RESUMO
Recombinant adeno-associated virus (rAAV) vectors are often produced in HEK293 or Spodoptera frugiperda (Sf)-based cell lines. We compared expression profiles of "oversized" (â¼5,000 bp) and "standard-sized" (4,600 bp) rAAV5-human α1-antitrypsin (rAAV5-hA1AT) vectors manufactured in HEK293 or Sf cells and investigated molecular mechanisms mediating expression decline. C57BL/6 mice received 6 × 1013 vg/kg of vector, and blood and liver samples were collected through week 57. For all vectors, peak expression (weeks 12-24) declined by 50% to week 57. For Sf- and HEK293-produced oversized vectors, serum hA1AT was initially comparable, but in weeks 12-57, Sf vectors provided significantly higher expression. For HEK293 oversized vectors, liver genomes decreased continuously through week 57 and significantly correlated with A1AT protein. In RNA-sequencing analysis, HEK293 vector-treated mice had significantly higher inflammatory responses in liver at 12 weeks compared with Sf vector- and vehicle-treated mice. Thus, HEK293 vector genome loss led to decreased transgene protein. For Sf-produced vectors, genomes did not decrease from peak expression. Instead, vector genome accessibility significantly decreased from peak to week 57 and correlated with transgene RNA. Vector DNA interactions with active histone marks (H3K27ac/H3K4me3) were significantly reduced from peak to week 57, suggesting that epigenetic regulation impacts transgene expression of Sf-produced vectors.
Assuntos
Epigênese Genética , Insetos , Humanos , Camundongos , Animais , Células HEK293 , Camundongos Endogâmicos C57BL , RNA , MamíferosRESUMO
Recently, growing attention has been directed toward stem cell metabolism, with the key observation that the plasticity of stem cells also reflects the plasticity of their energy substrate metabolism. There seems to be a clear link between the self-renewal state of stem cells, in which cells proliferate without differentiation, and the activity of specific metabolic pathways. Differentiation is accompanied by a shift from anaerobic glycolysis to mitochondrial respiration. This metabolic switch of differentiating stem cells is required to cover the energy demands of the different organ-specific cell types. Among other metabolic signatures, amino acid and carbohydrate metabolism is most prominent in undifferentiated embryonic stem cells, whereas the fatty acid metabolic signature is unique in cardiomyocytes derived from embryonic stem cells. Identifying the specific metabolic pathways involved in pluripotency and differentiation is critical for further progress in the field of developmental biology and regenerative medicine. The recently generated knowledge on metabolic key processes may help to generate mature stem cell-derived somatic cells for therapeutic applications without the requirement of genetic manipulation. In the present review, the literature about metabolic features of stem cells and their cardiovascular cell derivatives as well as the specific metabolic gene signatures differentiating between stem and differentiated cells are summarized and discussed.
Assuntos
Metabolismo Energético/fisiologia , Miócitos Cardíacos/metabolismo , Células-Tronco/metabolismo , Aminoácidos/metabolismo , Animais , Metabolismo dos Carboidratos/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Ácidos Graxos/metabolismo , Humanos , CamundongosRESUMO
BACKGROUND: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs) requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs) of different murine ESC lines. METHODS: Two wild-type (D3 and R1) and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7) were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP) and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC) promoter. Action potentials (APs) were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. RESULTS: Spontaneous AP frequency and AP duration (APD) as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. CONCLUSION: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.
