RESUMO
Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.
Assuntos
Técnicas de Reprogramação Celular/métodos , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Transcriptoma , Animais , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos/genética , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/genética , Células-Tronco Pluripotentes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus Sendai/genéticaRESUMO
Functional smooth muscle engineering requires isolation and expansion of smooth muscle cells (SMCs), and this process is particularly challenging for visceral smooth muscle tissue where progenitor cells have not been clearly identified. Herein we showed for the first time that efficient SMCs can be obtained from human amniotic fluid stem cells (hAFSCs). Clonal lines were generated from c-kit(+) hAFSCs. Differentiation toward SM lineage (SMhAFSCs) was obtained using a medium conditioned by PDGF-BB and TGF-ß1. Molecular assays revealed higher level of α smooth muscle actin (α-SMA), desmin, calponin, and smoothelin in SMhAFSCs when compared to hAFSCs. Ultrastructural analysis demonstrated that SMhAFSCs also presented in the cytoplasm increased intermediate filaments, dense bodies, and glycogen deposits like SMCs. SMhAFSC metabolism evaluated via mass spectrometry showed higher glucose oxidation and an enhanced response to mitogenic stimuli in comparison to hAFSCs. Patch clamp of transduced hAFSCs with lentiviral vectors encoding ZsGreen under the control of the α-SMA promoter was performed demonstrating that SMhAFSCs retained a smooth muscle cell-like electrophysiological fingerprint. Eventually SMhAFSCs contractility was evident both at single cell level and on a collagen gel. In conclusion, we showed here that hAFSCs under selective culture conditions are able to give rise to functional SMCs.
Assuntos
Líquido Amniótico/citologia , Diferenciação Celular , Linhagem da Célula , Células-Tronco Fetais/citologia , Células-Tronco Multipotentes/citologia , Miócitos de Músculo Liso/citologia , Actinas/genética , Actinas/metabolismo , Potenciais de Ação , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Meios de Cultivo Condicionados/farmacologia , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Desmina/genética , Desmina/metabolismo , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/metabolismo , Células-Tronco Fetais/fisiologia , Glucose/metabolismo , Glicogênio/metabolismo , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/fisiologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fator de Crescimento Transformador beta/farmacologia , CalponinasRESUMO
The data deluge produced by next-generation sequencing (NGS) technologies is an appealing feature for clinical virologists that are involved in the diagnosis of emerging viral infections, molecular epidemiology of viral pathogens, drug-resistance testing, and also like to do some basic and clinical research. Indeed, NGS platforms are being implemented in many clinical and research laboratories, as the costs of these platforms are progressively decreasing. We provide here some suggestions for virologists who are planning to implement a NGS platform in their clinical laboratory and an overview on the potential applications of these technologies in diagnostic virology.