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1.
Nano Lett ; 22(1): 135-144, 2022 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-34967636

RESUMO

Current three-dimensional (3D) cell culture systems mainly rely on static cell culture and lack the ability to thoroughly manage cell intrinsic behaviors and biological characteristics, leading to unsatisfied cell activity. Herein, we have developed photoactive 3D-printed hypertensile metamaterials based dynamic cell culture system (MetaFold) for guiding cell fate. MetaFold exhibited high elasticity and photothermal conversion efficiency due to its metapattern architecture and micro/nanoscale polydopamine coating, allowing for responding to mechanical and light stimulation to construct dynamic culture conditions. In addition, MetaFold possessed excellent cell adhesion capability and could promote cell viability and function under dynamic stimulation, thereby maximizing cell activity. Importantly, MetaFold could improve the differentiation efficacy of stem cells into cardiomyocytes and even their maturation, offering high-quality precious candidates for cell therapy. Therefore, we present a dual stimuli-responsive dynamic culture system, which provides a physiologically realistic environment for cell culture and biological study.


Assuntos
Impressão Tridimensional , Alicerces Teciduais , Técnicas de Cultura de Células , Diferenciação Celular , Células-Tronco
2.
Nano Lett ; 21(13): 5540-5546, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34161107

RESUMO

Induced pluripotent stem cells (iPSCs) hold promise in revolutionizing medicine; however, their application potential is limited because of low reprogramming efficiency. Mesenchymal-to-epithelial transition (MET) has been proved to involve reprogramming of somatic cells into iPSCs, making it a promising target for enhancing generation of iPSCs. Here, we nanoengineered N-cadherin-blocking peptide ADH-1 with gold nanoparticles, generating a multivalent N-cadherin antagonist (ADH-AuNPs), for improving reprogramming efficiency through driving cell MET. ADH-AuNPs exhibited good biocompatibility and showed higher N-cadherin inhibitory activity than ADH-1 due to multivalency, thereby enhancing cell-state reprogramming toward epithelial lineages. Particularly, ADH-AuNPs improved reprogramming efficiency by more than 7-fold after introduction of four Yamanaka factors. Importantly, ADH-AuNPs generated iPSCs displayed high stemness and pluripotency in vitro and in vivo. Therefore, we provide a cooperative strategy for promoting the iPSC generation efficacy.


Assuntos
Caderinas/antagonistas & inibidores , Reprogramação Celular , Transição Epitelial-Mesenquimal , Células-Tronco Pluripotentes Induzidas , Nanopartículas Metálicas , Animais , Caderinas/genética , Fibroblastos , Ouro , Camundongos
3.
Genome Res ; 23(2): 248-59, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23080539

RESUMO

Pluripotent stem cells evade replicative senescence, whereas other primary cells lose their proliferation and differentiation potential after a limited number of cell divisions, and this is accompanied by specific senescence-associated DNA methylation (SA-DNAm) changes. Here, we investigate SA-DNAm changes in mesenchymal stromal cells (MSC) upon long-term culture, irradiation-induced senescence, immortalization, and reprogramming into induced pluripotent stem cells (iPSC) using high-density HumanMethylation450 BeadChips. SA-DNAm changes are highly reproducible and they are enriched in intergenic and nonpromoter regions of developmental genes. Furthermore, SA-hypomethylation in particular appears to be associated with H3K9me3, H3K27me3, and Polycomb-group 2 target genes. We demonstrate that ionizing irradiation, although associated with a senescence phenotype, does not affect SA-DNAm. Furthermore, overexpression of the catalytic subunit of the human telomerase (TERT) or conditional immortalization with a doxycycline-inducible system (TERT and SV40-TAg) result in telomere extension, but do not prevent SA-DNAm. In contrast, we demonstrate that reprogramming into iPSC prevents almost the entire set of SA-DNAm changes. Our results indicate that long-term culture is associated with an epigenetically controlled process that stalls cells in a particular functional state, whereas irradiation-induced senescence and immortalization are not causally related to this process. Absence of SA-DNAm in pluripotent cells may play a central role for their escape from cellular senescence.


