RESUMO
Human induced pluripotent stem cell (iPSC) lines were generated from peripheral blood mononuclear cells (PBMCs) isolated from two related patients diagnosed with either idiopathic ventricular fibrillation or catecholaminergic polymorphic ventricular tachycardia, carrying an unknown variant in the RYR2 gene, c.14201A>G (p.Y4734C) and one healthy related individual. Reprogramming was done using a commercially available Epi5 Reprogramming Kit. The pluripotency of the iPSC lines was verified by the expression of pluripotency markers and by their capacity to differentiate into all three embryonic germ layers in vitro. These iPSC lines are available for functional analysis and in vitro studies of RYR2 channelopathy.
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Thin composite films comprising two primary representatives of conducting polymers, poly(3, 4-ethylenedioxythiophene) (PEDOT) and polypyrrole (PPy), with eco-friendly cellulose nanocrystals (CNC) were prepared through electrochemical polymerization. The combination of CNC and PEDOT (or PPy) results in the formation of films with highly different surface topography and thickness. Intriguingly, different surface conductivity of PEDOT and PPy was revealed by atomic force microscopy albeit that the electrochemical properties were rather similar. The biological properties of the composites in contact with prospective human induced pluripotent stem cells (hiPSC) and cardiomyocytes derived from hiPSC demonstrated good cytocompatibility of both composites and their potential in engineering of electro-sensitive tissues. The as-prepared conducting, eco-friendly and cytocompatible composites are thus promising candidates for biomedical applications where stimuli-responsivity is a crucial cell-instructive property.
Assuntos
Células-Tronco Pluripotentes Induzidas , Nanopartículas , Humanos , Polímeros/química , Celulose/química , Engenharia Tecidual , Estudos Prospectivos , Pirróis/químicaRESUMO
Dual specificity phosphatase 7 (DUSP7) is a protein belonging to a broad group of phosphatases that can dephosphorylate phosphoserine/phosphothreonine as well as phosphotyrosine residues within the same substrate. DUSP7 has been linked to the negative regulation of mitogen activated protein kinases (MAPK), and in particular to the regulation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). MAPKs play an important role in embryonic development, where their duration, magnitude, and spatiotemporal activity must be strictly controlled by other proteins, among others by DUSPs. In this study, we focused on the effect of DUSP7 depletion on the in vitro differentiation of mouse embryonic stem (ES) cells. We showed that even though DUSP7 knock-out ES cells do retain some of their basic characteristics, when it comes to differentiation, they preferentially differentiate towards neural cells, while the formation of early cardiac mesoderm is repressed. Therefore, our data indicate that DUSP7 is necessary for the correct formation of neuroectoderm and cardiac mesoderm during the in vitro differentiation of ES cells.
Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Células-Tronco Embrionárias Murinas , Animais , Fosfatase 1 de Especificidade Dupla/metabolismo , Fosfatases de Especificidade Dupla/genética , Mesoderma/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Fosfosserina , Fosfotreonina , FosfotirosinaRESUMO
Organic semiconductors are constantly gaining interest in regenerative medicine. Their tunable physico-chemical properties, including electrical conductivity, are very promising for the control of stem-cell differentiation. However, their use for combined material-based and electrical stimulation remains largely underexplored. Therefore, we carried out a study on whether a platform based on the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) can be beneficial to the differentiation of mouse embryonic stem cells (mESCs). The platform was prepared using the layout of a standard 24-well cell-culture plate. Polyethylene naphthalate foil served as the substrate for the preparation of interdigitated gold electrodes by physical vapor deposition. The PEDOT:PSS pattern was fabricated by precise screen printing over the gold electrodes. The PEDOT:PSS platform was able to produce higher electrical current with the pulsed-direct-current (DC) electrostimulation mode (1 Hz, 200 mV/mm, 100 ms pulse duration) compared to plain gold electrodes. There was a dominant capacitive component. In proof-of-concept experiments, mESCs were able to respond to such electrostimulation by membrane depolarization and elevation of cytosolic calcium. Further, the PEDOT:PSS platform was able to upregulate cardiomyogenesis and potentially inhibit early neurogenesis per se with minor contribution of electrostimulation. Hence, the present work highlights the large potential of PEDOT:PSS in regenerative medicine.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Diferenciação Celular , Condutividade Elétrica , Células-Tronco Embrionárias Murinas/citologia , Polímeros/farmacologia , Poliestirenos/química , Animais , Técnicas de Cultura de Células , Eletrodos , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Polímeros/químicaRESUMO
An innovative multi-step phase separation process was used to prepare tissue culture for the polystyrene-based, hierarchically structured substrates, which mimicked in vivo microenvironment and architecture. Macro- (pore area from 3000 to 18,000 µm2; roughness (Ra) 7.2 ± 0.1 µm) and meso- (pore area from 50 to 300 µm2; Ra 1.1 ± 0.1 µm) structured substrates covered with micro-pores (area around 3 µm2) were prepared and characterised. Both types of substrate were suitable for human-induced pluripotent stem cell (hiPSC) cultivation and were found to be beneficial for the induction of cardiomyogenesis in hiPSC. This was confirmed both by the number of promoted proliferated cells and the expressions of specific markers (Nkx2.5, MYH6, MYL2, and MYL7). Moreover, the substrates amplified the fluorescence signal when Ca2+ flow was monitored. This property, together with cytocompatibility, make this material especially suitable for in vitro studies of cell/material interactions within tissue-mimicking environments.