Assuntos
Células-Tronco Embrionárias/citologia , Miócitos Cardíacos/citologia , Acetiltransferases/genética , Acetiltransferases/metabolismo , Potenciais de Ação/efeitos dos fármacos , Agonistas Adrenérgicos beta/farmacologia , Animais , Carbacol/farmacologia , Diferenciação Celular , Linhagem Celular , Eletrodos , Fenômenos Eletrofisiológicos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Isoproterenol/farmacologia , Camundongos , Agonistas Muscarínicos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , Regiões Promotoras GenéticasRESUMO
BACKGROUND: The ability to recapitulate mature adult phenotypes is critical to the development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as models of disease. The present study examines the characteristics of the transient outward current (Ito) and its contribution to the hiPSC-CM action potential (AP). METHOD: Embryoid bodies were made from a hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record APs from beating-clusters (BC) and patch-clamp techniques were used to record Ito in single hiPSC-CM. mRNA levels of Kv1.4, KChIP2 and Kv4.3 were quantified from BCs. RESULTS: BCs exhibited spontaneous beating (60.5±2.6 bpm) and maximum-diastolic-potential (MDP) of 67.8±0.8 mV (n=155). A small 4-aminopyridine-sensitive phase-1-repolarization was observed in only 6/155 BCs. A robust Ito was recorded in the majority of cells (13.7±1.9 pA/pF at +40 mV; n=14). Recovery of Ito from inactivation (at -80 mV) showed slow kinetics (τ1=200±110 ms (12%) and τ2=2380±240 ms (80%)) accounting for its minimal contribution to the AP. Transcript data revealed relatively high expression of Kv1.4 and low expression of KChIP2 compared to human native ventricular tissues. Mathematical modeling predicted that restoration of IK1 to normal levels would result in a more negative MDP and a prominent phase-1-repolarization. CONCLUSION: The slow recovery kinetics of Ito coupled with a depolarized MDP account for the lack of an AP notch in the majority of hiPSC-CM. These characteristics reveal a deficiency for the development of in vitro models of inherited cardiac arrhythmia syndromes in which Ito-induced AP notch is central to the disease phenotype.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Potenciais da Membrana/fisiologia , Modelos Biológicos , Miócitos Cardíacos/metabolismo , Potássio/metabolismo , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Proteínas Interatuantes com Canais de Kv/metabolismo , Canal de Potássio Kv1.4/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/citologia , Canais de Potássio Shal/metabolismoRESUMO
Brachyury(+) mesodermal cell population with purity over 79% was obtained from differentiating brachyury embryonic stem cells (ESC) generated with brachyury promoter driven enhanced green fluorescent protein and puromycin-N-acetyltransferase. A comprehensive transcriptomic analysis of brachyury(+) cells enriched with puromycin application from 6-day-old embryoid bodies (EBs), 6-day-old control EBs and undifferentiated ESCs led to identification of 1573 uniquely up-regulated and 1549 uniquely down-regulated transcripts in brachyury(+) cells. Furthermore, transcripts up-regulated in brachyury(+) cells have overrepresented the Gene Ontology annotations (cell differentiation, blood vessel morphogenesis, striated muscle development, placenta development and cell motility) and Kyoto Encyclopedia of Genes and Genomes pathway annotations (mitogen-activated protein kinase signaling and transforming growth factor beta signaling). Transcripts representing Larp2 and Ankrd34b are notably up-regulated in brachyury(+) cells. Knockdown of Larp2 resulted in a significantly down-regulation BMP-2 expression, and knockdown of Ankrd34b resulted in alteration of NF-H, PPARγ and PECAM1 expression. The elucidation of transcriptomic signatures of ESCs-derived brachyury(+) cells will contribute toward defining the genetic and cellular identities of presumptive mesodermal cells. Furthermore, there is a possible involvement of Larp2 in the regulation of the late mesodermal marker BMP-2. Ankrd34b might be a positive regulator of neurogenesis and a negative regulator of adipogenesis.
Assuntos
Células-Tronco Embrionárias/metabolismo , Proteínas Fetais/metabolismo , Proteínas com Domínio T/metabolismo , Linfócitos T/metabolismo , Transcriptoma , Acetiltransferases/metabolismo , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Células Cultivadas , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Camundongos , Proteínas de Neurofilamentos/metabolismo , PPAR gama/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Antígeno SS-BRESUMO
Personalized regenerative medicine and biomedical research have been galvanized and revolutionized by human pluripotent stem cells in combination with recent advances in genomics, artificial intelligence, and genome engineering. More recently, we have witnessed the unprecedented breakthrough life-saving translation of mRNA-based vaccines for COVID-19 to contain the global pandemic and the investment in billions of US dollars in space exploration projects and the blooming space-tourism industry fueled by the latest reusable space vessels. Now, it is time to examine where the translation of pluripotent stem cell research stands currently, which has been touted for more than the last two decades to cure and treat millions of patients with severe debilitating degenerative diseases and tissue injuries. This review attempts to highlight the accomplishments of pluripotent stem cell research together with cutting-edge genomics and genome editing tools and, also, the promises that have still not been transformed into clinical applications, with cardiovascular research as a case example. This review also brings to our attention the scientific and socioeconomic challenges that need to be effectively addressed to see the full potential of pluripotent stem cells at the clinical bedside.