Assuntos
Senescência Celular/genética , Metilação de DNA , Células-Tronco Pluripotentes/metabolismo , Adulto , Idoso , Linhagem Celular Transformada , Células Cultivadas , Senescência Celular/efeitos da radiação , Metilação de DNA/efeitos da radiação , Epigênese Genética/efeitos da radiação , Raios gama/efeitos adversos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos da radiação , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos da radiação , Pessoa de Meia-Idade , Modelos Biológicos , Células-Tronco Pluripotentes/efeitos da radiação
4.
Mol Ther ; 21(1): 240-50, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23032973

RESUMO

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is an epigenetic phenomenon. It has been suggested that iPSC retain some tissue-specific memory whereas little is known about interindividual epigenetic variation. We have reprogrammed mesenchymal stromal cells from human bone marrow (iP-MSC) and compared their DNA methylation profiles with initial MSC and embryonic stem cells (ESCs) using high-density DNA methylation arrays covering more than 450,000 CpG sites. Overall, DNA methylation patterns of iP-MSC and ESC were similar whereas some CpG sites revealed highly significant differences, which were not related to parental MSC. Furthermore, hypermethylation in iP-MSC versus ESC occurred preferentially outside of CpG islands and was enriched in genes involved in epidermal differentiation indicating that these differences are not due to random de novo methylation. Subsequently, we searched for CpG sites with donor-specific variation. These "epigenetic fingerprints" were highly enriched in non-promoter regions and outside of CpG islands-and they were maintained upon reprogramming. In conclusion, iP-MSC clones revealed relatively little intraindividual variation but they maintained donor-derived epigenetic differences. In the absence of isogenic controls, it would therefore be more appropriate to compare iPSC from different donors rather than a high number of different clones from the same patient.


Assuntos
Células Clonais , Metilação de DNA , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Ilhas de CpG , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Cell Physiol Biochem ; 28(4): 579-92, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22178870

RESUMO

BACKGROUND/AIMS: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. METHODS: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. RESULTS: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca(2+) release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca(2+)-induced Ca(2+)-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. CONCLUSION: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Potenciais de Ação , Cálcio/metabolismo , Catecolaminas/metabolismo , Diferenciação Celular , Colforsina/metabolismo , AMP Cíclico/metabolismo , Eletrocardiografia , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Cariotipagem , Mutação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Fenótipo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologia
6.
J Inflamm Res ; 14: 2851-2863, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34234510

RESUMO

BACKGROUND: Microglia play an essential role in the central nervous system immune response. The transcription factor myocyte enhancer factor-2 D (MEF2D) is known to participate in stress regulation in various cell types and is easily activated in microglia. MEF2D has been shown to transcriptionally regulate several cytokine genes in immune cells and directly regulates the inflammatory response, suggesting that MEF2D may act as a key stimulus response regulator of microglia and is involved in the regulation of brain microhomeostasis. To uncover the molecular mechanism of MEF2D in the inflammatory system, in the present study, we investigated the global effect of MEF2D in activated microglia and explored its potential regulatory network. METHODS: Experiments with a recombinant lentiviral vector containing either shRNA or overexpressing MEF2D were performed in the murine microglial BV2 cell line. Transcriptome sequencing and global gene expression patterns were analysed in lipopolysaccharide-stimulated shMEF2D BV2 cells. Pro- and anti-inflammatory factors were assessed by Western blot, qPCR or ELISA, and microglial activity was assessed by phagocytosis and morphologic analysis. The direct binding of MEF2D to the promoter region of interferon regulatory factor 7 (IRF7) was tested by ChIP-qPCR. The interferon-stimulated genes (ISGs) were tested by qPCR. RESULTS: MEF2D actively participated in the inflammatory response of BV2 microglial cells. Stably expressed RNAi-induced silencing of MEF2D disrupted the microglial immune balance in two ways: (1) the expression of proinflammatory factors, such as NLRP3, IL-1ß, and iNOS was promoted; and (2) the type-I interferon signalling pathway was markedly inhibited by directly modulating IRF7 transcription. In contrast, overexpression of MEF2D significantly reduced the expression of NLRP3 and iNOS under LPS stimulation and alleviated the level of immune stress in microglia. CONCLUSION: These findings demonstrate that MEF2D plays an important role in regulating inflammatory homeostasis partly through transcriptional regulation of the type-I interferon signalling pathway.

