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INTRODUCTION: Chemo-responsiveness in pancreatic cancer is known to be dependent on fibrosis and vascularity. The purpose of this study was to assess vascular enhancement in advanced pancreatic adenocarcinoma with or without liver metastasis in computed tomography (CT) and to analyze the correlation between enhancement patterns and chemo-responsiveness. METHODS: Patients were assigned to either a responder group (partial response or stable disease) or a non-responder group (progressive disease) according to chemo-responsiveness assessed by CT before and after gemcitabine-based chemotherapy. Hounsefield unit (HU) was measured in pancreatic mass and the largest metastatic liver mass using region of interest (ROI). HU differences (ΔHU) between arterial and pre-contrast phase were calculated. RESULTS: Of the 101 study subjects, 78(77.2%) were assigned to the pancreas responder group {mean ΔHU (±SD), 36.7(±21.6)} and 23(22.8%) to the pancreas non-responder group {mean ΔHU (±SD), 20.6(±9.9)} (p = 0.001 for ΔHUs). Of the 46 study subjects with liver metastasis, 25(54.3%) were assigned to the liver metastasis responder group {mean ΔHU (±SD), 36.9(±21.0} and 21(45.7%) to the liver metastasis non-responder group {mean ΔHU (±SD), 17.1 (±24.0)}, (p = 0.005 for ΔHUs). CONCLUSION: CT determined mass vascular enhancement patterns may predict chemoresponse in advanced pancreatic cancer.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Pâncreas/irrigação sanguínea , Neoplasias Pancreáticas/tratamento farmacológico , Tomografia Computadorizada por Raios X , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/diagnóstico por imagem , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/secundário , Desoxicitidina/uso terapêutico , Feminino , Seguimentos , Humanos , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Pâncreas/diagnóstico por imagem , Pâncreas/patologia , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/patologia , Curva ROC , Estudos Retrospectivos , Resultado do Tratamento , GencitabinaRESUMO
The potential use of induced pluripotent stem cells (iPSCs) in personalized regenerative medicine applications may be augmented by transgenics, including the expression of constitutive cell labels, differentiation reporters, or modulators of disease phenotypes. Thus, there is precedence for reproducible transgene expression amongst iPSC sub-clones with isogenic or diverse genetic backgrounds. Using virus or transposon vectors, transgene integration sites and copy numbers are difficult to control, and nearly impossible to reproduce across multiple cell lines. Moreover, randomly integrated transgenes are often subject to pleiotropic position effects as a consequence of epigenetic changes inherent in differentiation, undermining applications in iPSCs. To address this, we have adapted popular TALEN and CRISPR/Cas9 nuclease technologies in order to introduce transgenes into pre-defined loci and overcome random position effects. AAVS1 is an exemplary locus within the PPP1R12C gene that permits robust expression of CAG promoter-driven transgenes. Gene targeting controls transgene copy number such that reporter expression patterns are reproducible and scalable by â¼2-fold. Furthermore, gene expression is maintained during long-term human iPSC culture and in vitro differentiation along multiple lineages. Here, we outline our AAVS1 targeting protocol using standardized donor vectors and construction methods, as well as provide practical considerations for iPSC culture, drug selection, and genotyping.
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Cromossomos Humanos Par 19/genética , Engenharia Genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Sequência de Bases , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Clonagem Molecular , Dependovirus , Genes Reporter , Loci Gênicos , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Parvovirinae/genética , Regiões Promotoras Genéticas , Transfecção , TransgenesRESUMO
GATA factors establish transcriptional networks that control fundamental developmental processes. Whereas the regulator of hematopoiesis GATA-1 is subject to multiple posttranslational modifications, how these modifications influence GATA-1 function at endogenous loci is unknown. We demonstrate that sumoylation of GATA-1 K137 promotes transcriptional activation only at target genes requiring the coregulator Friend of GATA-1 (FOG-1). A mutation of GATA-1 V205G that disrupts FOG-1 binding and K137 mutations yielded similar phenotypes, although sumoylation was FOG-1 independent, and FOG-1 binding did not require sumoylation. Both mutations dysregulated GATA-1 chromatin occupancy at select sites, FOG-1-dependent gene expression, and were rescued by tethering SUMO-1. While FOG-1- and SUMO-1-dependent genes migrated away from the nuclear periphery upon erythroid maturation, FOG-1- and SUMO-1-independent genes persisted at the periphery. These results illustrate a mechanism that controls trans-acting factor function in a locus-specific manner, and differentially regulated members of the target gene ensemble reside in distinct subnuclear compartments.
