RESUMEN
The self-renewal capacity of germline derived stem cells (GSCs) makes them an ideal source for research and use in clinics. Despite the presence of active gene network similarities between embryonic stem cells (ESCs) and GSCs, there are unanswered questions regarding the roles of evolutionary conserved genes in GSCs. To determine the reprogramming potential of germ cell- specific genes, we designed a polycistronic gene cassette expressing Stella, Oct4 and Nanos2 in a lentiviral-based vector. Deep transcriptome analysis showed the activation of a set of pluripotency and germ-cell-specific markers and the downregulation of innate immune system. The global shut down of antiviral genes included MHC class I, interferon response genes and dsRNA 2'-5'-oligoadenylate synthetase are critical pathways that has been affected . Individual expression of each factor highlighted suppressive effect of Nanos2 on genes such as Isg15 and Oasl2. Collectively, to our knowledge this is the first report showing that Nanos2 could be considered as an immunosuppressive factor. Furthermore, our results demonstrate suppression of endogenous retrotransposons that harbor immune response but further analysis require to uncover the correlation between transposon suppression and immune response in germ cell development.
Asunto(s)
Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Inmunidad Innata/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Animales , Reprogramación Celular , Proteínas Cromosómicas no Histona , Elementos Transponibles de ADN/genética , Regulación hacia Abajo/genética , Retrovirus Endógenos/metabolismo , Redes Reguladoras de Genes , Células HEK293 , Humanos , Ratones Endogámicos BALB C , Modelos Biológicos , Regiones Promotoras Genéticas/genética , ARN Largo no Codificante/genéticaRESUMEN
Germline stem cells (GSCs) are attractive biological models because of their strict control on pluripotency gene expression, and their potential for huge epigenetic changes in a short period of time. Few data exists on the cooperative impact of GSC-specific genes on differentiated cells. In this study, we over-expressed 3 GSC-specific markers, STELLA, OCT4 and NANOS2, collectively designated as (SON), using the novel polycistronic lentiviral gene construct FUM-FD, in HEK293T cells and evaluated promoter activity of the Stra8 GSC marker gene We could show that HEK293T cells expressed pluripotency and GSC markers following ectopic expression of the SON genes. We also found induction of pluripotency markers after serum starvation in non-transduced HEK293T cells. Expression profiling of SON-expressing and serum-starved cells at mRNA and protein level showed the potential of SON factors and serum starvation in the induction of ESRRB, NANOG, OCT4 and REX1 expression. Additionally, the data indicated that the mouse Stra8 promoter could only be activated in a subpopulation of HEK293T cells, regardless of SON gene expression. We conclude that heterogeneous population of the HEK293T cells might be easily shifted towards expression of the pluripotency markers by ectopic expression of the SON factors or by growth in serum depleted media.
Asunto(s)
Técnicas de Reprogramación Celular/métodos , Células HEK293/citología , Células HEK293/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/citología , Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Diferenciación Celular/fisiología , Proteínas Cromosómicas no Histona , Humanos , Células Madre Pluripotentes/metabolismoRESUMEN
OBJECTIVES: Cell-based therapeutic approaches have witnessed significant developments during the last decade especially after approval of MSCs based treatment of graft versus host disease. Several cell-based approaches have shown immunomodulatory behavior during regeneration following the unknown cascade of events but the exact mechanisms are yet to be defined. Clinical applications of cell-based drugs are hampered all over the world because of incomplete understanding of molecular mechanisms requiring the application of mechanistic approaches to solving the mystery. Current work has given us the idea that Nanos2 enhances the cellular pluripotency characteristics while down-regulating the innate immunity genes, simultaneously. MATERIALS AND METHODS: The immunomodulatory behavior of cells was studied against cells carrying the ectopic expression of Nanos2 in comparison with Stella and Oct4 individually and simultaneously using SON vector (Stella, Nanos2 and Oct4). RESULTS: It was observed that overexpression of Nanos2 leads to down-regulation of Interferon-Stimulated Genes (ISGs)-mRNAs such as Ifitm1, lsg15, Oas2, and Oas12. Nanos2 overexpressing MEF cells have shown restrictive inflammatory effects when cells were treated with inflammatory stimuli such as LPS and Poly (I:C). CONCLUSION: From our recent findings in line with many others, it can be concluded that Nanos2 acts as a coin with two sides, regulating pluripotency and immunity together which enhances resistance against inflammatory stimuli. Nanos2 could be a potential candidate as a molecular drug for management of inflammation and immunomodulation but it requires a comprehensive comparative expression analysis of innate immunity genes in vitro and in vivo.