Role of OCT4 in reprogramming human skin keratinocytes

描述（由申请人提供）：2006年，Yamanka证明，通过携带两种胚胎基因Oct4和Sox2以及两种癌症基因Klf4和c-myc的鸡尾酒的逆转录病毒载体转导，小鼠皮肤成纤维细胞可以被重编程为多能性。虽然成功了，但这种方法有几个主要的缺点：基因是通过整合逆转录病毒传递的，当整合到不合适的DNA位置时，可能会导致癌症。这种方法需要传递多个基因，增加了产生有害影响的可能性。转导细胞在小鼠体内形成肿瘤。细胞是通过耐药性来选择的，这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，即瞬时转染单个胚胎基因Oct4。我们的方法旨在诱导内源性基因的表达，模仿正常哺乳动物发育过程中的胚胎表达模式。由于Oct4被认为是发育过程中多能状态的主要调控因子，我们推断，如果角化细胞被重编程为es样细胞，Oct4的表达应该会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在时间上重新激活。此外，当暴露于神经元分化培养基中时，细胞可以重定向为神经元细胞类型。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通过Oct4的短暂表达进行重编程。本研究的目的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，并了解人类角质形成细胞中OCT4表达的调节机制。我们假设OCT4的短暂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编程为多能干细胞。为了验证我们的假设，我们提出了以下具体目标：1)通过OCT4的瞬时表达对成人皮肤角质形成细胞进行重编程，2)研究重编程人角质形成细胞在体内的分化潜力，3)研究重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再激活，然后首先在体外通过改变培养基中的成分来检测这些细胞的分化潜力，然后在体内通过注射到小鼠囊胚中来检测这些细胞的分化潜力，以及畸胎瘤的形成。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）（如超氧化物）水平的变化。

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.