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Generation of Genetically Stable Human Direct-Conversion-Derived Neural Stem Cells Using Quantity Control of Proto-oncogene Expression.

ABSTRACT: As the human lifespan has increased due to developments in medical technology, the number of patients with neurological diseases has rapidly increased. Therefore, studies on effective treatments for neurological diseases are becoming increasingly important. To perform these studies, it is essential to obtain a large number of patient-derived neural cells. The purpose of the present study was to establish a technology that allows the high-efficiency generation of genetically stable, direct-conversion-derived neural stem cells (dcNSCs) through the expression of a new combination of reprogramming factors, including a proto-oncogene. Specifically, human c-MYC proto-oncogene and the human SOX2 gene were overexpressed in a precisely controlled manner in various human somatic cells. As a result, the direct conversion into multipotent dcNSCs occurred only when the cells were treated with an MOI of 1 of hc-MYC proto-oncogene and hSOX2 retrovirus. When MOIs of 5 or 10 were utilized, distinct results were obtained. In addition, the pluripotency was bypassed during this process. Notably, as the MOI used to treat the cells increased, expression of the p53 tumor suppressor gene, which is typically a reprogramming hurdle, increased proportionately. Interestingly, p53 was genetically stable in dcNSCs generated through direct conversion into a low p53 expression state. In the present study, generation of genetically stable dcNSCs using direct conversion was optimized by precisely controlling the overexpression of a proto-oncogene. This method could be utilized in future studies, such as in vitro drug screening using generated dcNSCs. In addition, this method could be effectively utilized in studies on direct conversion into other types of target cells.

SUBMITTER: Daekee K

PROVIDER: S-EPMC6365637 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Generation of Genetically Stable Human Direct-Conversion-Derived Neural Stem Cells Using Quantity Control of Proto-oncogene Expression.

Daekee Kwon K Mi-Jung Han H Minjun Ji J Hee-Jin Ahn A Kwang-Won Seo S Kyung-Sun Kang K

Molecular therapy. Nucleic acids 20181220

As the human lifespan has increased due to developments in medical technology, the number of patients with neurological diseases has rapidly increased. Therefore, studies on effective treatments for neurological diseases are becoming increasingly important. To perform these studies, it is essential to obtain a large number of patient-derived neural cells. The purpose of the present study was to establish a technology that allows the high-efficiency generation of genetically stable, direct-conver ...[more]

PMID: 30731320

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Project description:Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production.IMPORTANCE Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines.

Dataset Information

Generation of Genetically Stable Human Direct-Conversion-Derived Neural Stem Cells Using Quantity Control of Proto-oncogene Expression.

Publications

Generation of Genetically Stable Human Direct-Conversion-Derived Neural Stem Cells Using Quantity Control of Proto-oncogene Expression.

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