Assuntos
Doenças Cardiovasculares/terapia , Genômica , Células-Tronco Pluripotentes/transplante , Inteligência Artificial , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Sistema Cardiovascular/citologia , Sistema Cardiovascular/crescimento & desenvolvimento , Diferenciação Celular , Descoberta de Drogas , Edição de Genes , Humanos , Modelos Biológicos , Células-Tronco Pluripotentes/citologia , Medicina de Precisão , Medicina Regenerativa , Segurança , Pesquisa Translacional BiomédicaRESUMO
Early mammalian heart development is characterized by transient expression of alpha-smooth muscle actin (Acta2). To date, cardiomyocytes expressing Acta2 in the early stages of in vivo development have not been characterized. To functionally characterize Acta2-expressing cardiomyocytes, we used a transgenic ES cell line expressing both the puromycin acetyl transferase (Pac) and enhanced green fluorescent protein (EGFP) cassettes under the control of the Acta2 promoter. The onset of Acta2 expression occurred in parallel with the appearance of beating areas, indicating the formation of cardiomyocytes. Antibiotic selection resulted in a high yield of cardiomyocytes and smooth muscle cells. The green fluorescent beating areas stained positively for multiple cardiomyocyte markers. Comparative electrophysiological analysis including fetal and alpha-MHC-expressing ES cell-derived cardiomyocyte controls showed that Acta2-positive cardiomyocytes contained pacemaker-, atrial- and ventricular-like phenotypes. Interestingly, the proportion of ventricular-like cells was much higher in the Acta2-positive cardiomyocytes population than in control alpha-MHC-expressing cardiomyocytes (75 % and 12 %, respectively). The findings of the present study provide a novel approach for the identification and enrichment of Acta2-positive cardiomyocytes, especially of the ventricular phenotype under in vitro conditions.
Assuntos
Actinas/isolamento & purificação , Actinas/metabolismo , Células-Tronco Embrionárias/citologia , Músculo Liso/metabolismo , Miócitos Cardíacos/metabolismo , Acetiltransferases/genética , Actinas/genética , Animais , Linhagem Celular , Separação Celular , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Camundongos , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransgenesRESUMO
BACKGROUND: Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF). Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency. RESULTS: By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i) LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [Pluri genes], ii) LIF induced genes [Lifind genes] whose expression is differentially regulated depending upon cell context and iii) genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of Pluri and Lifind genes, and revealed their differential modulation by Oct4 or Nanog master genes. Individual knock down of a selection of Pluri and Lifind genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed. CONCLUSION: We have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment), which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG) is still expressed and which might be down-regulated together for driving cells towards differentiation.
Assuntos
Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Fator Inibidor de Leucemia/metabolismo , Camundongos/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Análise por Conglomerados , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/metabolismoRESUMO
Induced pluripotent stem cell (iPSC)-based disease modelling and the cell replacement therapy approach have proven to be very powerful and instrumental in biomedical research and personalized regenerative medicine as evidenced in the past decade by unraveling novel pathological mechanisms of a multitude of monogenic diseases at the cellular level and the ongoing and emerging clinical trials with iPSC-derived cell products. iPSC-based disease modelling has sparked widespread enthusiasm and has presented an unprecedented opportunity in high throughput drug discovery platforms and safety pharmacology in association with three-dimensional multicellular organoids such as personalized organs-on-chips, gene/base editing, artificial intelligence and high throughput "omics" methodologies. This critical review summarizes the progress made in the past decade with the advent of iPSC discovery in biomedical applications and regenerative medicine with case examples and the current major challenges that need to be addressed to unleash the full potential of iPSCs in clinical settings and pharmacology for more effective and safer regenerative therapy.
