Role of OCT4 in reprogramming human skin keratinocytes

描述（由申请人提供）：2006年，Yamanka证明小鼠皮肤成纤维细胞可以通过用携带两种胚胎基因Oct 4和Sox 2以及两种癌症基因Klf 4和c-myc的混合物的逆转录病毒载体转导而重编程为多能性。虽然成功，但这种方法有几个主要缺点：基因是使用整合逆转录病毒传递的，当整合到不合适的DNA位置时会导致癌症。这种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另一种方法重新编程，瞬时转染单个胚胎基因Oct 4。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看到的胚胎表达模式。由于Oct 4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，Oct 4的表达应该重新激活胚胎靶基因。我们发现了内源性Oct 4靶基因Sox 2、Nanog、Utf 1和雷克斯-1的暂时再激活。此外，当暴露于神经元分化培养基时，细胞可以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通过单独的Oct 4的瞬时表达来重编程。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OCT 4表达的机制。我们假设OCT 4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性胚胎因子。为了验证我们的假设，我们提出了以下具体目的：1）通过OCT 4的瞬时表达来重编程成人皮肤角质形成细胞，2）研究重编程的人角质形成细胞在体内的分化潜力，以及3）研究重编程所影响的内源性变化。我们计划将OCT 4瞬时转染入人皮肤角质形成细胞，研究内源性胚胎因子的再激活，然后首先通过改变培养基中的组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在体内检查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性基因OCT 4，SOX 2和NANOG的甲基化状态，然后评估重编程细胞中活性氧（ROS）水平的变化，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，都是由主要器官中的细胞死亡引起的。为了治疗这些疾病，丢失的细胞必须被替换，最好是用自体细胞。这样的细胞将不得不被诱导以改变它们的谱系并移植到新的环境中。来自表皮（皮肤的外层）的角质形成细胞提供优于其他类型的成体细胞的优点，因为它们可以被分离而对个体几乎没有伤害，并且由于皮肤是身体中最大的器官，它们大量存在。然而，它们必须重新编程以形成皮肤以外的组织。因此，如果我们能用一种简单的机制诱导角质形成细胞作为干细胞，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单一基因，那么这将克服两个重大障碍，使用癌症诱导基因和使用癌症诱导病毒。

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.