástase Neoplásica , Neoplasias/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase. The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype, and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into 'safe harbor' sites in the genomes of autologous, patient-derived iPS cells.
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Reprogramação Celular , Elementos de DNA Transponíveis , Técnicas de Introdução de Genes , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Vetores Genéticos , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Integrases/metabolismo , Fator 4 Semelhante a Kruppel , Camundongos , Transposases/metabolismoRESUMO
During progressive tubulointerstitial fibrosis, renal tubular epithelial cells transform into α-smooth muscle actin (SMA)-expressing myofibroblasts via epithelial-to-mesenchymal transition (EMT). SMA expression is regulated by transforming growth factor (TGF)-ß1 and cell contact disruption, through signaling events targeting the serum response factor-myocardin-related transcription factor (MRTF) complex. MRTFs are important regulators of fibrosis, tumor cell invasion, and metastasis. Consistent with the role of MRTFs in tumor progression, suppressor of cancer cell invasion (SCAI) was recently identified as a negative regulator of MRTF. Herein, we studied the role of SCAI in a fibrotic EMT model established on LLC-PK1 cells. SCAI overexpression prevented SMA promoter activation induced by TGF-ß1. When co-expressed, it inhibited the stimulatory effects of MRTF-A, MRTF-B or the constitutive active forms of RhoA, Rac1, or Cdc42 on the SMA promoter. SCAI interfered with TGF-ß1-induced SMA, connective tissue growth factor, and calponin protein expression; it rescued TGF-ß1-induced E-cadherin down-regulation. IHC studies on human kidneys showed that SCAI expression is reduced during fibrosis. Kidneys of diabetic rats and mice with unilateral ureteral obstruction depicted significant loss of SCAI expression. In parallel with the decrease of SCAI protein expression, diabetic rat and mouse kidneys with unilateral ureteral obstruction showed SMA expression, as evidenced by using Western blot analysis. Finally, TGF-ß1 treatment of LLC-PK1 cells attenuated SCAI protein expression. These data suggest that SCAI is a novel transcriptional cofactor that regulates EMT and renal fibrosis.
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Transição Epitelial-Mesenquimal , Rim/metabolismo , Rim/patologia , Fatores de Transcrição/metabolismo , Actinas/genética , Animais , Caderinas/genética , Caderinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibrose , Humanos , Células LLC-PK1 , Camundongos , Proteínas dos Microfilamentos/metabolismo , Regiões Promotoras Genéticas/genética , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Suínos , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/farmacologia , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , CalponinasRESUMO
Angiotensin II (ANG II), the major effector molecule of the renin-angiotensin system (RAS), is a powerful vasoactive mediator associated with hypertension and renal failure. In this study the permeability changes and its morphological attributes in endothelial cells of human umbilical vein (HUVECs) were studied considering the potential regulatory role of ANG II. The effects of ANG II were compared with those of vascular endothelial growth factor (VEGF). Permeability was determined by 40 kDa FITC-Dextran and electrical impedance measurements. Plasmalemmal vesicle-1 (PV-1) mRNA levels were measured by PCR. Endothelial cell surface was studied by atomic force microscopy (AFM), and caveolae were visualized by transmission electron microscopy (TEM) in HUVEC monolayers. ANG II (10(-7) M), similarly to VEGF (100 ng/ml), increased the endothelial permeability parallel with an increase in the number of cell surface openings and caveolae. AT1 and VEGF-R2 receptor blockers (candesartan and ZM-323881, respectively) blunted these effects. ANG II and VEGF increased the expression of PV-1, which could be blocked by candesartan or ZM-323881 pretreatments and by the p38 mitogem-activated protein (MAP) kinase inhibitor SB-203580. Additionally, SB-203580 blocked the increase in endothelial permeability and the number of surface openings and caveolae. In conclusion, we have demonstrated that ANG II plays a role in regulation of permeability and formation of cell surface openings through AT1 receptor and PV-1 protein synthesis in a p38 MAP kinase-dependent manner in endothelial cells. The surface openings that increase in parallel with permeability may represent transcellular channels, caveolae, or both. These morphological and permeability changes may be involved in (patho-) physiological effects of ANG II.