Assuntos
Doença , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Descoberta de Drogas , Instabilidade Genômica , Humanos , Medicina RegenerativaRESUMO
BACKGROUND: Heart failure (HF) is a leading cause of mortality and is associated with cardiac remodeling. Vulnerability to atrial fibrillation (AF) has been shown to be greater in the early stages of HF, whereas ventricular tachycardia/fibrillation develop during late stages. Here, we explore changes in gene expression that underlie the differential development of fibrosis and structural alterations that predispose to atrial and ventricular arrhythmias. OBJECTIVE: To study transcriptomic changes associated with the development of cardiac arrhythmias in early and late stages of heart failure. METHODS: Dogs were tachy-paced from right ventricle (RV) for 2-3 or 5-6 weeks (early and late HF). We performed transcriptomic analysis of right atria (RA) and RV isolated from control dogs and those in early and late HF. Transcripts with mean relative log2-fold change ≥2 were included in the differential analysis with significance threshold adjusted to p<0.05. RESULTS: Early HF remodeling was more prominent in RA with enrichment of extracellular matrix, circulatory system, wound healing and immune response pathways; many of these processes were not present in RA in late HF. RV showed no signs of remodeling in early HF but enrichment of extracellular matrix and wound healing in late HF. CONCLUSION: Our transcriptomic data indicate significant fibrosis-associated transcriptional changes in RA in early HF and in RV in late HF, with strong atrial predominance. These alterations in gene expression are consistent with the development of arrhythmogenesis in atria in early but not late HF and in the ventricle in late but not early HF.
Assuntos
Fibrilação Atrial/genética , Proteínas da Matriz Extracelular/genética , Insuficiência Cardíaca/genética , Marca-Passo Artificial/veterinária , Taquicardia Ventricular/genética , Transcriptoma , Animais , Fibrilação Atrial/diagnóstico por imagem , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Modelos Animais de Doenças , Cães , Ecocardiografia , Proteínas da Matriz Extracelular/metabolismo , Fibrose , Perfilação da Expressão Gênica , Átrios do Coração/diagnóstico por imagem , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Hemodinâmica/genética , Imunidade Inata/genética , Análise em Microsséries , Taquicardia Ventricular/diagnóstico por imagem , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologia , Fatores de Tempo , Cicatrização/genéticaRESUMO
Cardiovascular diseases are the leading cause of death globally. The pluripotency and indefinite proliferative capacity of embryonic stem (ES) cells make them a promising candidate for the cell replacement therapy where the damaged cells are replaced by the functional cells derived from stem cells in vitro. Emerging results with human ES cells for the myocardial repair are encouraging, but this approach is still in its infancy and is under extensive investigation. The daily upcoming experimental observations are reinforcing the solid hope that ES cells will be the potential source for use in cell replacement therapy. Although this demands serious considerations ethically and on practical applicability, the newly upcoming discoveries show that the adult human fibroblasts can be reprogrammed to embryonic stem cell like cells (called induced pluripotent stem cells) which can be used for cell replacement therapies. Remarkably, this obviates the need for the embryo destruction and overcomes related immunological problems which are the long time hurdles for the ES cell based cell replacement therapy. This review weighs the actual stand off in the human ES cells based cell replacement therapy for the treatment of cardiovascular degenerative diseases with a special emphasis on the hurdles and challenges to be resolved before the onset of clinical trials and the potential of the recently reported "induced pluripotent stem cells" for their use in cell replacement therapy.