Assuntos
Materiais Biocompatíveis/química , Diferenciação Celular , Fluorescência , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Poliestirenos/química , Proliferação de Células , HumanosRESUMO
The active role of biomaterials in the regeneration of tissues and their ability to modulate the behavior of stem cells in terms of their differentiation is highly advantageous. Here, polypyrrole, as a representantive of electro-conducting materials, is found to modulate the behavior of embryonic stem cells. Concretely, the aqueous extracts of polypyrrole induce neurogenesis within embryonic bodies formed from embryonic stem cells. This finding ledto an effort to determine the physiological cascade which is responsible for this effect. The polypyrrole modulates signaling pathways of Akt and ERK kinase through their phosphorylation. These effects are related to the presence of low-molecular-weight compounds present in aqueous polypyrrole extracts, determined by mass spectroscopy. The results show that consequences related to the modulation of stem cell differentiation must also be taken into account when polypyrrole is considered as a biomaterial.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Corpos Embrioides/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Polímeros/farmacologia , Pirróis/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Linhagem Celular , Corpos Embrioides/citologia , Expressão Gênica/efeitos dos fármacos , Camundongos , Estrutura Molecular , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Fator de Transcrição PAX6/genética , Polímeros/química , Pirróis/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genéticaRESUMO
Novel composite films combining biocompatible polysaccharides with conducting polyaniline (PANI) were prepared via the in-situ polymerization of aniline hydrochloride in the presence of sodium hyaluronate (SH) or chitosan (CH). The composite films possess very good cytocompatibility in terms of adhesion and proliferation of two lines of human induced pluripotent stem cells (hiPSC). Moreover, the cardiomyogenesis and even formation of beating clusters were successfully induced on the films. The proportion of formed cardiomyocytes demonstrated excellent properties of composites for tissue engineering of stimuli-responsive tissues. The testing also demonstrated antibacterial activity of the films against E. coli and PANI-SH was able to reduce bacterial growth from 2 × 105 to < 1 cfu cm-2. Physicochemical characterization revealed that the presence of polysaccharides did not notably influence conductivities of the composites being â¼1 and â¼2 S cm-1 for PANI-SH and PANI-CH respectively; however, in comparison with neat PANI, it modified their topography making the films smoother with mean surface roughness of 4 (PANI-SH) and 14 nm (PANI-CH). The combination of conductivity, antibacterial activity and mainly cytocompatibility with hiPSC opens wide application potential of these polysaccharide-based composites.