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Angiotensina II/fisiologia , Permeabilidade Capilar/fisiologia , Proteínas de Transporte/biossíntese , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/biossíntese , Angiotensina II/metabolismo , Permeabilidade Capilar/genética , Cavéolas/metabolismo , Células Cultivadas , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Transporte Proteico/genética , Receptor Tipo 1 de Angiotensina/biossíntese , Receptor Tipo 1 de Angiotensina/fisiologiaRESUMO
Epithelial-mesenchymal transition (EMT) is a process of cellular plasticity regulated by complex signaling networks. Under physiological conditions, it plays an important role in wound healing and organ repair. Its importance for human disease is given by its central role in chronic fibroproliferative diseases and cancer, which represent leading causes of death worldwide. In tumors, EMT is involved in primary tumor growth, metastasis and therapy resistance. It is therefore a major requisite to investigate and understand the role of EMT and the mechanisms leading to EMT in order to tackle these diseases therapeutically. Forward genetic screens link genome modifications to phenotypes, and have been successfully employed to identify oncogenes, tumor suppressor genes and genes involved in metastasis or therapy resistance. In particular, transposon-based insertional mutagenesis screens and CRISPR-based screens are versatile and easy-to-use tools applied in recent years to discover and identify novel cancer-related mechanisms. Here, we review the contribution of forward genetic screens to our understanding of how EMT is regulated and how it is involved in various aspects of cancer. Based on the current literature, we propose these methods as additional tools to investigate EMT.
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Since first proposed as a new tool for gene targeting and genome editing, CRISPR technology has quickly advanced into the clinical stage. Initial studies highlight the potential for CRISPR-Cas9-mediated therapeutic approaches in human medicine to correct incurable genetic diseases and enhance cell-based therapeutic approaches. While acknowledging the opportunities this technology brings for the treatment of patients with severe diseases, timely development of these innovative medicinal products requires regulatory oversight and adaptation of regulatory requirements to ensure the safety and efficacy of medicinal products based on CRISPR technology. We briefly present the current regulatory framework applicable for CRISPR-Cas-based developments as advanced therapy medicinal products. Moreover, scientific- and regulatory-driven considerations relevant for advancing product development toward clinical trial applications in Germany are highlighted by discussing the key aspects of quality and nonclinical and clinical development requirements.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Ensaios Clínicos como Assunto , Marcação de Genes , HumanosRESUMO
Metastasis, a complex, multistep process, is responsible for the overwhelming majority of cancer-related deaths. Despite its devastating consequences, it is not possible to effectively treat cancer that has spread to vital organs, the mechanisms leading to metastasis are still poorly understood, and the catalog of metastasis promoting genes is still incomprehensive. To identify new driver genes of metastasis development, we performed an in vitro Sleeping Beauty transposon-based forward genetic screen in nonmetastatic SKBR3 human breast cancer cells. Boyden chamber-based matrix invasion assays were used to harvest cells that acquired a de novo invasive phenotype. Using targeted RNA sequencing data from 18 pools of invasive cells, we carried out a gene-centric candidate gene prediction and identified established and novel metastasis driver genes. Analysis of these genes revealed their association with metastasis related processes and we further established their clinical relevance in metastatic breast cancer. Two novel candidate genes, G protein-coupled receptor kinase interacting ArfGAP 2 (GIT2) and muscle-associated receptor tyrosine kinase (MUSK), were functionally validated as metastasis driver genes in a series of in vitro and in vivo experimental metastasis models. We propose that our robust and scalable approach will be a useful addition to the toolkit of methodologic resources used to identify genes driving cancer metastasis. IMPLICATIONS: Novel metastasis drivers were identified in a human breast cancer cell line by performing an in vitro, Sleeping Beauty transposon-based forward genetic screen and an RNA fusion-based candidate gene prediction.
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Neoplasias da Mama , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Elementos de DNA Transponíveis/genética , Feminino , Humanos , Mutagênese , Mutagênese Insercional , Proteínas Tirosina Quinases/genética , RNA , Receptores Acoplados a Proteínas G/genéticaRESUMO
BACKGROUND/AIMS: Epithelial-mesenchymal transition of tubular cells into alpha-smooth muscle actin (SMA)-expressing myofibroblasts is a central mechanism in tubulointerstitial fibrosis. Previously, a 'two-hit' model was proposed for epithelial-mesenchymal transition wherein an initial injury of the intercellular contacts and TGF-beta(1) are both required for SMA protein expression in LLC-PK1 cells. The Rho-Rho kinase-myosin light chain-myocardin-related transcription factor (MRTF)-serum response factor (SRF) pathway and Rac1, p21-activated kinase (PAK) and p38 were described as important regulators of MRTF localization and SMA expression. Cdc42 is another small G protein situated upstream of PAK and p38, and is activated upon cell contact disassembly. Here, we investigated its potential role in the regulation of MRTF nuclear shuttling and in the regulation of the SMA promoter. RESULTS: Transfection of a constitutive active (CA) Cdc42 construct alone induced the activation of the SMA promoter. The dominant negative (DN) Cdc42 construct prevented the activation of the promoter induced by cell contact disassembly, and reduced the combined effect of cell contact disruption and TGF-beta(1). SRF showed a marked nuclear accumulation in CA Cdc42-transfected cells. Cdc42 induced the nuclear translocation of MRTF, while DN Cdc42 inhibited its nuclear translocation induced by cell contact disassembly. Blocking PAK, MRTF and p38 by the corresponding DN constructs blunted the effects of CA Cdc42 on the SMA promoter. CONCLUSION: Cdc42 is involved in the regulation of SMA promoter activation through PAK, p38, MRTF and SRF. Cdc42 may be an important regulator of MRTF cellular localization.
