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Derivation and Analysis of Murine Extraembryonic Endoderm Cell Lines

Derivation and Analysis of Murine Extraembryonic Endoderm Cell Lines
Zhong Yixiang
Bert Binas
Issue Date
At the blastocyst stage, mammalian pre-implantation embryos contain the three dedicated precursors of the three principal lineages of the conceptus: epiblast, primitive endoderm (PrE), and trophectoderm, which later form the fetus (all embryonic germ layers), extraembryonic endoderm (ExEn) and placental trophoblast, respectively. Each of these in vivo cell types has close or at least approximate in vitro counterparts. However, compared to the cell lines that represent the epiblast (embryonic stem [ES] cells) and trophoblast lineages (trophoblast stem cells), cell lines representing the early ExEn lineage are less understood. Therefore, my graduate project has been devoted to the ExEn lineage. Several cell lines have been presented that represent the early ExEn lineage, but their exact relationship with the in vivo cell types and each other has not been clarified. Before the work on this thesis, so-called mouse ExEn stem (XEN) cells, rat ExEn precursor-like (XENP) cells, and mouse naïve ExEn progenitor (nEnd) cells have been shown to exhibit essential features (such as gene expression and development potential) of the early ExEn lineage. In this study, I derived an additional type of mouse ExEn stem cells – named mouse primitive XEN (pXEN) cells – and performed a comparison with the other ExEn cell types and cell lines. The first part of my project was devoted to isolate and describe the mouse pXEN cells. As a starting point, it was known that the committed PrE cells of the mouse, which express the key transcription factor Oct4, depend on LIF/LIF-like factor-driven Jak/Stat signaling, and initially exhibit lineage plasticity. The previously described rat blastocyst-derived XENP cells also show these features, but equivalent mouse blastocyst-derived cell lines were lacking. In this thesis, I present mouse blastocyst-derived cell lines, named pXEN cells, which share these and additional characteristics with the rat XENP cells, but not with the previously known mouse blastocyst-derived XEN cells and the mouse ES cell-derived nEnd cells. Otherwise, pXEN cells are highly similar to XEN cells (but not nEnd cells) by morphology, lineage-intrinsic differentiation potential, and multi-gene expression profile. I also find that compared to the XEN cells, the pXEN cell profile correlates better with the blastocyst stage. Finally, I show that pXEN cells easily convert into XEN-like cells but not vice versa. The findings indicate that (i) pXEN cells are more representative than XEN cells of the blastocyst stage
(ii) mouse pXEN, rather than XEN, cells are homologs of rat XENP cells, which our laboratory proposes to call rat pXEN cells. The second part of my project was devoted to a more detailed comparison of the known ExEn-like stem cells and their presumed in vivo correlates. Thus, the above-described work, including my own work, had revealed that the existing ExEn cell lines (mouse XEN, rat and mouse pXEN, mouse nEnd cells) exhibit key features of the early ExEn and exhibit the respective lineage markers and developmental potential. By analysis of transcriptome data, I show: (i) Compared to ES cells, all ExEn cell lines are less in vivo like. (ii) All ExEn cell lines show post-implantation features that are biased towards either a mesenchymal/parietal endoderm (PE)-like phenotype (XEN, pXEN cells) or an epithelial/visceral endoderm (VE)-like phenotype (nEnd cells). The epithelial/VE and mesenchymal/PE features appear to be related to Wnt and Tgf-β signaling, respectively. (iii) The pXEN and nEnd cell lines additionally exhibit pre-implantation (pluripotency-related) features. (iv) One – but unstable - in vitro phenotype, the early nEnd cells, is most similar to the peri-sorting ExEn stage. These results are compatible with cell line models of either mixed or hybrid early ExEn phenotypes. Taken together, these findings indicate that closely related but distinct ExEn cell phenotypes result from different niches, which will have to be better understood in order to derive a continuous cell line that faithfully represents the committed or indeed any distinct ExEn stage. In conclusion, my work established a new in vitro stem cell type, mouse pXEN cells, and revealed that these cells represent the pre-implantation ExEn phenotype more closely than the previously isolated mouse XEN and mouse nEnd cells. However, my work also revealed that neither the first committed step of the ExEn lineage, nor indeed any pre- or peri-implantation stage of the ExEn lineage, has already been obtained in a continuous, self-renewable form in vitro. Hence, my work also highlights the need to better define the niche/s that would have the ability to maintain distinct cell types of the early ExEn lineage in homogenous form in vitro.
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GRADUATE SCHOOL[S](대학원) > MOLECULAR & LIFE SCIENCE(분자생명과학과) > Theses (Ph.D.)
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