Assuntos
Antibacterianos/química , Materiais Biocompatíveis/química , Quitosana/química , Ácido Hialurônico/química , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Nanocompostos/química , Compostos de Anilina/química , Antibacterianos/farmacologia , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Condutividade Elétrica , Escherichia coli/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Polimerização , Staphylococcus aureus/efeitos dos fármacos , Propriedades de Superfície , Engenharia Tecidual/métodosRESUMO
12-O-Tetradecanoylphorbol-13-acetate (TPA) is the most widely used diacylglycerol (DAG) mimetic agent and inducer of protein kinase C (PKC)-mediated cellular response in biomedical studies. TPA has been proposed as a pluripotent cell differentiation factor, but results obtained have been inconsistent. In the present study we show that TPA can be applied as a cardiomyogenesis-promoting factor for the differentiation of mouse embryonic stem (mES) cells in vitro. The mechanism of TPA action is mediated by the induction of extracellular signal-regulated kinase (ERK) activity and the subsequent phosphorylation of GATA4 transcription factor. Interestingly, general mitogens (FGF, EGF, VEGF and serum) or canonical WNT signalling did not mimic the effect of TPA. Moreover, on the basis of our results, we postulate that a TPA-sensitive population of cardiac progenitor cells exists at a certain time point (after days 6-8 of the differentiation protocol) and that the proposed treatment can be used to increase the multiplication of ES cell-derived cardiomyocytes.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Miócitos Cardíacos/citologia , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Animais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , FosforilaçãoRESUMO
Conducting polymers (CP) can be used as pH- and/or electro-responsive components in various bioapplications, for example, in 4D smart scaffolds. The ability of CP to maintain conductivity under physiological conditions is, therefore, their crucial property. Unfortunately, the conductivity of the CP rapidly decreases in physiological environment, as their conducting salts convert to non-conducting bases. One of the promising solutions how to cope with this shortcoming is the use of alternative "doping" process that is not based on the protonation of CP with acids but on interactions relying in acidic hydrogen bonding. Therefore, the phosphonates (dimethyl phosphonate, diethyl phosphonate, dibutyl phosphonate, or diphenyl phosphonate) were used to re-dope two most common representatives of CP, polyaniline (PANI) and polypyrrole (PPy) bases. As a result, PANI doped with organic phosphonates proved to have significantly better stability of conductivity under different pH. It has also been shown that cytotoxicity of studied materials determined on embryonic stem cells and their embryotoxicity, determined as the impact on cardiomyogenesis and erythropoiesis, depend both on the polymer and phosphonate types used. With the exception of PANI doped with dibutyl phosphonate, all PPy-based phosphonates showed better biocompatibility than the phosphonates based on PANI.
Assuntos
Compostos de Anilina/química , Materiais Biocompatíveis/química , Organofosfonatos/química , Polímeros/química , Pirróis/química , Compostos de Anilina/farmacologia , Animais , Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Condutividade Elétrica , Concentração de Íons de Hidrogênio , Camundongos , Células-Tronco Embrionárias Murinas , Polímeros/farmacologia , Pirróis/farmacologiaRESUMO
Embryonic neural stem cells (NSCs), comprising neuroepithelial and radial glial cells, are indispensable precursors of neurons and glia in the mammalian developing brain. Since the process of neurogenesis occurs in a hypoxic environment, the question arises of how NSCs deal with low oxygen tension and whether it affects their stemness. Genes from the hypoxia-inducible factors (HIF) family are well known factors governing cellular response to hypoxic conditions. In this study, we have discovered that the endogenous stabilization of hypoxia-inducible factor 1α (Hif1α) during neural induction is critical for the normal development of the NSCs pool by preventing its premature depletion and differentiation. The knock-out of the Hif1α gene in mESC-derived neurospheres led to a decrease in self-renewal of NSCs, paralleled by an increase in neuronal differentiation. Similarly, neuroepithelial cells differentiated in hypoxia exhibited accelerated neurogenesis soon after Hif1α knock-down. In both models, the loss of Hif1α was accompanied by an immediate drop in neural repressor Hes1 levels while changes in Notch signaling were not observed. We found that active Hif1α/Arnt1 transcription complex bound to the evolutionarily conserved site in Hes1 gene promoter in both neuroepithelial cells and neural tissue of E8.5 - 9.5 embryos. Taken together, these results emphasize the novel role of Hif1α in the regulation of early NSCs population through the activation of neural repressor Hes1, independently of Notch signaling.