Assuntos
Actinas/genética , Células Epiteliais/citologia , Túbulos Renais/patologia , Mesoderma/citologia , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/fisiologia , Transativadores/metabolismo , Proteína cdc42 de Ligação ao GTP/fisiologia , Animais , Células LLC-PK1 , SuínosRESUMO
Epithelial-mesenchymal-myofibroblast transition (EMT), a key feature in organ fibrosis, is regulated by the state of intercellular contacts. Our recent studies have shown that an initial injury of cell-cell junctions is a prerequisite for transforming growth factor-beta1 (TGF-beta1)-induced transdifferentiation of kidney tubular cells into alpha-smooth muscle actin (SMA)-expressing myofibroblasts. Here we analyzed the underlying contact-dependent mechanisms. Ca(2+) removal-induced disruption of intercellular junctions provoked Rho/Rho kinase (ROK)-mediated myosin light chain (MLC) phosphorylation and Rho/ROK-dependent SMA promoter activation. Importantly, myosin-based contractility itself played a causal role, because the myosin ATPase inhibitor blebbistatin or a nonphosphorylatable, dominant negative MLC (DN-MLC) abolished the contact disruption-triggered SMA promoter activation, eliminated the synergy between contact injury and TGF-beta1, and suppressed SMA expression. To explore the responsible mechanisms, we investigated the localization of the main SMA-inducing transcription factors, serum response factor (SRF), and its coactivator myocardin-related transcription factor (MRTF). Contact injury enhanced nuclear accumulation of SRF and MRTF. These processes were inhibited by DN-Rho or DN-MLC. TGF-beta1 strongly facilitated nuclear accumulation of MRTF in cells with reduced contacts but not in intact epithelia. DN-myocardin abrogated the Ca(2+)-removal- +/- TGF-beta1-induced promoter activation. These studies define a new mechanism whereby cell contacts regulate epithelial-myofibroblast transition via Rho-ROK-phospho-MLC-dependent nuclear accumulation of MRTF.
Assuntos
Comunicação Celular , Células Epiteliais/citologia , Fibroblastos/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miosinas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/genética , Animais , Células CHO , Cálcio/metabolismo , Núcleo Celular/metabolismo , Cricetinae , Cricetulus , Citoplasma/metabolismo , Humanos , Túbulos Renais/citologia , Músculo Liso/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Quinases Associadas a rhoRESUMO
Back in 1995, a landmark paper was published, which shaped the fibrosis literature for many years to come. During the characterization of a fibroblast-specific marker (FSP1) in the kidneys, an observation was made, which gave rise to the hypothesis that "fibroblasts in some cases arise from the local conversion of epithelium." In the following years, epithelial-mesenchymal transition was in the spotlight of fibrosis research, especially in the kidney. However, the hypothesis came under scrutiny following some discouraging findings from lineage tracing experiments and clinical observations. In this review, we provide a timely overview of the current position of the epithelial-mesenchymal transition hypothesis in the context of fibrosis (with a certain focus on renal fibrosis) and highlight some of the potential hurdles and pitfalls preventing therapeutic breakthroughs targeting fibrotic epithelial-mesenchymal transition.
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Genetic modification of induced pluripotent stem (iPS) cells may be necessary for the generation of effector cells for cellular therapies. Hereby, it can be important to induce transgene expression at restricted and defined time windows, especially if it interferes with pluripotency or differentiation. To achieve this, inducible expression systems can be used such as the tetracycline-inducible retroviral vector system, however, retroviral expression can be subjected to epigenetic silencing or to position-effect variegation. One strategy to overcome this is the incorporation of ubiquitous chromatin opening elements (UCOE®'s) into retroviral vectors to maintain a transcriptionally permissive chromatin state at the integration site. In this study, we developed Tet-inducible all-in-one gammaretroviral vectors carrying different sized UCOE®'s derived from the A2UCOE. The ability to prevent vector silencing by preserving the Tet-regulatory potential was investigated in different cell lines, and in murine and human iPS cells. A 670-bp fragment spanning the CBX3 promoter region of A2UCOE (U670) was the most potent element in preventing silencing, and conferred the strongest expression from the vector in the induced state. While longer fragments of A2UCOEs also sustained expression, vector titers and induction efficiencies were impaired. Finally, we demonstrate that U670 can be used for constitutive expression of the transactivator in the all-in-one vector for faithful regulation of transgenes by doxycycline, including the thrombopoietin receptor Mpl conferring cytokine-dependent cell growth.