Assuntos
Células-Tronco Embrionárias/citologia , Cardiopatias/terapia , Miócitos Cardíacos/citologia , Transplante de Células-Tronco/tendências , Diferenciação Celular/fisiologia , HumanosRESUMO
The Melanoma-associated Antigen gene family (MAGE) generally encodes for tumour antigens. We had identified that one of the MAGE gene members, Mageb16 was highly expressed in undifferentiated murine embryonic stem cells (ESCs). While the role of Mageb16 in stemness and differentiation of pluripotent stem cells is completely unknown, here, in our current study, we have demonstrated that Mageb16 (41 kDa) is distributed in cytosol and/or in surface membrane in undifferentiated ESCs. A transcriptome study performed at differentiated short hairpin RNA (shRNA)-mediated Mageb16 knockdown (KD) ESCs and scrambled control (SCR) ESCs until a period of 22 days, revealed that Mageb16 KD ESCs mainly differentiated towards cells expressing mesodermal and cardiovascular lineage - gene markers. Gene markers of other mesoderm-oriented biological processes such as adipogenesis, osteogenesis, limb morphogenesis and spermatogenesis were also significantly enriched in the differentiated Mageb16 KD ESCs. The expression levels of contractile genes were higher in differentiated Mageb16 KD ESCs when compared to differentiated SCR and wild ESCs, suggesting a higher cardiomyogenic potential of Mageb16 depleted ESCs. Further analysis indicates that regulative epigenetic networks and nucleocytoplasmic modifications induced by the depletion of Mageb16, may play a probable role in differentiation.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Neoplasias/deficiência , Células-Tronco Pluripotentes/metabolismo , Animais , Antígenos de Neoplasias/genética , Membrana Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Proteínas de Neoplasias/genética , Células-Tronco Pluripotentes/citologia , RNA Interferente Pequeno/metabolismo , TranscriptomaRESUMO
In vivo, cardiomyocytes comprise a heterogeneous population of contractile cells defined by unique electrophysiologies, molecular markers and morphologies. The mechanisms directing myocardial cells to specific sub-lineages remain poorly understood. Here we report that overexpression of TGFß-Activated Kinase (TAK1/Map3k7) in mouse embryonic stem (ES) cells faithfully directs myocardial differentiation of embryoid body (EB)-derived cardiac cells toward the sinoatrial node (SAN) lineage. Most cardiac cells in Map3k7-overexpressing EBs adopt markers, cellular morphologies, and electrophysiological behaviors characteristic of the SAN. These data, in addition to the fact that Map3k7 is upregulated in the sinus venous-the source of cells for the SAN-suggest that Map3k7 may be an endogenous regulator of the SAN fate.
Assuntos
Diferenciação Celular/genética , MAP Quinase Quinase Quinases/genética , Miócitos Cardíacos/citologia , Nó Sinoatrial/citologia , Animais , Células Cultivadas , Vetores Genéticos , Lentivirus/genética , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The role of striatin interacting protein 2 (Strip2) in differentiation of embryonic stem cells (ESCs) is still under debate. Strip2-silenced murine (KD) ESCs were differentiated for 4, 8, 12, and 16 days. We show that Strip2 is distributed in the perinucleus or nuclei of wild-type (WT) undifferentiated ESCs, but is localized in high-density nuclear bodies in differentiated cells. CellNet analysis of microarray gene expression data for the KD and scrambled control (SCR) embryoid bodies (EBs), as well as immunostainings of key pluripotent factors, demonstrated that differentiation of KD ESCs is repressed. This occurs even in 16-day-old EBs, which possessed a high tumorigenic potential. Correlated with very high expression levels of epigenetic regulator genes, Hat1 and Dnmt3, enzymatic activities of the histone acetyltransferase type B (Hat1) and DNA (cytosine-5)-methyltransferase 3 beta (Dnmt3b) were higher in differentiated 16-day-old KD EBs than in SCR or WT EBs. The expression levels of let-7, 290, and 302 microRNA families were opposed in KD ESCs, while KD EBs had levels comparable to WT and SCR ESCs during differentiation. Strip2 is critical for the regular differentiation of ESCs. Moreover, Strip2 deficient ESCs showed a dysregulation of epigenetic regulators and microRNAs regulating pluripotency.
RESUMO
Epidemiological studies have repeatedly demonstrated a correlation between nutrition, development and the severity of malignant and non-malignant proliferative diseases such as cancer and atherosclerosis. Therefore, the prevention of chronic proliferative diseases through dietary intervention is currently receiving considerable attention. Until now, much of the research is being focused on the cellular and molecular action mechanisms of dietary small molecules explaining their beneficial effects. Dietary chemicals may affect gene expression in several human diseases. However, significant progress has been made and several molecular action mechanisms have been proposed. Alteration of genetical pathways by nutrition, also called "Nutrigenomics", may offer a new approach for understanding the beneficial effects of dietary compounds on the development of severe polygenic diseases, such as cardiovascular disease, diabetes and hypertension. This review focuses on the nutritional genomics of dietary chemicals with a special emphasis on catechins. Catechins belong to the flavonoid family, which are polyphenolic compounds available in foods of plant origin. Several epidemiological studies have reported that consumption of flavonoids, and especially catechins might function as chemopreventive agents against cancer and cardiovascular diseases.
Assuntos
Regulação da Expressão Gênica , Genômica/métodos , Neoplasias/prevenção & controle , Fenômenos Fisiológicos da Nutrição , Animais , Arildialquilfosfatase/efeitos dos fármacos , Arildialquilfosfatase/genética , Catequina/análogos & derivados , Catequina/farmacologia , Suplementos Nutricionais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Proteômica/métodos , Transdução de SinaisRESUMO
In vitro culturing of mammalian cells provides an elegant platform to study cell signaling, interactions, and metabolism as well as proliferation and differentiation processes. Often, these cells are cultured and maintained in sera obtained from animals such as horses, cows, and rabbits. The sera used for this purpose fluctuates in composition from individual animals and, hence, influences the cellular growth and differentiation at different magnitudes. This poses a need to use a substitute for sera in cell culture systems to overcome the observed variations. Here, we present and compare protocols for culturing of embryonic stem (ES) cells in serum-free conditions, derivation of germ layers, and cardiac differentiation of ES cells in both serum-free and serum-containing culture conditions. Differentiated embryoid bodies by serum-free protocols produce significantly increased frequencies of clusters of cardiac cells beating stronger than found in serum-containing media. Therefore, we conclude that the use of serum replacement media (SRM) in our experiments led to more specific differentiation but reduced proliferation because these SRMs contained reduced essential substances like growth factors and hormones. Unlike serum media, SRMs have a well-defined composition and are highly reproducible. Hence, SRM will be the ideal substitute for serum-containing media.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Meios de Cultura Livres de Soro , Miócitos Cardíacos/fisiologia , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Masculino , Camundongos , Miócitos Cardíacos/citologia , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
In vitro differentiation of embryonic stem (ES) cells results in generation of tissue-specific somatic cells and may represent a powerful tool for general understanding of cellular differentiation and development in vivo. Culturing of most ES cell lines requires murine embryonic fibroblasts (MEF), which may influence adventitiously the genetic differentiation program of ES cells. We compared the expression profile of key developmental genes in the MEF-independent CGR8 ES cell line and in the MEF-dependent D3 ES cell line. Using neomycin-resistant MEFs we demonstrated that MEFs are able to contaminate the D3 ES cells even after removing the MEFs. Subsequently, optimal differentiation conditions were established for the differentiation of CGR8 ES cells into various germ layer cells. Detailed gene expression studies in differentiating CGR8 cells were done by RT-PCR analysis and by microarray analysis demonstrating a general trend of the assessed genes to be expressed either in 3 days- or 10-days old embryoid bodies (EBs) when compared to undifferentiated ES cells. Subsets within the various functional gene classes were defined that are specifically up- or down-regulated in concert. Interestingly, the present results demonstrate that developmental processes toward germ layer formation are irreversible and mostly independent of the culture conditions. Notably, apoptotic and mitochondrial ribosomal genes were down- and up-regulated in 10-days old EBs, respectively, whereas compared to the 3-days old EBs whereas the activity of the extracellular signal-regulated kinase (ERK) 1/2 decreased with progressive development. This article defines a platform for ES cell differentiation and gene expression studies.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco/fisiologia , Animais , Apoptose/genética , Blastocisto/citologia , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Marcadores Genéticos , Masculino , Proteínas de Membrana/genética , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Animais , Família Multigênica , Fator 3 de Transcrição de Octâmero/genética , Fosforilação , Células-Tronco/citologia , Fator de Crescimento Transformador beta/genéticaRESUMO
The prevention of cancer through dietary intervention is currently receiving considerable attention. Several epidemiological studies substantiate that green tea has a protective effect against a variety of malignant proliferative disorders such as lung cancer, breast cancer and prostate cancer. This preventive potential of green tea against cancer is attributed to the biologically active flavonoids called catechins. Epigallocatechin 3-o-gallate, the major catechin found in green tea, mediates diverse physiological and pharmacological actions in bringing about the regression of the tumors and also lowers the risk of nonmalignant cardiovascular proliferative diseases. Much of the current research is being focused on how these catechins specifically bring about the regression of the experimentally induced tumors both in vitro and in vivo. These catechins exert diverse physiological effects against proliferative diseases by several mechanisms, most of which are not completely characterized. This review summarizes the mechanisms by which these catechins play an essential role in regulating the process of carcinogenesis, with a special emphasis on how these catechins antagonize the growth factor-induced proliferative disorders.
Assuntos
Catequina/análogos & derivados , Catequina/uso terapêutico , Substâncias de Crescimento/metabolismo , Neoplasias/prevenção & controle , Animais , Catequina/metabolismo , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/fisiologia , Fator de Crescimento Derivado de Plaquetas/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
AIMS: Heart failure (HF) is associated with development of AF and life-threatening ventricular tachycardia and fibrillation (VT/VF). Vulnerability to development of AF and VT/VF at different stages of HF and the underlying pathophysiological mechanisms are poorly defined. The present study was designed to determine the time-course of development of electrical and structural remodelling of the atria and ventricles, and their contribution to induction of AF and VT/VF in a canine model of HF. METHODS AND RESULTS: Dogs were ventricular tachypaced (VTP) for 2-3 weeks or 5-6 weeks ('early' and 'late' HF, respectively). Electrophysiological studies were performed in isolated atrial and ventricular preparations and correlated with cardiac dimensions and haemodynamic parameters recorded in vivo. Vulnerability to programmed electrical stimulation-induced AF was greater in early vs. late stages of HF (78% vs. 38%). In contrast, VT/VF was inducible in late but not in early stages of HF (38% vs. 0%). The temporal distinction in atrial and ventricular arrhythmia susceptibility was associated with a much more rapid development of electrical and structural remodelling in atria. Vulnerability to AF developed following moderate electro-structural remodelling and waned with further progression to severe remodelling, which averted rapid atrial activation. CONCLUSIONS: A temporal window of vulnerability for AF appears relatively early during development of VTP-induced HF in dogs, whereas VT/VF vulnerability is observed at more advanced stages of HF. These findings, if confirmed in humans, may have clinical implications with regard to prognosis and approach to therapy of patients with HF.
Assuntos
Fibrilação Atrial/etiologia , Fibrilação Atrial/fisiopatologia , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/fisiopatologia , Animais , Fibrilação Atrial/diagnóstico por imagem , Biomarcadores/sangue , Modelos Animais de Doenças , Progressão da Doença , Cães , Ecocardiografia , Eletrocardiografia , Insuficiência Cardíaca/diagnóstico por imagem , Hemodinâmica , Fatores de TempoRESUMO
A potential application of embryonic and inducible pluripotent stem cells for the therapy of degenerative diseases involves pure somatic cells, free of tumorigenic undifferentiated embryonic and inducible pluripotent stem cells. In complex collections of chemicals with pharmacological potential we expect to find molecules able to induce specific pluripotent stem cell death, which could be used in some cell therapy settings to eliminate undifferentiated cells. Therefore, we have screened a chemical library of 1120 small chemicals to identify compounds that induce specifically apoptotic cell death in undifferentiated mouse embryonic stem cells (ESCs). Interestingly, three compounds currently used as clinically approved drugs, nortriptyline, benzethonium chloride and methylbenzethonium chloride, induced differential effects in cell viability in ESCs versus mouse embryonic fibroblasts (MEFs). Nortriptyline induced apoptotic cell death in MEFs but not in ESCs, whereas benzethonium and methylbenzethonium chloride showed the opposite effect. Nortriptyline, a tricyclic antidepressant, has also been described as a potent inhibitor of mitochondrial permeability transition, one of two major mechanisms involved in mitochondrial membrane permeabilization during apoptosis. Benzethonium chloride and methylbenzethonium chloride are quaternary ammonium salts used as antimicrobial agents with broad spectrum and have also been described as anticancer agents. A similar effect of benzethonium chloride was observed in human induced pluripotent stem cells (hiPSCs) when compared to both primary human skin fibroblasts and an established human fibroblast cell line. Human fibroblasts and hiPSCs were similarly resistant to nortriptyline, although with a different behavior. Our results indicate differential sensitivity of ESCs, hiPSCs and fibroblasts to certain chemical compounds, which might have important applications in the stem cell-based therapy by eliminating undifferentiated pluripotent stem cells from stem cell-derived somatic cells to prevent tumor formation after transplantation for therapy of degenerative diseases.