Assuntos
Células-Tronco Neurais , Animais , Diferenciação Celular , Linhagem Celular , Hipóxia , NeurogêneseRESUMO
The cytocompatibility of cardiomyocytes derived from embryonic stem cells and neural progenitors, which were seeded on the surface of composite films made of graphene oxide (GO) and polypyrrole (PPy-GO) or poly(3,4-ethylenedioxythiophene) (PEDOT-GO) are reported. The GO incorporated in the composite matrix contributes to the patterning of the composite surface, while the electrically conducting PPy and PEDOT serve as ion-to-electron transducers facilitating electrical stimulation/sensing. The films were fabricated by a simple one-step electropolymerization procedure on electrically conducting indium tin oxide (ITO) and graphene paper (GP) substrates. Factors affecting the cell behaviour, i.e. the surface topography, wettability, and electrical surface conductivity, were studied. The PPy-GO and PEDOT-GO prepared on ITO exhibited high surface conductivity, especially in the case of the ITO/PPy-GO composite. We found that for cardiomyocytes, the PPy-GO and PEDOT-GO composites counteracted the negative effect of the GP substrate that inhibited their growth. Both the PPy-GO and PEDOT-GO composites prepared on ITO and GP significantly decreased the cytocompatibility of neural progenitors. The presented results enhance the knowledge about the biological properties of electroactive materials, which are critical for tissue engineering, especially in context stimuli-responsive scaffolds.
Assuntos
Condutividade Elétrica , Eletroquímica , Grafite/farmacologia , Miócitos Cardíacos/citologia , Células-Tronco Neurais/citologia , Polímeros/farmacologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/química , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Polímeros/química , Pirróis/química , Água/químicaRESUMO
The activation of p38alpha kinase mediates cell response to various extracellular factors including many interleukins and growth factors important for haematopoiesis. The role of p38alpha kinase was previously analysed in particular haematopoietic cells. In this study and for the first time, the role of p38alpha kinase in haematopoiesis was studied using a model of continuous haematopoietic development in pluripotent embryonic stem cells in vitro. The expression of transcripts associated with haematopoiesis and the potential for the formation of specific haematopoietic cell colonies were compared between wild-type and mutant p38alpha gene-depleted cells. The absence of p38alpha kinase led to the inhibition of hemangioblast formation during the first step of haematopoiesis. Later, during differentiation, due to the lack of p38alpha kinase, erythrocyte maturation was impaired. Mutant p38α-/- cells also exhibited decreased potential with respect to the expansion of granulocyte colony-forming units. This effect was reversed in the absence of erythropoietin as shown by colony-forming unit assay in media for colony-forming unit granulocytes/macrophages. p38alpha kinase thus plays an important role in the differentiation of common myeloid precursor cells into granulocyte lineages.
RESUMO
P38alpha kinase plays an important role in the regulation of both cell stress response and cell fate. In this study, we report that p38alpha kinase-deficient embryonic stem cells exhibit a higher production of reactive oxygen species (ROS) in contrast to their wild-type counterpart. Analysis of the expressions of NADPH oxidases (NOXs) and dual oxidases, crucial enzymes involved in intracellular ROS formation, shows NOX2/gp91phox is over-expressed in p38alpha deficient cells. The particular increase in superoxide formation was confirmed by the specific detection of hydroethidine derivate 2-hydroxyethidium. ROS formation decreased when the level of NOX2 was silenced by siRNA in p38alpha deficient cells. These data suggest the importance of p38alpha kinase in the regulation of ROS metabolism in embryonic stem cells and the significance of the observed phenomena of cancer cell-like phenotypes, which is discussed.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , NADPH Oxidase 2/metabolismo , Superóxidos/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Mitocôndrias/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/genética , NADPH Oxidase 2/genéticaRESUMO
Conducting polymers (CP), namely polyaniline (PANI) and polypyrrole (PPy), are promising materials applicable for the use as biointerfaces as they intrinsically combine electronic and ionic conductivity. Although a number of works have employed PANI or PPy in the preparation of copolymers, composites, and blends with other polymers, there is no systematic study dealing with the comparison of their fundamental biological properties. The present study, therefore, compares the biocompatibility of PANI and PPy in terms of cytotoxicity (using NIH/3T3 fibroblasts and embryonic stem cells) and embryotoxicity (their impact on erythropoiesis and cardiomyogenesis within embryonic bodies). The novelty of the study lies not only in the fact that embryotoxicity is presented for the first time for both studied polymers, but also in the elimination of inter-laboratory variations within the testing, such variation making the comparison of previously published works difficult. The results clearly show that there is a bigger difference between the biocompatibility of the respective polymers in their salt and base forms than between PANI and PPy as such. PANI and PPy can, therefore, be similarly applied in biomedicine when solely their biological properties are considered. Impurity content detected by mass spectroscopy is presented. These results can change the generally accepted opinion of the scientific community on better biocompatibility of PPy in comparison with PANI.