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Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Expressão Gênica , Vetores Genéticos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Lentivirus/genética , Tetraciclina/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citocinas/metabolismo , Doxiciclina/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Fosfoglicerato Quinase/metabolismo , Regiões Promotoras Genéticas , Receptores de Trombopoetina/metabolismo , Ativação Transcricional/genética , TransgenesRESUMO
Human stem cells harbor significant potential for basic and clinical translational research as well as regenerative medicine. Currently ~ 3000 adult and ~ 30 pluripotent stem cell-based, interventional clinical trials are ongoing worldwide, and numbers are increasing continuously. Although stem cells are promising cell sources to treat a wide range of human diseases, there are also concerns regarding potential risks associated with their clinical use, including genomic instability and tumorigenesis concerns. Thus, a deeper understanding of the factors and molecular mechanisms contributing to stem cell genome stability are a prerequisite to harnessing their therapeutic potential for degenerative diseases. Chemical and physical factors are known to influence the stability of stem cell genomes, together with random mutations and Copy Number Variants (CNVs) that accumulated in cultured human stem cells. Here we review the activity of endogenous transposable elements (TEs) in human multipotent and pluripotent stem cells, and the consequences of their mobility for genomic integrity and host gene expression. We describe transcriptional and post-transcriptional mechanisms antagonizing the spread of TEs in the human genome, and highlight those that are more prevalent in multipotent and pluripotent stem cells. Notably, TEs do not only represent a source of mutations/CNVs in genomes, but are also often harnessed as tools to engineer the stem cell genome; thus, we also describe and discuss the most widely applied transposon-based tools and highlight the most relevant areas of their biomedical applications in stem cells. Taken together, this review will contribute to the assessment of the risk that endogenous TE activity and the application of genetically engineered TEs constitute for the biosafety of stem cells to be used for substitutive and regenerative cell therapies.
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Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells were co-transfected with pFAR4-inverted terminal repeats (ITRs) CMV-PEDF-BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats, and transfected primary cells (transfected RPE [tRPE] and transfected IPE [tIPE] cells) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinases 2 and 9 (MMP-2/9), and microglial/macrophage markers were measured. Injection with tRPE/IPE cells significantly reduced the leakage area at 7 and 14 days and the CNV area at 7 days. There was a significant increase in PEDF and the PEDF/VEGF ratio with tRPE cells and a reduction in the MMP-2 activity. Our data demonstrated that ex vivo non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.
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We investigated the mechanism whereby cell contact injury stimulates the alpha-smooth muscle actin (SMA) promoter, a key process for epithelial-mesenchymal transition (EMT) during organ fibrosis. Contact disruption by low-Ca(2+) medium (LCM) activated Rac, PAK and p38 MAPK, and triggered the nuclear accumulation of myocardin-related transcription factor (MRTF), an inducer of the SMA promoter. Dominant negative (DN) Rac, DN-PAK, DN-p38, or the p38 inhibitor SB203580 suppressed the LCM-induced nuclear accumulation of MRTF and the activation of the SMA promoter. These studies define novel pathway(s) involving Rac, PAK, and p38 in the regulation of MRTF and the contact-dependent induction of EMT.
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Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/metabolismo , Quinases Ativadas por p21/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Proteínas rac de Ligação ao GTP/fisiologia , Animais , Células LLC-PK1 , Microscopia de Fluorescência , Fosforilação , Transporte Proteico , SuínosRESUMO
Detection of HPV infections requires a robust time-effective single-step method for efficient screening. A molecular beacon-based one-step multiplex real-time PCR system was developed to detect 15 high-risk (HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and 5 low-risk HPV types (HPV types 6, 11, 42, 43, 44). Molecular beacons detecting high-risk types are 5'-FAM-3'-DABCYL-labelled, molecular beacons for low-risk detection are 5'-TET-3'-DABCYL-labelled, while the internal control added before sample DNA extraction is detected by a 5'-FAM-TexasRed-3'-DABCYL wavelength-shifting molecular beacon. Accordingly, fluorescent data for HPV detection are collected at 530 nm for high-risk types, 560 nm in case of low-risk types and the reaction internal control is detected at 610 nm on a Roche LightCycler 2.0 instrument. The sensitivity for detected types varies between 22 and 700 copies/reaction. The clinical performance was tested on 161 clinical sample DNAs. The MB-RT PCR results were compared to the typing results obtained by the L1F/L1R PCR and hybridization-based system described previously, and the concordance rate between the two systems was 89.44%. The favorable characteristics shown by this multiplex single-step real-time HPV detection system make this promising approach worthy for further development and application for clinical screening.