Different flavors of pluripotency

Pluripotent stem cells have the ability to give rise to derivatives of all the germ layers and hold therefore great promise for future applications like cell replacement therapies and drug screenings, for example. Pluripotent stem cells can be expanded indefinitely which leads to an almost unlimited source of cells. These unique characteristics allow for the biochemical study of developmental processes in vitro that are not feasible in vivo due to restricted cell numbers within one embryo. The recent derivation of induced pluripotent stem cells (iPSC) from somatic cells unlocked a second source for pluripotent stem cells and fueled the hope for treatment of numerous diseases. But a comprehensive understanding of pluripotency per se is necessary to fully exploit the possibilities of these patient specific pluripotent stem cells while limiting the accompanying risks. This thesis focuses on different aspects of pluripotency in mouse and human pluripotent stem cells. The use of bacterial artificial chromosome (BAC) recombineering is a common practice to generate transgenic reporter cell lines that faithfully recapitulate the tissue specific expression profile of the gene of interest. We describe here the development of an efficient system for the generation of BAC-based targeting constructs. This system allows for the quick and straightforward generation of a BAC targeting vector and subsequent derivation of recombined BACs to that allows for the manipulation of the mammalian genome. This second part of this thesis concentrates on different aspects of the pluripotent stem cell state. Murine and human pluripotent stem cells show vast differences regarding their colony morphology, growth factor dependency and molecular characteristics and are therefore considered being in two different pluripotent stem cell states: naïve and primed pluripotency respectively. We set out to analyze the influence of the growth factor environment on the outcome of the reprogramming event. We first focused on permissive mouse strains and showed that murine iPS cells from the B6/129 hyrbid strain derived under bFGF and ActivinA signaling are more similar to regular naïve pluripotent stem cells then to primed pluripotent stem cells or EpiSCs. In a second set of experiments we analyzed the opposite scenario: the derivation of human pluripotent stem cells under naïve pluripotent conditions. We used induction of pluripotency and showed that cells can be derived with characteristics of naïve pluripotent stem cells regarding their morphology, growth characteristics and growth factor environment. These cells can facilitate homologous recombination and therefore allow the generation of transgenic human cell lines. The cell are not pluripotent but can be converted into primed pluripotent stem cells via an easy growth factor switch thereby allowing the derivation of transgenic hES like cells from patient derived somatic cells.