7.
Adv Mater ; 32(48): e2005295, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33118267

RESUMO

Despite multiple treatment options being available, many critical challenges are still ongoing in the treatment of oral squamous cell carcinoma (OSCC). Particularly, the major hurdle is to avoid facial disfigurement and oral function disability during treatment. Herein, nanoengineered mesenchymal stem cells (MSCs) are developed as a supersonosensitizer, named M/LPV/O2 , for improving nondestructive sonodynamic therapy (SDT) against OSCC along with good therapeutic compliance. M/LPV/O2 is composed of an MSCs membrane functionalized liposomal formulation of oxygen-loading perfluorocarbon and sonosensitizer verteporfin (M/LPV/O2 ), which can not only increase circulation and targeting efficacy but also supply oxygen to overcome tumor-hypoxia-associated resistance in SDT, resulting in enhanced therapeutic outcomes in vitro and in vivo. It is identified that M/LPV/O2 effectively stimulates the generation of reactive oxygen species even in hypoxic conditions, and consequently tremendously induces cancer cell death. In addition, M/LPV/O2 displays good tumor accumulation and penetration under ultrasound stimulation, and efficiently induces tumor inhibition and even abrogation, leading to prolonged survival of tumor-bearing mice. Importantly, M/LPV/O2 -based SDT exhibits minimal systemic adverse effects and successfully maintains oral functions with no facial tissue damage. Therefore, these studies provide a promising therapeutic strategy for OSCC, which has a potential to enhance life quality and compliance after treatment.


Assuntos
Células-Tronco Mesenquimais/citologia , Neoplasias Bucais/terapia , Terapia por Ultrassom/métodos , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Camundongos , Neoplasias Bucais/patologia , Hipóxia Tumoral
8.
PLoS One ; 13(1): e0190364, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29293594

RESUMO

Recombinant protein therapeutics have become important components of the modern medicine. Majority of them are produced with mammalian cells that are cultured either through adherent culturing, in which cells are cultured on substrates, or suspension culturing, in which cells are suspended and cultured in agitated cell culture medium in a culture vessel. The adherent cell culturing method is limited by its low yield. In suspension culturing, cells need extensive genetic manipulation to grow as single cells at high density, which is time- and labor-consuming. Here, we report a new method, which utilizes a thermoreversible hydrogel as the scaffold for culturing protein-expressing cells. The hydrogel scaffolds not only provide 3D spaces for the cells, but also act as physical barriers to prevent excessive cellular agglomeration and protect cells from the hydrodynamic stresses. As a result, cells can grow at high viability, high growth rate, and extremely high yield even without genetic manipulations. The cell yield in the hydrogels is around 20 times of the suspension culturing. In addition, the protein productivity per cell per day in the hydrogel is higher than the adherent culturing method. This new method is simple, scalable and defined. It will be of great value for both the research laboratories and pharmaceutical industry for producing proteins.


Assuntos
Hidrogéis , Proteína Wnt3A/biossíntese , Animais , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Meios de Cultura , Humanos , Camundongos , Alicerces Teciduais , Proteína Wnt3A/genética
9.
Sci Rep ; 5: 8883, 2015 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-25748532

RESUMO

Drug discovery and development are hampered by high failure rates attributed to the reliance on non-human animal models employed during safety and efficacy testing. A fundamental problem in this inefficient process is that non-human animal models cannot adequately represent human biology. Thus, there is an urgent need for high-content in vitro systems that can better predict drug-induced toxicity. Systems that predict cardiotoxicity are of uppermost significance, as approximately one third of safety-based pharmaceutical withdrawals are due to cardiotoxicty. Here, we present a cardiac microphysiological system (MPS) with the attributes required for an ideal in vitro system to predict cardiotoxicity: i) cells with a human genetic background; ii) physiologically relevant tissue structure (e.g. aligned cells); iii) computationally predictable perfusion mimicking human vasculature; and, iv) multiple modes of analysis (e.g. biological, electrophysiological, and physiological). Our MPS is able to keep human induced pluripotent stem cell derived cardiac tissue viable and functional over multiple weeks. Pharmacological studies using the cardiac MPS show half maximal inhibitory/effective concentration values (IC50/EC50) that are more consistent with the data on tissue scale references compared to cellular scale studies. We anticipate the widespread adoption of MPSs for drug screening and disease modeling.