Assuntos
Fator de Transcrição GATA1/metabolismo , Hematopoese/fisiologia , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica , Camundongos , Mutação , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
BACKGROUND: High-quality bowel preparation is necessary for colonoscopy. A few studies have been conducted to investigate improvement in bowel preparation quality through patient education. However, the effect of patient education on bowel preparation has not been well studied. METHODS: A randomized and prospective study was conducted. All patients received regular instruction for bowel preparation during a pre-colonoscopy visit. Those scheduled for colonoscopy were randomly assigned to view an educational video instruction (video group) on the day before the colonoscopy, or to a non-video (control) group. Qualities of bowel preparation using the Ottawa Bowel Preparation Quality scale (Ottawa score) were compared between the video and non-video groups. In addition, factors associated with poor bowel preparation were investigated. RESULT: A total of 502 patients were randomized, 250 to the video group and 252 to the non-video group. The video group exhibited better bowel preparation (mean Ottawa total score: 3.03 ± 1.9) than the non-video group (4.21 ± 1.9; P < 0.001) and had good bowel preparation for colonoscopy (total Ottawa score <6: 91.6 % vs. 78.5 %; P < 0.001). Multivariate analysis revealed that males (odds ratio [OR] = 1.95, P = 0.029), diabetes mellitus patients (OR = 2.79, P = 0.021), and non-use of visual aids (OR = 3.09, P < 0.001) were associated with poor bowel preparation. In the comparison of the colonoscopic outcomes between groups, the polyp detection rate was not significantly different between video group and non-video group (48/250, 19.2 % vs. 48/252, 19.0 %; P = 0.963), but insertion time was significantly short in video group (5.5 ± 3.2 min) than non-video group (6.1 ± 3.7 min; P = 0.043). CONCLUSION: The addition of an educational video could improve the quality of bowel preparation in comparison with standard preparation method. TRIAL REGISTRATION: Clinical Research Information Service KCT0001836 . The date of registration: March, 08(th), 2016, Retrospectively registered.
Assuntos
Catárticos/administração & dosagem , Colonoscopia/métodos , Enema , Educação de Pacientes como Assunto/métodos , Gravação em Vídeo , Adulto , Pólipos do Colo/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Estudos ProspectivosRESUMO
BACKGROUND/AIMS: This study investigated the safety of endoscopic variceal ligation (EVL) under conscious sedation with midazolam and sequential flumazenil after procedure in these patients. METHODS: A total of 279 patients who underwent secondary prophylactic EVL at our institution between April 2012 and June 2014, were enrolled. Conscious sedation was achieved using intravenous midazolam, and flumazenil was routinely used as an antidote immediately after EVL. Patients with sleep (n = 165) and non-sleep (n = 55) endoscopy were matched using propensity score analysis (3:1). Frequencies of overt hepatic encephalopathy (HEP) and patient' satisfactions with EVL were compared between the 2 groups. RESULTS: Of the 279 patients, 155 (55.6%) were of Child-Turcotte-Pugh (CTP) class, B or C, and 224 (80.3%) patients underwent sleep endoscopy. After propensity score analysis, overt HEP was observed in 1 (0.4%) of the 165 patients in the sedated group, but not found in any in the non-sedated group. Patient' satisfaction with EVL was better in the sedated group (p < 0.001). Twenty-nine (65.9%) of the 44 patients with CTP class C underwent sleep endoscopy, and only one (3.4%) experienced overt HEP. CONCLUSIONS: Prophylactic EVL under conscious sedation using midazolam and flumazenil is probably safe in cirrhotic patients without experience of HEP, even in those of CTP class C.
