RESUMO
Adenoviral early region 1A (E1A) is a viral gene that can promote cellular proliferation and de-differentiation in mammalian cells, features required for the reprogramming of somatic cells to a pluripotent state. E1A has been shown to interact with OCT4, and as a consequence, to increase OCT4 transcriptional activity. Indeed, E1A and OCT4 are sufficient to revert neuroepithelial hybrids to pluripotency, as demonstrated in previous cell fusion experiments. However, the role that E1A might play in the generation of induced pluripotent stem cells (iPSCs) has not been investigated yet. In this report, we show that E1A can generate iPSCs in combination with OCT4 and KLF4, thus replacing exogenous SOX2. The generated iPSCs are bona fide pluripotent cells as shown by in vitro and in vivo tests. Overall, our study suggests that E1A might replace SOX2 through enhancing OCT4 transcriptional activity at the early stages of reprogramming.
Assuntos
Reprogramação Celular , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Ativação Transcricional , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/farmacologia , Animais , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Fibroblastos , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Camundongos , Fatores de Transcrição SOXB1/farmacologiaRESUMO
Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD), which is due to mutations in the CYBB (gp91phox) gene, a component of NADPH oxidase, accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function, we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5' homology arm (HA) of 8 kb and 3'HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3'HA. Both, BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion, we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general.