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

基本信息

批准号：
7996059
负责人：
JACKIE R BICKENBACH
金额：
$ 32.08万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
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/7996059
关键词：
Adult Affect Alzheimer&apos 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 Feline Immunodeficiency Virus 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

项目摘要

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，Yamanka 证明，通过使用携带两个胚胎基因 Oct4 和 Sox2 以及两个癌症基因 Klf4 和 c-myc 混合物的逆转录病毒载体转导，可以将小鼠皮肤成纤维细胞重新编程为多能性。。虽然这种方法很成功，但有几个主要缺点：基因是使用整合逆转录病毒传递的，当整合到不适当的 DNA 位置时，可能会导致癌症。该方法需要传递多个基因，从而增加了产生有害影响的可能性。转导的细胞在小鼠体内形成肿瘤。利用耐药性来选择细胞，这可以改变细胞本身。 2006 年晚些时候，我们发表了可以使用另一种方法对小鼠皮肤角质形成细胞进行重新编程，即瞬时转染单个胚胎基因 Oct4。我们的方法旨在诱导模仿正常哺乳动物发育过程中胚胎表达模式的内源基因的表达。由于 Oct4 被认为是发育过程中多能状态的主要调节因子，因此我们推断，如果角质形成细胞被重编程为 ES 样细胞，那么 Oct4 的表达应该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 暂时重新激活。此外，当暴露于神经元分化培养基时，细胞可以被重定向到神经元细胞类型。因此，与小鼠皮肤成纤维细胞不同，小鼠皮肤角质形成细胞可以仅通过Oct4的瞬时表达进行重编程。本提案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类角质形成细胞，并了解调节人类角质形成细胞中 OCT4 表达的机制。我们假设 OCT4 的瞬时表达足以重新激活将人皮肤角质形成细胞重编程为多能干细胞所需的内源胚胎因子。为了检验我们的假设，我们提出以下具体目标：1）通过 OCT4 的瞬时表达重新编程成人皮肤角质形成细胞，2）研究重新编程的人类角质形成细胞在体内的分化潜力，以及 3）研究重新编程影响的内源性变化。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，然后首先通过改变培养基中的成分在体外检查这些细胞的分化潜力，然后通过注射到小鼠囊胚中以及畸胎瘤的形成进行体内检查。最后，我们将研究重新激活的内源多能性基因 OCT4、SOX2 和 NANOG 的甲基化状态，然后评估重编程细胞中活性氧 (ROS)（例如超氧化物）水平的变化。公共卫生相关性：许多疾病，包括糖尿病、阿尔茨海默病和心脏病，都是由主要器官细胞死亡引起的。为了治疗这些疾病，必须替换丢失的细胞，最好是用自体细胞替换。必须诱导这些细胞改变其谱系并移植到新的环境中。来自表皮（皮肤的外层）的角质形成细胞比其他类型的成体细胞具有优势，因为它们可以在对个体几乎没有伤害的情况下分离出来，并且由于皮肤是体内最大的器官，因此它们存在于大量细胞中。数量。然而，它们必须重新编程才能形成皮肤以外的组织。因此，如果我们能够用一种简单的机制来诱导角质形成细胞充当干细胞，这将具有临床意义。此外，如果我们能够确定角质形成细胞只需要在短时间内引入单个基因，那么这将克服两个重大障碍，即使用癌症诱导基因和使用癌症诱导病毒。