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Antídotos/administração & dosagem , Sedação Consciente , Esofagoscopia/métodos , Flumazenil/administração & dosagem , Hipnóticos e Sedativos , Midazolam , Procedimentos Cirúrgicos Profiláticos/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Varizes Esofágicas e Gástricas/cirurgia , Esofagoscopia/psicologia , Feminino , Hemorragia Gastrointestinal/prevenção & controle , Humanos , Ligadura , Cirrose Hepática/complicações , Masculino , Pessoa de Meia-Idade , Satisfação do Paciente , Procedimentos Cirúrgicos Profiláticos/psicologiaRESUMO
Activated hepatic stellate cells (HSCs) play a detrimental role in liver fibrosis progression. Natural killer (NK) cells are known to selectively recognize abnormal or transformed cells via their receptor activation and induce target cell apoptosis and, therefore, can be used as a potential therapeutic strategy for liver cirrhosis. In this study, we examined the therapeutic effects of NK cells in the carbon tetrachloride (CCl4)-induced liver cirrhosis mouse model. NK cells were isolated from the mouse spleen and expanded in the cytokine-stimulated culture medium. Natural killer group 2, member D (NKG2D)-positive NK cells were significantly increased after a week of expansion in culture. The intravenous injection of NK cells significantly alleviated liver cirrhosis by reducing collagen deposition, HSC marker activation, and macrophage infiltration. For in vivo imaging, NK cells were isolated from codon-optimized luciferase-expressing transgenic mice. Luciferase-expressing NK cells were expanded, activated and administrated to the mouse model to track them. Bioluminescence images showed increased accumulation of the intravenously inoculated NK cells in the cirrhotic liver of the recipient mouse. In addition, we conducted QuantSeq 3' mRNA sequencing-based transcriptomic analysis. From the transcriptomic analysis, 33 downregulated genes in the extracellular matrix (ECM) and 41 downregulated genes involved in the inflammatory response were observed in the NK cell-treated cirrhotic liver tissues from the 1532 differentially expressed genes (DEGs). This result indicated that the repetitive administration of NK cells alleviated the pathology of liver fibrosis in the CCl4-induced liver cirrhosis mouse model via anti-fibrotic and anti-inflammatory mechanisms. Taken together, our research demonstrated that NK cells could have therapeutic effects in a CCl4-induced liver cirrhosis mouse model. In particular, it was elucidated that extracellular matrix genes and inflammatory response genes, which were mainly affected after NK cell treatment, could be potential targets.
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The dynamic packaging of DNA into chromatin is a fundamental step in the control of diverse nuclear processes. Whereas certain transcription factors and chromosomal components promote the formation of higher-order chromatin loops, the co-regulator machinery mediating loop assembly and disassembly is unknown. Using mice bearing a hypomorphic allele of the BRG1 chromatin remodeler, we demonstrate that the Brg1 mutation abrogated a cell type-specific loop between the beta-globin locus control region and the downstream beta major promoter, despite trans-acting factor occupancy at both sites. By contrast, distinct loops were insensitive to the Brg1 mutation. Molecular analysis with a conditional allele of GATA-1, a key regulator of hematopoiesis, in a novel cell-based system provided additional evidence that BRG1 functions early in chromatin domain activation to mediate looping. Although the paradigm in which chromatin remodelers induce nucleosome structural transitions is well established, our results demonstrating an essential role of BRG1 in the genesis of specific chromatin loops expands the repertoire of their functions.
Assuntos
DNA Helicases/genética , Mutação , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Alelos , Animais , Células CHO , Cromatina/química , Cromatina/metabolismo , Cricetinae , Cricetulus , DNA/química , Fator de Transcrição GATA1/metabolismo , Hematopoese , Camundongos , Nucleossomos/metabolismo , Globinas beta/metabolismoRESUMO
Natural killer (NK) cell immunotherapies for cancer can complement existing T cell therapies while benefiting from advancements already made in the immunotherapy field. For NK cell manufacturing, induced pluripotent stem cells (iPSCs) offer advantages including eliminating donor variation and providing an ideal platform for genome engineering. At the same time, extracellular vesicles (EVs) have become a major research interest, and purified NK cell extracellular vesicles (NKEVs) have been shown to reproduce the key functions of their parent NK cells. NKEVs have the potential to be developed into a standalone therapeutic with reduced complexity and immunogenicity compared to cell therapies. This review explores the role iPSC technology can play in both NK cell manufacturing and NKEV development.
