Description of the work performed since the beginning of the project
The five pluripotency markers from rabbit (OCT4, SOX2, KLF4, cMYC and NANOG) were identified and isolated. For this step phylogenetic analysis were performed, and based on the sequence analysis degenerative primers were used to isolate the cDNA of the pluripotency markers.
To generate rabbit induced pluripotent stem cells (rbiPSC) two different reprogramming methods were used: lentiviral and protein transduction. Two different lentiviral constructs were used; one contained the mouse pluripotency markers (OSKM) and the eGFP as reporter protein under the control of the EF1alpha promoter. In the other lentiviral construct the codon optimized human pluripotency factors were present (OKSM) and contained the dTomato as reporter protein under the control of a retroviral promoter, which theoretically silenced. By using the human factors containing lentiviral construct, two rbiPSC lines were generated and characterized for their pluripotency marker expression pattern and their in vitro differentiation ability towards the three germ layer lineages.
The generation of rbIPSCs by protein transduction was just partially successful as the obtained colonies after 2-3 passages started to differentiate and died.
Porcine iPS cells have been obtained via collaboration between the Roelen group at Utrecht University and the Hyttel group at Copenhagen University generated several porcine iPS cell lines. The differences among these cell lines are due to the method of generation, in particular whether the cells where generated in the presence of leukemia inhibitory factor (LIF) or fibroblast growth factors (FGF), and two types of reporter construct: Venus and mCherry. The cells have been cultured under various conditions with various signalling pathways inhibitors.
As an alternative strategy it is investigated whether reprogramming of porcine somatic cells can occur by chemical means only making use of small molecule inhibitors. This would facilitate the generation of porcine iPS cells in a non-transgenic fashion.
Description of the main results achieved so far
The consortium identified and isolated the 5 rabbit pluripotency markers: OCT4, SOX2, KLF4, cMYC and NANOG; and successfully generated 2 rabbit induced pluripotent stem cells (rbiPSC) and characterized them for pluripotency marker expression pattern by IHC and verified their ability to differentiate in vitro towards the three germ layer lineages.
The acquired porcine iPS cells have been cultured under various conditions. RNA from these cells has been isolated and gene expression of pluripotency genes (OCT4, NANOG, SOX2) will be examined.
Porcine Cumulus cells have been cultured in the presence of small molecule inhibitors for 5 days followed by culture with several additional factors for 7 days. The cells have been analysed by staining for alkaline phosphatase activity and qRT-PCR to examine expression levels of genes that are markers of pluripotency or differentiation.