Role of OCT4 in reprogramming human skin keratinocytes

OCT4在人皮肤角质形成细胞重编程中的作用

• 批准号: 7754673
• 负责人: JACKIE R BICKENBACH
• 金额: $ 33.41万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-02 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7754673
• 关键词: Adult; Affect; Alzheimer' s Disease; Area; Autologous; B-Lymphocytes; Biological Assay; Cancer Etiology; Categories; Cause of Death; Cell Lineage; Cells; Chromatin; Cues; DNA; Data; Dermal; Development; Diabetes Mellitus; Disease; Drug resistance; Drug usage; Elderly; Elements; Embryo; Engineering; Environment; Epidermis; Fibroblasts; Gene Delivery; Gene Expression; Gene Proteins; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Gentamicins; Germ Layers; Goals; Heart Diseases; Histones; Human; In Vitro; Individual; Injection of therapeutic agent; Link; Location; Lymphocyte; Malignant Neoplasms; Methylation; Mus; Neuronal Differentiation; Neurons; Nude Mice; Oncogenes; Organ; Oxidation-Reduction; Pattern; Pluripotent Stem Cells; Production; Proteins; Publications; Publishing; Reactive Oxygen Species; Research; Retroviral Vector; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Role; Skin; Staining method; Stains; Stem cells; Superoxides; Techniques; Teratoma; Testing; Therapeutic; Time; Tissue Stains; Tissues; Transfection; Translating; Virus; Western Blotting; adult stem cell; bisulfite; blastocyst; c-myc Genes; cell type; cellular transduction; chromatin immunoprecipitation; clinically significant; design; embryonic stem cell; experience; human tissue; in vivo; keratinocyte; pluripotency; promoter; public health relevance; retroviral transduction; small hairpin RNA; stem; transcription factor; tumor

DESCRIPTION (provided by applicant): In 2006, Yamanka demonstrated that mouse skin fibroblasts could be reprogrammed to become pluripotent through transduction with a retroviral vector carrying a cocktail of two embryonic genes, Oct4 and Sox2, and two cancer genes, Klf4 and c-myc. Although successful, this type of approach has several major drawbacks: Genes were delivered using the integrating retrovirus, which can cause cancer when integrated into an inappropriate DNA location. The approach required the delivery of multiple genes, enhancing the possibility of deleterious effects. The transduced cells formed tumors in mice. Cells were selected using drug resistance, which can impart changes in the cells themselves. Later in 2006, we published that mouse skin keratinocytes could be reprogrammed using an alternative approach, transient transfection of the single embryonic gene Oct4. Our approach was designed to induce expression of endogenous genes that mimicked embryonic expression patterns seen during normal mammalian development. Since Oct4 is considered to be the master regulator of the pluripotent state during development, we reasoned that expression of Oct4 should reactivate embryonic target genes if the keratinocyte was reprogrammed into an ES-like cell. We found a temporal reactivation of the endogenous Oct4 target genes, Sox2, Nanog, Utf1, and Rex-1. Furthermore, the cells could be redirected toward a neuronal cell type when exposed to neuronal differentiation medium. Thus, unlike mouse skin fibroblasts, mouse skin keratinocytes could be reprogrammed by transient expression of Oct4 alone. The goals in this proposal are to demonstrate that our findings using mouse keratinocytes can be translated to human keratinocytes, and to understand the mechanisms that regulate OCT4 expression in human keratinocytes. We hypothesize that transient expression of OCT4 is sufficient to reactivate endogenous embryonic factors required to reprogram human skin keratinocytes into pluripotent stem cells. To test our hypothesis, we propose the following Specific Aims: 1) Reprogram adult human skin keratinocytes by transient expression of OCT4, 2) Investigate the differentiation potential of reprogrammed human keratinocytes in vivo, and 3) Investigate endogenous changes effected by reprogramming. We plan to transiently transfect OCT4 into human skin keratinocytes, investigate reactivation of endogenous embryonic factors, then examine the differentiation potential of these cells first in vitro by altering components in the medium, then in vivo by injection into mouse blastocysts, and formation of teratomas. Finally, we will investigate the methylation states of the reactivated endogenous pluripotency genes OCT4, SOX2, and NANOG, then assess changes in the levels of reactive oxygen species (ROS), such as superoxide, in the reprogrammed cells. PUBLIC HEALTH RELEVANCE: Many diseases, including diabetes, Alzheimer's, and heart disease, are caused by the death of cells in major organs. To treat these diseases, the lost cells must be replaced, ideally with autologous cells. Such cells will have to be induced to change their lineage and engraft into a new environment. Keratinocytes from the epidermis, the outer layer of the skin, offer advantages over other types of adult cells in that they can be isolated with little harm to the individual and, since the skin is the largest organ in the body, they are present in large quantities. However, they must be reprogrammed to form tissues other than skin. Therefore, if we can use a simple mechanism to induce keratinocytes to act as stem cells, this would have clinical significance. Furthermore, if we can determine that keratinocytes need only to have a single gene introduced for a short period of time, then this would overcome two significant obstacles, using cancer-inducing genes and using cancer inducing viruses.

