Generation of Human iPS Cells via Non-integrating Vectors

通过非整合载体生成人类 iPS 细胞

• 批准号: 7708251
• 负责人: WEN-SHU WU
• 金额: $ 18.81万
• 依托单位: MAINEHEALTH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7708251
• 关键词: Address; Adenoviruses; Biological Assay; Cell Culture Techniques; Cell Differentiation process; Cell Nucleus; Cell Transplantation; Cell fusion; Cells; Chromosomes; Cleaved cell; Cloning Vectors; Culture Media; DNA Methylation; Disease; Dose; Embryo; Ensure; Equilibrium; Ethics; Fibrinogen; Fibroblasts; Gene Delivery; Gene Expression Profile; Gene Silencing; Gene Transfer; Generations; Genetic; Genome; Hepatocyte; Human; Hybrid Cells; Immune; In Vitro; Infection; Laboratories; Link; Lung; Mediating; Methodology; Methods; Modification; Monitor; Mus; Mutation; Oocytes; Open Reading Frames; Patients; Peptides; Pluripotent Stem Cells; Preclinical Drug Evaluation; Process; Protocols documentation; Regenerative Medicine; Research; Site; Somatic Cell; Source; Stem cells; System; Technology; Testing; Therapeutic; Time; Tissue Engineering; Toxicology; Transgenes; Tumor-Suppressor Gene Inactivation; Viral; Viral Insertional Mutageneses; Virus; Western Blotting; Zinc; base; c-myc Genes; clinical application; egg; embryonic stem cell; expression vector; fusion gene; human embryonic stem cell; improved; induced pluripotent stem cell; mouse genome; nuclear reprogramming; penis foreskin; pluripotency; promoter; public health relevance; self-renewal; somatic cell nuclear transfer; stem; stem cell differentiation; tool; transcription factor; transgene expression; tumor; vector

DESCRIPTION (provided by applicant): Stem cells have great therapeutic potential for treatment of various diseases, and alternative methods for generation of pluripotent cells are needed. A particularly exciting method for the generation of patient-specific (or isogenic) pluripotent stem cells is somatic cell reprogramming. Three reprogramming approaches have been investigated: i) the somatic cell nuclear transfer (SCNT) approach; ii) cell fusion-based approach; iii) generation of induced pluripotent stem (iPS) cells by reprogramming of somatic cells via the defined transcription factors (Oct3/4 and Sox2 with Klf4 and c-Myc or Nanog and Lin28). The first two approaches are limited by extremely low efficiency and the requirement for fresh human oocytes or formation of somatic/embryonic stem (ES) hybrid cells, thereby limiting their further clinical applications. The last approach does not require embryos or oocytes, and provides a potential alternative to the current source of ES cells, thereby offering the possibility of generating patient- and disease-specific pluripotent stem cells. These cells may be valuable for not only personalized cell transplantation therapy without immune rejection but also for understanding disease mechanisms, drug screening, tissue engineering, and toxicology. iPS cells were generated mainly from mouse somatic cells, and these cells are similar to ES cells in terms of gene expression profile, DNA methylation, and perhaps most significantly self-renewal and differentiation potential. However, these iPS cells are significantly genetically modified with many mutations in the genome, and mice derived from iPS cells develop tumors, mainly through reactivation of c-Myc and inactivation of tumor suppressor genes resulting from viral insertional mutagenesis. Recently, mouse iPS cells were successfully generated from fibroblasts and liver cells via adenovirus-mediated gene delivery of the four transcription factors. However, the efficiency is extremely low, ranging from less than 0.0001% to 0.001%. 'Genetically clean' human iPS cells have not been generated, maybe because of inadequate reprogramming efficiency. We recently improved the generation of iPS cells by forced expression of four reprogramming factors from one single open reading frame (ORF) that was linked by self-cleaving 2A sequences. This improved system significantly increased reprogramming efficiency (up to 100 times) and enabled multiple reprogramming factors to be expressed in a single vector. Therefore, we intend to test whether forced expression of the reprogramming factors from a single ORF via non-integrating vectors enhances generation of 'genetically clean' human iPS cells from somatic cells. To address this question, we propose the following two Specific Aims: Aim 1: To generate human iPS cells from somatic cells by inducible expression of the reprogramming factors from a single ORF via a self-replication episomal vector; Aim 2: To generate human iPS cells from somatic cells by adenovirus-mediated gene transfer of the reprogramming factors in a single ORF. PUBLIC HEALTH RELEVANCE: This project will generate 'genetically clean' human ES-like pluripotent stem cells by somatic cell reprogramming approaches. Therefore, this is an alternative to the use of human embryonic stem cells for regenerative medicine applications.

描述（由申请人提供）：干细胞具有治疗各种疾病的巨大治疗潜力，并且需要用于产生多能细胞的替代方法。一种特别令人兴奋的产生患者特异性（或同基因）多能干细胞的方法是体细胞重编程。已经研究了三种重编程方法：i）体细胞核转移（SCNT）方法; ii）基于细胞融合的方法; iii）通过经由限定的转录因子（Oct 3/4和Sox 2与Klf 4和c-Myc或Nanog和Lin 28）重编程体细胞来产生诱导的多能干（iPS）细胞。前两种方法受到效率极低以及需要新鲜人卵母细胞或形成体细胞/胚胎干（ES）杂交细胞的限制，从而限制了它们的进一步临床应用。最后一种方法不需要胚胎或卵母细胞，并提供了一个潜在的替代目前的ES细胞来源，从而提供了产生患者和疾病特异性多能干细胞的可能性。这些细胞不仅对无免疫排斥的个性化细胞移植治疗有价值，而且对理解疾病机制、药物筛选、组织工程和毒理学也有价值。iPS细胞主要由小鼠体细胞产生，这些细胞在基因表达谱、DNA甲基化以及可能最重要的自我更新和分化潜力方面与ES细胞相似。然而，这些iPS细胞被显著地遗传修饰，在基因组中具有许多突变，并且源自iPS细胞的小鼠主要通过c-Myc的再活化和由病毒插入诱变引起的肿瘤抑制基因的失活而发展肿瘤。最近，通过腺病毒介导的四种转录因子的基因递送，成功地从成纤维细胞和肝细胞产生小鼠iPS细胞。然而，效率极低，范围从小于0.0001%到0.001%。“基因清洁”的人类iPS细胞尚未产生，可能是因为重编程效率不足。我们最近通过从一个由自切割2A序列连接的单个开放阅读框（ORF）强制表达四个重编程因子来改进iPS细胞的产生。这种改进的系统显著提高了重编程效率（高达100倍），并使多个重编程因子能够在单个载体中表达。因此，我们打算测试通过非整合载体从单个ORF强制表达重编程因子是否增强从体细胞产生“遗传上清洁的”人iPS细胞。为了解决这个问题，我们提出了以下两个具体的目的：目的1：从体细胞产生人iPS细胞诱导表达的重编程因子从一个单一的ORF通过自我复制附加型载体;目的2：从体细胞产生人iPS细胞的腺病毒介导的基因转移的重编程因子在一个单一的ORF。公共卫生相关性：该项目将通过体细胞重编程方法产生“基因清洁”的人类ES样多能干细胞。因此，这是一种替代人类胚胎干细胞用于再生医学应用的方法。