Studies on the isolation of murine and ovine embryonic stem cells

• Main Author: Wells, David Norman
• Published: University of Edinburgh 1991
• Subjects: 571.53
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.663638

Techniques for the isolation and genetic manipulation of embryonic stem cells (ES cells) in the mouse are well established. However, little is understood regarding the mechanisms which enable pluripotent cells from the early mouse embryo to be diverted experimentally from their normal fate of differentiation <i>in vivo</i> and maintained instead in culture, as stable ES cell lines. Such knowledge might aid the establishment of ES cells from other mammalian species; particularly from farm animals, with the potential applications of genetically manipulating the stem cells to modify production traits. The objective of this thesis was to investigate various factors which may influence the potential of murine and ovine embryos to yield ES cells. Mouse genotype influences the capacity of embryos to give rise to ES cells. A significantly greater proportion of day 3.5 <i>p.c.</i> mouse embryos from the inbred 129/Sv-<i>CP</i> strain yielded ES cell lines compared to crossbred F<SUB>2</SUB> (C57BL/6 X CBA/Ca) embroys. The effect of mouse genotype was first observed in the significantly greater proportion of 129/Sv-<i>CP</i> embryos that gave rise to ES-like colonies at the first passage, following the disaggregation of inner cell mass (ICM) outgrowths. The efficiency of ES cell isolation was significantly increased by culturing murine blastocyst-stage embryos which had been induced experimentally, to enter a period of implantational delay for five days. Typically, murine ES cells have been derived from the day 3.5 <i>p.c.</i> ICM, although day 2.5 <i>p.c.</i> morulae have also been used. Here, a study describes the isolation of pluripotent ES cell lines from the primitive ectoderm of day 5.5 <i>p.c.</i> egg cylinder-stage mouse embroys. Although this study has shown that, in the mouse, primitive ectodermal cells influenced by endoderm can be established as ES cells in culture, the frequency is significantly lower than from the ICM. ES cells isolated from the three different embryonic stages equivalent to three days of development, have the same morphological characteristics. The brief exposure of day 3.5 <i>p.c.</i> embryos from the 129/Sv-<i>CP</i> mouse strain to treatments expected to perturb gene expression, heat shock or puromycin-containing medium, significantly increased the frequency of ES cell isolation. These effects resulted from increases in the proportion of embryos giving rise to ES-like colonies at the first passage which, in the case of heat shocked embryos, were also less likely to differentiate in subsequent passages. ES cell lines derived from embryos exposed to heat shock or puromycin have retained the capacity for <i>in vivo</i> development, as the stem cells colonised the germline in some chimaeric mice. These results suggest that the isolation of ES cells depends upon reversible, epigenetic changes in the pattern of gene expression and that the heat shock and puromycin treatments utilised here, may have helped to promote these changes <i>in vitro</i>. It is suggested that heat shock and implantational delay may act through a similar mechanism; whereby the treatments may have interfered with the transcriptional activation of genes responsible for differentiation of the ICM.