The therapeutic potential of embryonic stem (ES) cells has garnered great interest but the need to destroy early stage embryos in order to obtain these cells has raised many ethical concerns. The recent discovery that four transcription factors: Oct4, Sox2, c-Myc and Klf4, can reprogram somatic fibroblasts into an embryonic-like pluripotent state has provided a potential alternative to the current source of ES cells. In order to advance the potential of this discovery, the goal of this proposal is to understand the molecular mechanisms that enable these four transcription factors to induce pluripotency in mouse embryonic fibroblasts. We will address this question using cells derived from mice harboring a neomycin selectable marker in the locus of the pluripotency-maintaining transcription factor Oct4. In this context, neomycin resistance is conferred only when the Oct4 gene is activated, thereby providing a source of somatic cells that can be used to select for 'reprogrammed'ES-like clones. Oct4-neo selectable mouse embryonic fibroblasts have been utilized successfully in our lab to generate pluripotent cells by introducing Oct4, Sox2, c-Myc and Klf4 into these cells. Using Oct4-neo selectable cells, we aim to identify genetic pathways that are critical to the function of c-Myc and Klf4 as reprogramming factors. By transforming mouse embryonic fibroblasts with c-Myc and Klf4, we will generate c-Myc/Klf4 clones that can be reprogrammed by simply adding Oct4 and Sox2. Gene targets of c-Myc and Klf4 in these clones will be identified at a global level using genome-wide chromatin immunoprecipitation analysis (CHIP-chip) and specific changes will be confirmed using more traditional techniques such as Northern analysis and western blotting. The goal is to generate a correlation between specific gene expression patterns and the efficiency of reprogramming. In parallel, we will transduce other cell types with the four aforementioned transcription factors to determine whether there are cell-specific differences in the requirements for cellular reprogramming. Additionally, we aim to demonstrate the therapeutic potential of this reprogramming method by rescuing the hematopoietic defect of Rag2-deficient mice with Rag2-corrected reprogrammed cells isolated from generated RAG2ko/Oct4-neo mice. This proposal intends to delineate further the molecular mechanisms involved in the generation and maintenance of pluripotency in order to enhance the therepeutic potential of stem cells.
|Welstead, G Grant; Creyghton, Menno P; Bilodeau, Steve et al. (2012) X-linked H3K27me3 demethylase Utx is required for embryonic development in a sex-specific manner. Proc Natl Acad Sci U S A 109:13004-9|