Dissecting Molecular Similarities and Differences Between Pluripotent Stem Cell Lines

Traditionally, pluripotent stem cells are derived from preimplantation embryos and fetal germ cells, which give rise to embryonic stem cells (ESCs) and embryonic germ cells (EGCs), respectively, In contrast, induced pluripotent stem cells (iPSCs) are derived from somatic cells upon overexpression of defined transcription factors such as Oct4, Sox2, Klf4 and c-Myc. Despite their origin from different cell types, all of these pluripotent stem cell lines share the ability to self-renew indefinitely in culture while retaining the capacity to differentiate into derivatives of all three germ layers. Because pluripotent cells provide a useful tool in basic research and cell therapy, it is critical to understand the molecular similarities and differences between ESCs, EGCs and iPSCs. The studies presented in this thesis aim to address the equivalence of different pluripotent cell types. In the first study, we performed a systematic comparison of DNA methylation and gene expression patterns between isogenic mouse ESCs and EGCs. Surprisingly, we found that global DNA methylation patterns were indistinguishable between ESC and EGC lines of the same sex, while female cell lines exhibited global hypomethylation compared to male cell lines. Mechanistically, upregulation of the X-linked gene, dual specificity phosphatase 9 (Dusp9) in female cells attenuated MAP kinase signaling, resulting in global DNA hypomethylation via the reduction of Dnmt3a and Dnmt3b protein levels. In the second study, we compared isogenic, transgene-free hESC and hiPSC lines to determine whether molecular differences exist between hiPSC and hESC lines when controlling for genetic background and reprogramming methodology. Strikingly, transcriptional variation between different genetic backgrounds was greater than variation observed between cell types (i.e., hiPSCs compared to hESCs). Moreover, the few transcriptional differences observed between isogenic hESC and hiPSC lines had no apparent functional consequences and these genes were not identified during the comparison of a larger set of independently derived non-isogenic hESC/hiPSC lines. We conclude that hESCs and hiPSCs are highly similar on a transcriptional and functional level and cannot be distinguished by a defined gene expression signature. Together, our data demonstrated that sex rather than cell type of origin drives global epigenetic and transcriptional patterns in conventional mouse pluripotent cell lines. These results provide fundamental insights into the epigenetic regulatory mechanisms that govern pluripotency. Additionally, our comparison of isogenic hiPSCs and hESCs supports the view that cellular reprogramming technologies faithfully reset the transcriptional pattern of somatic cells and establish a pluripotent state that is molecularly and functionally equivalent to embryo-derived stem cells. These findings may provide the basis for future mechanistic studies and help to translate iPSC technologies into a therapeutic setting.
Medical Sciences