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Vesículas Extracelulares , Células-Tronco Pluripotentes Induzidas , Neoplasias , Humanos , Imunoterapia , Células-Tronco Pluripotentes Induzidas/fisiologia , Células Matadoras Naturais , Neoplasias/terapiaRESUMO
It is known that C3 is required for optimal expansion of T cells during acute viral infections. However, it is not yet determined whether T cell responses to intracellular bacterial infections require C3. Therefore, we have investigated the requirement for C3 to elicit potent T cell responses to Listeria monocytogenes (LM). We show that expansion of Ag-specific CD8 and CD4 T cells during a primary response to LM was markedly reduced in the absence of C3 activity. Further studies indicated that, unlike in an influenza virus infection, the regulation of LM-specific T cell responses by C3 might not involve the downstream effector C5a. Moreover, reduced T cell responses to LM was not linked to defective maturation of dendritic cells or developmental anomalies in the peripheral T cell compartment of C3-deficient mice. Experiments involving adoptive transfer of C3-deficient CD8 T cells into the C3-sufficient environment of wild-type mice showed that these T cells do not have intrinsic proliferative defects, and a paracrine source of C3 will suffice for clonal expansion of CD8 T cells in vivo. However, stimulation of purified C3-deficient CD8 T cells by plastic-immobilized anti-CD3 showed that C3 promotes T cell proliferation directly, independent of its effects on APC. On the basis of these findings, we propose that diminished T cell responses to LM in C3-deficient mice might be at least in part due to lack of direct effects of C3 on T cells. These studies have furthered our understanding of C3-mediated regulation of T cell immunity to intracellular pathogens.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Proliferação de Células , Complemento C3/fisiologia , Listeriose/imunologia , Listeriose/patologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Complemento C3/deficiência , Complemento C3/genética , Listeria monocytogenes/imunologia , Listeriose/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/fisiologiaRESUMO
Alpha globin expression must be regulated properly to prevent the occurrence of alpha-thalassemias, yet many questions remain unanswered regarding the mechanism of transcriptional activation. Identifying factors that regulate chromatin structure of the endogenous alpha globin locus in developing erythroblasts will provide important mechanistic insight. Here, we demonstrate that the BRG1 catalytic subunit of SWI/SNF-related complexes co-immunoprecipitates with GATA-1 and EKLF in murine fetal liver cells in vivo and is recruited to the far-upstream major-regulatory element (MRE) and alpha2 promoter. Furthermore, based on our analysis of Brg1(null/ENU1) mutant mice, BRG1 regulates DNase I sensitivity, H3ac, and H3K4me2 but not CpG methylation at both sites. Most importantly, BRG1 is required for chromatin loop formation between the MRE and alpha2 promoter and for maximal RNA Polymerase II occupancy at the alpha2 promoter. Consequently, Brg1 mutants express alpha globin mRNA at only 5-10% of wild-type levels and die at mid-gestation. These data identify BRG1 as a chromatin-modifying factor required for nucleosome remodeling and transcriptional activation of the alpha globin locus. These data also demonstrate that chromatin looping between the MRE and alpha2 promoter is required as part of the transcriptional activation mechanism.
Assuntos
Cromatina/química , DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/química , Fatores de Transcrição/metabolismo , Ativação Transcricional , alfa-Globinas/genética , Animais , DNA Helicases/genética , Desoxirribonuclease I , Epigênese Genética , Camundongos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Elementos de Resposta , Fatores de Transcrição/genética , alfa-Globinas/metabolismoRESUMO
The discovery of human pluripotent stem cells (PSCs) at the turn of the century opened the door to a new generation of regenerative medicine research. Among PSCs, the donors available for induced pluripotent stem cells (iPSCs) are greatest, providing a potentially universal cell source for all types of cell therapies including cancer immunotherapies using natural killer (NK cells). Unlike primary NK cells, those prepared from iPSCs can be prepared with a homogeneous quality and are easily modified to exert a desired response to tumor cells. There already exist several protocols to genetically modify and differentiate iPSCs into NK cells, and each has its own advantages with regards to immunotherapies. In this short review, we detail the benefits of using iPSCs in NK cell immunotherapies and discuss the challenges that must be overcome before this approach becomes mainstream in the clinic.