描述（由申请人提供）：2006年，亚曼卡证明了小鼠皮肤成纤维细胞可以重新编程，从而通过转导的逆转录病毒载体，载有两个胚胎基因的鸡尾酒，即oct4和sox2，以及两个癌症基因，以及两个癌症基因，klf4和c-myc。尽管成功，但这种方法具有几个主要缺点：使用集成逆转录病毒输送基因，当整合到不适当的DNA位置中时，可能会导致癌症。该方法需要传递多个基因，从而增强了有害影响的可能性。转导的细胞在小鼠中形成肿瘤。使用耐药性选择细胞，这可以赋予细胞本身的变化。 2006年晚些时候，我们发表了小鼠皮肤角质形成细胞可以使用替代方法（单个胚胎基因oct4的瞬时转染）重编程。我们的方法旨在诱导内源基因的表达，该基因模仿正常哺乳动物发育过程中的胚胎表达模式。由于OCT4在发育过程中被认为是多能状态的主要调节剂，因此我们认为，如果将角质形成细胞重编程为ES样细胞，则OCT4的表达应重新激活胚胎靶基因。我们发现内源性OCT4靶基因Sox2，Nanog，UTF1和REX-1的时间重新激活。此外，当暴露于神经元分化培养基时，可以将细胞转向神经元细胞类型。因此，与小鼠皮肤成纤维细胞不同，单独的OCT4的瞬时表达可以重新编程小鼠皮肤角质形成细胞。该提案中的目标是证明我们使用小鼠角质形成细胞的发现可以转化为人角质形成细胞，并了解调节人角质形成细胞中OCT4表达的机制。我们假设OCT4的瞬时表达足以重新激活内源性胚胎因子，将人皮肤角质形成细胞重新编程为多能干细胞所需。为了检验我们的假设，我们提出了以下特定目的：1）通过OCT4的短暂表达重新编程成年人类皮肤角质形成细胞，2）研究体内重编程的人角质形成细胞的分化潜力，以及3）研究通过重新编程实现的内源性变化。我们计划将OCT4瞬时转染到人皮肤角质形成细胞中，研究内源性胚胎因子的重新激活，然后检查这些细胞在体外的分化潜力，通过改变培养基中的成分，然后通过体内注射到小鼠胚泡中，并形成Teratomas。最后，我们将研究重新激活的内源性多能基因Oct4，Sox2和Nanog的甲基化状态，然后评估重编程细胞中活性氧（ROS）的水平的变化（例如超氧化物）。 公共卫生相关性：许多疾病，包括糖尿病，阿尔茨海默氏症和心脏病，都是由主要器官中细胞死亡引起的。为了治疗这些疾病，必须用自体细胞代替丢失的细胞。这些细胞必须被诱导改变其谱系并植入新环境。皮肤表皮的角质形成细胞比其他类型的成年细胞具有优势，因为它们可以隔离而不会对个体造成伤害，并且由于皮肤是体内最大的器官，因此它们大量存在。但是，它们必须重新编程以形成皮肤以外的组织。因此，如果我们可以使用一种简单的机制诱导角质形成细胞充当干细胞，那么这将具有临床意义。此外，如果我们可以确定角质形成细胞只需要在短时间内引入一个基因，那么使用癌症诱导的基因并使用癌症诱导病毒，这将克服两个重要的障碍。