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.
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.
|Chinnathambi, Sathivel; Wiechert, Susan; Tomanek-Chalkley, Ann et al. (2012) Treatment with the cancer drugs decitabine and doxorubicin induces human skin keratinocytes to express Oct4 and the OCT4 regulator mir-145. J Dermatol 39:617-24|
|Ross, Caitlin; Alston, Myrissa; Bickenbach, Jackie R et al. (2011) Oxygen tension changes the rate of migration of human skin keratinocytes in an age-related manner. Exp Dermatol 20:58-63|
|Racila, D; Winter, M; Said, M et al. (2011) Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway. Gene Ther 18:294-303|