Assuntos
Imunoterapia , Células-Tronco Pluripotentes Induzidas/citologia , Células Matadoras Naturais/citologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Diferenciação Celular , Engenharia Genética , HumanosRESUMO
In this study, the interfacial reactions and mechanical properties of solder joints after multiple reflows were observed to evaluate the applicability of the developed materials for high-temperature soldering for automotive electronic components. The microstructural changes and mechanical properties of Sn-Cu solders regarding Al(Si) addition and the number of reflows were investigated to determine their reliability under high heat and strong vibrations. Using differential scanning calorimetry, the melting points were measured to be approximately 227, 230, and 231 °C for the SC07 solder, SC-0.01Al(Si), and SC-0.03Al(Si), respectively. The cross-sectional analysis results showed that the total intermetallic compounds (IMCs) of the SC-0.03Al(Si) solder grew the least after the as-reflow, as well as after 10 reflows. Electron probe microanalysis and transmission electron microscopy revealed that the Al-Cu and Cu-Al-Sn IMCs were present inside the solders, and their amounts increased with increasing Al(Si) content. In addition, the Cu6Sn5 IMCs inside the solder became more finely distributed with increasing Al(Si) content. The Sn-0.5Cu-0.03Al(Si) solder exhibited the highest shear strength at the beginning and after 10 reflows, and ductile fracturing was observed in all three solders. This study will facilitate the future application of lead-free solders, such as an Sn-Cu-Al(Si) solder, in automotive electrical components.
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GATA factors orchestrate hematopoiesis via multistep transcriptional mechanisms, but the interrelationships and importance of individual steps are poorly understood. Using complementation analysis with GATA-1-null cells and mice containing a hypomorphic allele of the chromatin remodeler BRG1, we dissected the pathway from GATA-1 binding to cofactor recruitment, chromatin loop formation, and transcriptional activation. Analysis of GATA-1-mediated activation of the beta-globin locus, in which GATA-1 assembles dispersed complexes at the promoters and the distal locus control region (LCR), revealed molecular intermediates, including GATA-1-independent and GATA-1-containing LCR subcomplexes, both defective in promoting loop formation. An additional intermediate consisted of an apparently normal LCR complex and a promoter complex with reduced levels of total RNA polymerase II (Pol II) and Pol II phosphorylated at serine 5 of the carboxy-terminal domain. Reduced BRG1 activity solely compromised Pol II and serine 5-phosphorylated Pol II occupancy at the promoter, phenocopying the LCR-deleted mouse. These studies defined a hierarchical order of GATA-1-triggered events at a complex locus and establish a novel mechanism of long-range gene regulation.
Assuntos
Diferenciação Celular , Cromatina/metabolismo , Fator de Transcrição GATA1/fisiologia , Regulação da Expressão Gênica , Sistema Hematopoético/fisiologia , Alelos , Animais , Linhagem Celular , Cromatina/química , Imunoprecipitação da Cromatina , DNA Helicases/genética , DNA Helicases/metabolismo , Células Eritroides/citologia , Células Eritroides/metabolismo , Fator de Transcrição GATA1/genética , Deleção de Genes , Globinas/genética , Região de Controle de Locus Gênico , Camundongos , Camundongos Knockout , Modelos Genéticos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Temperatura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The reciprocal expression of GATA-1 and GATA-2 during hematopoiesis is an important determinant of red blood cell development. Whereas Gata2 is preferentially transcribed early in hematopoiesis, elevated GATA-1 levels result in GATA-1 occupancy at sites upstream of the Gata2 locus and transcriptional repression. GATA-2 occupies these sites in the transcriptionally active locus, suggesting that a "GATA switch" abrogates GATA-2-mediated positive autoregulation. Chromatin immunoprecipitation (ChIP) coupled with genomic microarray analysis and quantitative ChIP analysis with GATA-1-null cells expressing an estrogen receptor ligand binding domain fusion to GATA-1 revealed additional GATA switches 77 kb upstream of Gata2 and within intron 4 at +9.5 kb. Despite indistinguishable GATA-1 occupancy at -77 kb and +9.5 kb versus other GATA switch sites, GATA-1 functioned uniquely at the different regions. GATA-1 induced histone deacetylation at and near Gata2 but not at the -77 kb region. The -77 kb region, which was DNase I hypersensitive in both active and inactive states, conferred equivalent enhancer activities in GATA-1- and GATA-2-expressing cells. By contrast, the +9.5 kb region exhibited considerably stronger enhancer activity in GATA-2- than in GATA-1-expressing cells, and other GATA switch sites were active only in GATA-1- or GATA-2-expressing cells. Chromosome conformation capture analysis demonstrated higher-order interactions between the -77 kb region and Gata2 in the active and repressed states. These results indicate that dispersed GATA factor complexes function via long-range chromatin interactions and qualitatively distinct activities to regulate Gata2 transcription.
