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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：
8197353
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 2013-08-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8197353
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 retroviral transduction small hairpin RNA stem transcription factor tumor

项目摘要

ABSTRACT 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.

摘要 2006年，Yamanka展示了小鼠皮肤成纤维细胞可以重新编程成为多能细胞通过携带两种胚胎基因Oct4和Sox2的逆转录病毒载体进行转导，以及两个癌症基因，KLF4和c-myc。虽然这种方法很成功，但它有几个主要缺点：基因是通过整合的逆转录病毒传递的，当整合到一个不恰当的DNA定位。这种方法需要多个基因的传递，增加了有害的影响。转导的细胞在小鼠体内形成了肿瘤。用抗药性来选择细胞，它可以使细胞本身发生变化。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另一种方法重新编程，即瞬时转染单胚胎基因 10月4日。我们的方法旨在诱导模仿胚胎的内源基因的表达。在正常的哺乳动物发育过程中看到的表达模式。因为10月4日被认为是大师赛在发育过程中，我们推测Oct4的表达应该重新激活如果角质形成细胞被重新编程为类ES细胞，则为胚胎靶基因。我们发现了一个临时的内源性Oct4靶基因Sox2、Nanog、Utf1和Rex-1的重新激活。此外，这些细胞可以当暴露在神经元分化介质中时，被重定向为神经细胞类型。因此，不同于 Oct4瞬时表达可对小鼠皮肤成纤维细胞、小鼠皮肤角质形成细胞重新编程独自一人。这项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翻译到人角质形成细胞，并了解OCT4在角质形成细胞中表达的调节机制人类角质形成细胞。我们假设OCT4的瞬时表达足以重新激活内源性将人类皮肤角质形成细胞重新编程为多能干细胞所需的胚胎因素。测试我们的假设，我们提出了以下具体目标：1)重新编程成人皮肤角质形成细胞 OCT4的表达，2)研究体内重编程的人角质形成细胞的分化潜能，以及3)研究重新编程对内生变化的影响。我们计划瞬时转导OCT4 进入人皮肤角质形成细胞，研究内源性胚胎因子的重新激活，然后检查通过改变培养液中的成分首先在体外培养这些细胞的分化潜能，然后通过在体内注射到小鼠的胚泡中，形成畸胎瘤。最后，我们将研究甲基化状态再激活的内源性多能性基因OCT4、SOX2和NANOG，然后评估在重新编程的细胞中，活性氧物种(ROS)的水平，如超氧化物。