Two-phase Analysis Of Molecular Pathways Underlying IPS Cell Induction

Induced pluripotent stem (iPS) cells can be reprogrammed from adult somatic cells by transduction with Oct4, Sox2, Klf4, and c-Myc. iPS cells, which bring patient's specific pluripotent stem cells to reality, is a big breakthrough in the stem cell research and regenerative medicine, but there are still several problems which block the way to clinical application:these include low efficiency of iPS cell induction, unexpected genome random modification and side effects of reprogramming factors. To solve these problems, the underlying mechanisms of reprogramming process need to be studied. However, the molecular cascades initiated by these factors remain poorly understood. Impeding their elucidation is the stochastic nature of the iPS cell induction process, which results in heterogeneous cell populations.After the comparison of iPS cell lines and embryonic stem (ES) cell lines, we found an intermediate phase from an abnormal iPS cell line. We hypotheses that a stable intermediate phase exists between somatic cell phase and pluripotent stem cell phase, and this stable phase may provide a homogeneous cell population for the analysis molecular mechanisms. In the first part of this thesis, we demonstrated that the reprogramming process can be synchronized by a two-phase induction:an initial stable intermediate phase following transduction with Oct4, Klf4, and c-Myc, and a final iPS cell phase following overexpression of Sox2.This two-phase iPS cell induction approach has enabled us to examine temporal gene expression profiles, permitting the identification of Sox2downstream genes critical for induction. After comparison of initial stable intermediate phase and final iPS cell phase, we have validated the feasibility of our new approach by employing the approach to confirm that downregulation of TGF-β signaling by Sox2proves essential to the reprogramming process. Thus, we present a novel means for dissecting the details underlying the induction of iPS cells, an approach with significant utility in this arena and the potential for wide-ranging implications in the study of other reprogramming mechanisms.