The two defining features of mouse embryonic stem (ES) cells are self-renewal and naive pluripotency, the ability to give rise to all cell lineages in the adult body. In addition to being a unique and interesting cell type, pluripotent ES cells have demonstrated their potential for continued advancements in biomedical science. Currently, there is an improved understanding in the chemical signals and the gene regulatory network responsible for the maintenance of ES cells in the naive pluripotent state. However, less is understood about how ES cells exit pluripotency. My main aim is to study the dynamics and the factors affecting the irreversible exit from pluripotency. Expression of the reporter Rex1-GFPd2, which is inactivated upon exit from naive pluripotency, was analyzed by quantitative long-term single-cell imaging over many generations. This technique allowed chemical, physical, and genealogical information to be recorded during the transition to exit. Culture conditions that provided homogeneous populations were used in all assays and these data were validated against bulk-culture data where appropriate. Changes in real-time cell behavior were seen in cell-cell contact, motility, and cell-cycle duration. Undifferentiated ES cells form tightly joined colonies, with cells that exhibit low motility and a constant cell-cycle duration. Exit is associated with increasing cell motility, decreased cell-cell contact, and an acceleration in cell proliferation. The onset of exit is associated with a sudden and irreversible inactivation of the Rex1-GFPd2 reporter. This inactivation is asynchronous, as it occurs at different times and in different generations during ES cell differentiation. However, examination of daughter cells generated from the same mother revealed a high level of synchronicity. Further investigation revealed that high levels of correlation in cell-cycle duration and Rex1-GFPd2 expression exist between differentiating sister and cousin cells, providing strong evidence that cell potency is inherited symmetrically in cell divisions during exit $\textit{in vitro}$. How cells change fate is a fundamental question in developmental biology. Knowing the cellular dynamics during the transition out of naive pluripotency is important for harnessing the potential of ES cells and understanding how cell fate decisions are made during embryonic development. The quantification of the timing of exit from naive pluripotency coupled with identifiable changes in cellular behaviors, such as motility, cell size, and cell-cycle duration, enhances the understanding of how cell fate changes are regulated during directed differentiation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:763839 |
| Date | January 2019 |
| Creators | Strawbridge, Stanley Eugene |
| Contributors | Smith, Austin ; Martello, Graziano ; Kugler, Hillel |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/287576 |
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