Assuntos
Compostos de Anilina , Corpos Embrioides/metabolismo , Eritropoese/efeitos dos fármacos , Teste de Materiais , Células-Tronco Embrionárias Murinas/metabolismo , Polímeros , Pirróis , Compostos de Anilina/efeitos adversos , Compostos de Anilina/farmacologia , Animais , Corpos Embrioides/patologia , Camundongos , Células-Tronco Embrionárias Murinas/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Células NIH 3T3 , Polímeros/efeitos adversos , Polímeros/farmacologia , Pirróis/efeitos adversos , Pirróis/farmacologiaRESUMO
Polyaniline cryogel is a new unique form of polyaniline combining intrinsic electrical conductivity and the material properties of hydrogels. It is prepared by the polymerization of aniline in frozen poly(vinyl alcohol) solutions. The biocompatibility of macroporous polyaniline cryogel was demonstrated by testing its cytotoxicity on mouse embryonic fibroblasts and via the test of embryotoxicity based on the formation of beating foci within spontaneous differentiating embryonic stem cells. Good biocompatibility was related to low contents of low-molecular-weight impurities in polyaniline cryogel, which was confirmed by liquid chromatography. The adhesion and growth of embryonic stem cells, embryoid bodies, cardiomyocytes, and neural progenitors prove that polyaniline cryogel has the potential to be used as a carrier for cells in tissue engineering or bio-sensing. The surface energy as well as the elasticity and porosity of cryogel mimic tissue properties. Polyaniline cryogel can therefore be applied in bio-sensing or regenerative medicine in general, and mainly in the tissue engineering of electrically excitable tissues.
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Compostos de Anilina/química , Materiais Biocompatíveis/química , Criogéis/química , Algoritmos , Animais , Técnicas de Cultura de Células , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Módulo de Elasticidade , Condutividade Elétrica , Fibroblastos , Teste de Materiais , Fenômenos Mecânicos , Camundongos , Modelos Teóricos , Porosidade , Engenharia TecidualRESUMO
The potentiation of the naturally limited regenerative capacity of the heart is dependent on an understanding of the mechanisms that are activated in response to pathological conditions such as hypoxia. Under these conditions, the expression of genes suggested to support cardiomyocyte survival and heart adaptation is triggered. Particularly important are changes in the expression of myosin heavy chain (MHC) isoforms. We propose here that alterations in the expression profiles of MHC genes are induced in response to hypoxia and are primarily mediated by hypoxia inducible factor (HIF). In in vitro models of mouse embryonic stem cell-derived cardiomyocytes, we showed that hypoxia (1% O2) or the pharmacological stabilization of HIFs significantly increased MHCbeta (Myh7) gene expression. The key role of HIF-1alpha is supported by the absence of these effects in HIF-1alpha-deficient cells, even in the presence of HIF-2alpha. Interestingly, ChIP analysis did not confirm the direct interaction of HIF-1alpha with putative HIF response elements predicted in the MHCalpha and beta encoding DNA region. Further analyses showed the significant effect of the mTOR signaling inhibitor rapamycin in inducing Myh7 expression and a hypoxia-triggered reduction in the levels of antisense RNA transcripts associated with the Myh7 gene locus. Overall, the recognized and important role of HIF in the regulation of heart regenerative processes could be highly significant for the development of novel therapeutic interventions in heart failure.
