Person:

Choi, Jiho

The generation of induced pluripotent stem cells (iPSCs) often results in aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster, compromising the ability to generate entirely iPSC-derived adult mice ('all-iPSC mice'). Here, we show that reprogramming in the presence of ascorbic acid attenuates hypermethylation of Dlk1-Dio3 by enabling a chromatin configuration that interferes with binding of the de novo DNA methyltransferase Dnmt3a. This approach allowed us to generate all-iPSC mice from mature B cells, which have until now failed to support the development of exclusively iPSC-derived postnatal animals. Our data show that transcription factor–mediated reprogramming can endow a defined, terminally differentiated cell type with a developmental potential equivalent to that of embryonic stem cells. More generally, these findings indicate that culture conditions during cellular reprogramming can strongly influence the epigenetic and biological properties of the resultant iPSCs.

Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo1. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential. Furthermore, we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L. Mechanistically, we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects, in part through the downregulation of DNA methyltransferases and their cofactors. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells. Taken together, our data suggest that, although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.

Development and differentiation are associated with profound changes to histone modifications, yet their in vivo function remains incompletely understood. Here, we generated mouse models expressing inducible histone H3 lysine-to-methionine mutants, which globally inhibit methylation at specific sites. Mice expressing H3K36M developed severe anemia with arrested erythropoiesis, a marked hematopoietic stem cell defect, and rapid lethality. By contrast, mice expressing H3K9M survived up to a year and showed expansion of multipotent progenitors, aberrant lymphopoiesis and thrombocytosis. Additionally, some H3K9M mice succumbed to aggressive T cell leukemia/lymphoma while H3K36M mutants exhibited differentiation defects in testis and intestine. Mechanistically, H3K36M and H3K9M reduced H3K36 and H3K9 trimethylation patterns genome-wide and altered chromatin accessibility and gene expression landscapes. Strikingly, discontinuation of transgene expression largely restored differentiation programs. Our work shows that individual chromatin modifications are required at several specific stages of differentiation and introduces powerful tools to interrogate their roles in vivo.

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.