Assuntos
Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Acetilação , Animais , Pareamento de Bases/genética , Sequência de Bases , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Sequência Conservada/genética , Desoxirribonuclease I/metabolismo , Elementos Facilitadores Genéticos/genética , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Receptores de Estrogênio/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
During somatic cell reprogramming to induced pluripotent stem cells (iPSCs), fibroblasts undergo dynamic molecular changes, including a mesenchymal-to-epithelial transition (MET) and gain of pluripotency; processes that are influenced by Yamanaka factor stoichiometry. For example, in early reprogramming, high KLF4 levels are correlated with the induction of functionally undefined, transiently expressed MET genes. Here, we identified the cell-surface protein TROP2 as a marker for cells with transient MET induction in the high-KLF4 condition. We observed the emergence of cells expressing the pluripotency marker SSEA-1+ mainly from within the TROP2+ fraction. Using TROP2 as a marker in CRISPR/Cas9-mediated candidate screening of MET genes, we identified the transcription factor OVOL1 as a potential regulator of an alternative epithelial cell fate characterized by the expression of non-iPSC MET genes and low cell proliferation. Our study sheds light on how reprogramming factor stoichiometry alters the spectrum of intermediate cell fates, ultimately influencing reprogramming outcomes.
Assuntos
Reprogramação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Fatores de Transcrição/metabolismo , Animais , Biomarcadores/metabolismo , Sistemas CRISPR-Cas/fisiologia , Moléculas de Adesão Celular/metabolismo , Proliferação de Células/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Regulação da Expressão Gênica/fisiologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Antígenos CD15/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes.
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Reparo do DNA por Junção de Extremidades , Edição de Genes , Células-Tronco Pluripotentes Induzidas/metabolismo , Alelos , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos/genética , Loci Gênicos , Células HEK293 , Humanos , Mutação/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismoRESUMO
Somatic cell reprogramming to induced pluripotent stem cells (iPSCs) is a revolutionary technology, with repercussions affecting modern functional genomics and regenerative medicine. Still, relatively little is known about the processes underlying this dramatic cellular and molecular metamorphosis. Reprogramming technology based on the implementation of piggyBac (PB) transposons has enabled studies of iPSC reprogramming mechanisms, shedding an increasing light on these processes. Unique characteristics of PB transposons such as efficient genomic integration, unlimited cargo capacity, robust gene expression, and even seamless excision highlight the importance of this transgenic tool in advancing stem cell biology. In this chapter, we provide a detailed overview of versatile primary iPSC generation from mouse somatic cells using PB transposons, and the subsequent establishment of robust secondary reprogramming systems. These protocols are highlighted with examples from recent studies as to how PB has been, and continues to be, conducive to the dissection of reprogramming processes at the cellular and molecular levels.