RESUMO
Hypoxia is involved in the regulation of stem cell fate, and hypoxia-inducible factor 1 (HIF-1) is the master regulator of hypoxic response. Here, we focus on the effect of hypoxia on intracellular signaling pathways responsible for mouse embryonic stem (ES) cell maintenance. We employed wild-type and HIF-1α-deficient ES cells to investigate hypoxic response in the ERK, Akt, and STAT3 pathways. Cultivation in 1% O2 for 24 h resulted in the strong dephosphorylation of ERK and its upstream kinases and to a lesser extent of Akt in an HIF-1-independent manner, while STAT3 phosphorylation remained unaffected. Downregulation of ERK could not be mimicked either by pharmacologically induced hypoxia or by the overexpression. Dual-specificity phosphatases (DUSP) 1, 5, and 6 are hypoxia-sensitive MAPK-specific phosphatases involved in ERK downregulation, and protein phosphatase 2A (PP2A) regulates both ERK and Akt. However, combining multiple approaches, we revealed the limited significance of DUSPs and PP2A in the hypoxia-mediated attenuation of ERK signaling. Interestingly, we observed a decreased reactive oxygen species (ROS) level in hypoxia and a similar phosphorylation pattern for ERK when the cells were supplemented with glutathione. Therefore, we suggest a potential role for the ROS-dependent attenuation of ERK signaling in hypoxia, without the involvement of HIF-1.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Regulação para Baixo , Camundongos , Transdução de SinaisRESUMO
Nanostructured TiO2 nanotubes (NTs) of diameters from 15 to 100nm were fabricated by an electrochemical anodization process. Biofilm-positive strains of Bacillus cereus and Pseudomonas aeruginosa behaved similarly on all TiO2 NTs as well as on native titanium (Ti) foil. The adhesion and growth of mesenchymal stem cells (MSc), embryonic stem cells (ESc), and pure cardiomyocytes derived from ESc exhibited significant differences. MSc as well as ESc were, in contrast to cardiomyocytes, able to adhere, and grow on TiO2 NTs. A correlation between NTs diameter and cell behaviour was however observed in the case of MSc and ESc. MSc were not in a physiological state in the case of 100nm TiO2 NTs, while ESc were not able to grow on 15nm TiO2 NTs. It can be stated that these differences can be assigned to different diameters of the NTs but not to the chemistry of the surface. This is the first study describing the comprehensive behaviour of both eukaryotic and prokaryotic cells on TiO2 NTs. On the basis of obtained results, it can be concluded that new generation of medical devices providing selective cell behaviour can be fabricated by optimizing the nanoscale morphology of TiO2.
Assuntos
Nanoestruturas , Bactérias , Biofilmes , Humanos , Nanotubos , Células-Tronco , TitânioRESUMO
The differentiation of pluripotent embryonic stem (ES) cells into various lineages in vitro represents an important tool for studying the mechanisms underlying mammalian embryogenesis. It is a key technique in studies evaluating the molecular mechanisms of cardiomyogenesis and heart development and also in embryotoxicology. Herein, modest modifications of the basic protocol for ES cell differentiation into cardiomyocytes were evaluated in order to increase the yield and differentiation status of developed cardiomyocytes. Primarily, the data show that ES cell cultivation in the form of non-adherent embryoid bodies (EBs) for 5 days compared to 8 days significantly improved cardiomyogenic differentiation. This is illustrated by the appearance of beating foci in the adherent EBs layer at earlier phases of differentiation from day 10 up to day 16 and by the significantly higher expression of genes characteristic of cardiomyogenic differentiation (sarcomeric alpha actinin, myosin heavy chain alpha and beta, myosin light chain 2 and 7, and transcriptional factor Nkx2.5) in EBs cultivated under non-adherent conditions for 5 days. The ratio of cardiomyocytes per other cells was also potentiated in EBs cultivated in non-adherent conditions for only 5 days followed by cultivation in adherent serum-free culture conditions. Nevertheless, the alteration in the percentage of beating foci among these two tested cultivation conditions vanished at later phases and also did not affect the total number of cardiomyocytes determined as myosin heavy chain positive cells at the end of the differentiation process on day 20. Thus, although these modifications of the conditions of ES cells differentiation may intensify cardiomyocyte differentiation, the final count of cardiomyocytes might not change. Thus, serum depletion was identified as a key factor that intensified cardiomyogenesis. Further, the treatment of EBs with N-acetylcysteine, a reactive oxygen species scavenger, did not affect the observed increase in cardiomyogenesis under serum depleted conditions. Interestingly, a mild induction of the ventricular-like phenotype of cardiomyocytes was observed in 5-day-old EBs compared to 8-day-old EBs. Overall, these findings bring crucial information on the mechanisms of ES cells differentiation into cardiomyocytes and on the establishment of efficient protocols for the cardiomyogenic differentiation of ES cells. Further, the importance of determining the absolute number of formed cardiomyocyte-like cells per seeded pluripotent cells in contrast to the simple quantification of the ratios of cells is highlighted.
Assuntos
Meios de Cultura Livres de Soro , Células-Tronco Embrionárias/citologia , Miócitos Cardíacos/citologia , Acetilcisteína/administração & dosagem , Actinina/genética , Animais , Células Cultivadas , Proteína Homeobox Nkx-2.5/genética , Técnicas In Vitro , Camundongos , Miosinas/genéticaRESUMO
Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells.