Reprogramming cells to pluripotency by defined factors

Ectopic expression of the transcription factors Oct4, Sox2, c-Myc, and Klf4, is sufficient to reprogram adult cells to a pluripotent state, generating induced pluripotent stem cells (iPSCs). These cells are considered the equivalent of embryonic stem cells (ESCs), but do not rely on embryonic material for derivation. The first description of mouse iPSCs in 2006 represented a landmark discovery; however, these cells demonstrated pluripotency in limited number of assays and had striking differences from ESCs that undermined their therapeutic potential. Several questions were raised by these findings, including whether it would be possible to generate iPSCs that were more similar to ESCs, whether human cells could be reprogrammed in a similar fashion, and why iPSC derivation was so inefficient.;The ability to derive pluripotent cells from adult tissues has been a pivotal step in regenerative medicine, and such insights into the process of reprogramming are paramount to the therapeutic success of iPSCs, a goal the field is rapidly moving towards.;The studies in this thesis have aimed to address these questions and represent key contributions to the field. In the first study, we discovered a novel method to obtain iPSCs, yielding cells that were nearly identical to ESCs; these findings formed the foundation for all subsequent work on iPSCs in the field. In the second study, we demonstrated that human fibroblasts could be reprogrammed with the same set of factors used in mouse. We also identified keratinocytes as a more suitable source of starting cells, as they reprogrammed faster and more efficiently than fibroblasts and could easily be obtained from patients. In order to address the inefficiency of reprogramming, we devised a strategy to homogeneously express all four reprogramming factors in a given population of cells. This system enabled 100-fold higher reprogramming efficiencies and has provided a unique platform for the identification of additional factors that influence the reprogramming process. In the third study, we discovered Tgfbeta signal inhibition as a potent cooperative factor, which yielded a 30-fold increase in efficiency in the presence of all four factors and could replace Sox2 and cMyc in the reprogramming of mouse fibroblasts.