Reprogramming of somatic cells by defined factors

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Nuclear reprogramming defines the dedifferentiation of adult cells into pluripotent embryonic cells and has enormous therapeutic potential as it allows generating genetically matched cells from patients for cell therapy. Reprogramming has so far been achieved by nuclear transfer into oocytes and by cell fusion between embryonic cells and somatic cells, two approaches that have serious technical or ethical limitations. Based on recently published observations, we have generated so-called induced pluripotent stem (iPS) cells directly from fibroblasts by retroviral overexpression of the transcription factors Oct4, Sox2, c-myc and Klf4. In contrast to the previously reported iPS cells, our iPS cells were indistinguishable from ES cells in their epigenetic state and developmental potential. Several crucial questions were raised by these findings; (i) what is the sequence of molecular changes that accompany nuclear reprogramming, (ii) what is the kinetics of reprogramming and does it require cell division, (iii) are different cell types at different differentiation stages equally amenable to reprogramming, and (iv) can human cells be reprogrammed into iPS cells? Resolving these questions will be critical for understanding the molecular nature of nuclear reprogramming and may lead to strategies that allow efficient reprogramming of patient?s cells into pluripotent cells. The current limitations to solve these questions are the low efficiency of direct reprogramming and the inability to follow reprogramming in real time. We will tackle these questions by generating ?reprogrammable mice? in which every single cell can be reversibly induced to express the four factors at levels necessary for reprogramming, and by attempting to reprogram human cells. The goals of this proposal are thus to determine (i) the robustness and kinetics of reprogramming, (ii) the hierarchy of transcriptional and epigenetic changes that accompany nuclear reprogramming, (iii) the responsiveness of different cell types to the four factors, and (iv) the feasibility of human reprogramming.