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Técnicas de Cultura de Células/métodos , Técnicas de Reprogramação Celular/métodos , Reprogramação Celular , Elementos de DNA Transponíveis/genética , Vetores Genéticos/genética , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Separação Celular/métodos , Rastreamento de Células/métodos , Células Cultivadas , Células Clonais/citologia , Criopreservação , Doxiciclina/farmacologia , Feminino , Fibroblastos/citologia , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Transgênicos , Proteína Homeobox Nanog , Proteína Vermelha FluorescenteRESUMO
Transgenics is a mainstay of functional genomics. Conditionally overexpressing genes of interest (GOIs) helps to reveal their roles in the control of complex biological processes. Complemented by findings in classic animal model systems, recent advances in human embryonic stem cell (hESC) and patient-specific induced pluripotent stem cell (hiPSC) differentiation have led to sophisticated in vitro models of human development and disease. Yet, as transgenic elements encoding inducible systems must be introduced de novo into each genetically unique human stem cell line, robust and straightforward solutions to gene delivery are required. Transposons are a family of mobile DNA elements that have been adapted as experimental tools for stable genomic integration of transgenes. The piggyBac (PB) transposon from Trichoplusia ni presents a number of benefits over classic viral or BAC transgenesis: ease of application, simple integration-site mapping, and the unique capacity for traceless excision. Moreover, their large capacity permits the consolidation of multiple transgene components in a single vector system. In this chapter, we outline the features of a panel of "All-in-One" PB transposons designed for drug-inducible gene expression and provide guidelines to establish and validate populations or clones of transgenic hiPSCs.
Assuntos
Técnicas de Cultura de Células/métodos , Técnicas de Reprogramação Celular/métodos , Reprogramação Celular , Elementos de DNA Transponíveis/genética , Expressão Gênica/genética , Vetores Genéticos/genética , Células-Tronco Pluripotentes Induzidas/citologia , Transgenes , Separação Celular/métodos , Rastreamento de Células/métodos , Células Cultivadas , Células Clonais/citologia , Doxiciclina/farmacologia , Fibroblastos/citologia , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Luciferases/genética , Proteínas Luminescentes/genética , Neomicina/farmacologia , Puromicina/farmacologia , Transgenes/genética , Proteína Vermelha FluorescenteRESUMO
Transarterial chemoembolization (TACE) is frequently used for treatment of unresectable hepatocellular carcinoma (HCC) and can also be used for case of liver metastases from rectal cancer. Although it is recognized as safe and effective treatment, various complications have been reported. However, post-TACE duodenal perforation with duodenal and esophageal ischemia has not been reported in the literature. A 43-year-old male had experienced duodenal perforation combined with duodenal and lower esophageal ischemia after 8 times of repeated TACE for recurrent and unresectable HCCs, that was confirmed on esophagogastroduodenoscopy and abdominal computed tomography. Interestingly, operative findings showed complete recovery of duodenal ischemia except perforation, and he recovered with just the segmental duodenectomy and gastrojejunostomy. We report a case of duodenal perforation with necrosis and esophageal ischemia after 8th TACE for unresectable HCC. Although this complication is rare and unexpected, it may result in severe sequelae requiring surgical repair.The careful procedure during TACE followed by post-TACE careful monitoring is required in patients with posthepatectomy or repeated TACE, especially in the case with unusual clinical manifestations.
Assuntos
Carcinoma Hepatocelular/terapia , Quimioembolização Terapêutica/efeitos adversos , Duodenopatias/etiologia , Esôfago/irrigação sanguínea , Perfuração Intestinal/etiologia , Isquemia/etiologia , Neoplasias Hepáticas/terapia , Adulto , Quimioembolização Terapêutica/métodos , Artéria Hepática , Humanos , MasculinoRESUMO
Pseudomembranous colitis (PMC) is a nosocomial and opportunistic infection caused by Clostridium difficile. PMC is related to the use of antibiotics leading to intestinal dysbiosis and an overgrowth of C. difficile. Metronidazole or vancomycin is considered to be the standard therapy for the management of PMC. However, PMC has a 15%-30% recurrence rate and can be refractory to standard treatments, resulting in morbidity and mortality. Here we describe a patient who experienced refractory PMC who was treated with fecal microbiota transplantation. A 69-year-old woman was admitted to the hospital with consistent abdominal pain and diarrhea, which had been present for 5 months. She was diagnosed with PMC by colonoscopy and tested positive for C. difficile toxin. Even though she took metronidazole for 10 days, followed by vancomycin for 4 weeks, her symptoms did not improve. Because of her recurrent and refractory symptoms, we decided to perform fecal microbiota transplantation. Fifty grams of fresh feces from a donor were obtained on the day of the procedure, mixed with 500 mL of normal saline, and then filtered. The filtered solution was administered to the patient's colon using a colonoscope. After the procedure, her symptoms rapidly improved and a follow-up colonoscopy showed that the PMC had resolved without recurrence.