Embryonic stem cells (ESCs) are pluripotent cells derived from the early embryo and
are able to differentiate into cells belonging to the three germ layers. They are a
valuable tool in research and for clinical use, but their applications are limited by
ethical and technical issues.
In 2006 a breakthrough report described the generation of induced pluripotent stem
cells (iPSCs). IPSCs are ESC-like cells generated from somatic cells by forcing the
ectopic expression of specific transcription factors. This circumvents the ethical
issues about the use of embryos in research and provides multiple opportunities to
understand the mechanisms behind pluripotency.
The aim of this project was to generate sheep iPSCs and characterise them. In order
to learn the technique I initially repeated the original iPSC methodology: the putative
mouse iPSCs I have generated display a morphology typical of ESCs, characterised
by a high nuclear to cytoplasmic ratio, and form colonies with neat edges and smooth
domes. These cells are positive to Nanog, a marker of pluripotency, and can give rise
to cells belonging to the mesodermal and the ectodermal lineages when differentiated
in vitro. Since the main aim of the thesis was the derivation of sheep pluripotent
cells, once established the protocol in mouse, I then moved to the generation of ovine
iPSC colonies. The cells I have generated have a morphology similar to that of
mouse ESCs, express markers of pluripotency such as alkaline phosphatase and
Nanog and can differentiate in vitro and in vivo into cells belonging to the three germ
layers. Additionally, these ovine iPSCs can contribute to live born chimeric lambs,
although at low level