Assuntos
Fármacos Cardiovasculares/administração & dosagem , Avaliação Pré-Clínica de Medicamentos/instrumentação , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Análise Serial de Tecidos/instrumentação , Bioensaio/instrumentação , Diferenciação Celular , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/instrumentação , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Dispositivos Lab-On-A-Chip , Miócitos Cardíacos/fisiologia
10.
Biores Open Access ; 3(6): 311-26, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25469316

RESUMO

Chronic granulomatous disease (CGD) is an inherited orphan disorder caused by mutations in one of the five genes encoding reduced nicotinamide-adenine-dinucleotide-phosphate oxidase subunits, which subsequently lead to impairment in the production of microbicidal reactive oxygen species (ROS). In order to offer several cell line models of CGD and therefore support research on pathophysiology and new therapeutic approaches, we optimized protocols to differentiate induced pluripotent stem cells (iPSCs) from wild-type, X(0)-, AR22(0)- and AR47(0)-CGD patient's fibroblasts into neutrophils and into macrophages. Aberrant genetic clones were discarded after chromosome karyotyping and array-comparative genomic hybridization analysis. All remaining iPSC lines showed human embryonic stem cell-like morphology, expressed all tested pluripotency markers and formed embryoid bodies that contained cells originating from all three primary germ layers. Furthermore, each CGD patient-specific iPSC line retained the gp91 (phox) , p47 (phox) , and p22 (phox) mutations found in the corresponding patient's neutrophils. The average production of CD34(+) progenitors was of 1.5×10(6) cells after 10 days of differentiation of 10×10(6) iPSCs. They were terminally differentiated into about 3×10(5) neutrophils or into 3×10(7) macrophages. Based on morphological, phenotypical, and functional criteria both phagocyte types were mature and indistinguishable from the native human neutrophils and macrophages. However, neutrophils and macrophages derived from X(0)-, AR22(0)-, and AR47(0)-CGD patient-specific iPSC lines lacked ROS production and the corresponding mutated proteins. To simplify the phagocytes' production upon request, progenitors can be cryopreserved. In conclusion, we describe a reproducible, simple, and efficient way to generate neutrophils and macrophages from iPSCs and provide a new cellular model for the AR22(0)-CGD genetic form that has not been described before.

11.
J Biol Chem ; 283(48): 33730-5, 2008 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-18826945

RESUMO

Induced pluripotent stem cell technology, also termed iPS, is an emerging approach to reprogram cells into an embryonic stem cell-like state by viral transduction with defined combinations of factors. iPS cells share most characteristics of embryonic stem cells, counting pluripotency and self-renewal, and have so far been obtained from mouse and humans, including patients with genetic diseases. Remarkably, autologous transplantation of cell lineages derived from iPS cells will eliminate the possibility of immunological rejection, as well as current ethical issues surrounding human embryonic stem cell research. However, before iPS can be used for clinical purposes, technical problems must be overcome. Among other considerations, full and homogeneous iPS reprogramming is an important prerequisite. However, despite the fact that cells from several mouse tissues can be successfully induced to iPS, the overall efficiency of chimera formation of these clones remains low even if selection for Oct4 or Nanog expression is applied. In this report, we demonstrate that cells from the mouse meningeal membranes express elevated levels of the embryonic master regulator Sox2 and are highly amenable to iPS. Meningeal iPS clones, generated without selection, are fully and homogeneously reprogrammed based on DNA methylation analysis and 100% chimera competent. Our results define a population of somatic cells that are ready to undergo iPS, thus highlighting a very attractive cell type for iPS research and application.


Assuntos
Desdiferenciação Celular/fisiologia , Quimera/embriologia , Meninges/citologia , Meninges/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Fatores de Transcrição SOXB1/biossíntese , Animais , Animais Recém-Nascidos , Metilação de DNA/fisiologia , Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Endogâmicos ICR , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/biossíntese , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , Transplante de Células-Tronco , Transplante Autólogo
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