Approaches for the derivation of induced pluripotent stem cells from cattle

Pluripotency describes the ability of a stem cell to form every cell type of the body. Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells has been achieved by forced expression of combinations of defined reprogramming factors, such as Oct4, Sox2, c-Myc, Klf4, Nanog and Lin28 in the mouse model and other species. The resulted murine iPS cells are morphologically and functionally similar to embryonic stem (ES) cells and can differentiate into virtually any cell type of the body including germ cells. These features makes iPS cells an attractive tool for basic and applied research in the fields of biomedicine and biotechnology. With regard to livestock species, the iPS technology promises to improve productive and reproductive traits. Viral methods of gene transfer for reprogramming ensure a high transduction efficiency, but have some disadvantages like i) limited cargo, ii) potential genotoxicity and iii) safety concerns. Here, I assessed a non-viral transposon method for reprogramming of bovine somatic cells to iPS cells. I compared two transposon systems namely, piggyBac (PB) and Sleeping Beauty (SB) employing different combinations of reprogramming factors. Advantages of the transposon systems are i) low costs, ii) larger cargo, iii) no safety concerns (transposons are non-infectious) and iv) potential of seamless removal of transposon cassettes. The first aim included to test and to compare the suitability of the transposon systems for reprogramming in the well-defined mouse species. Then the suitability of transposon iPS cells for targeted differentiation into a terminally differentiated cell type was assessed. Finally, bovine iPS cells were derived by employing optimized transposon conditions. Initially the PB and SB transposon systems have been tested for the derivation of iPS cells from cells of inbred (BL6) and outbred (NMRI) mice, respectively. The murine fibroblasts derived from an inbred BL/6 mouse line carrying a pluripotency reporter, Oct4-EGFP, allowed to following reprogramming by fluorescence microscopy. The reprogramming PB transposon encoded the cDNAs of reprogramming factors OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG, each separated by self-cleaving peptide sequences and driven by the chimeric CAGGS promoter, whereas SB transposon encoded for the same transcription factors excluding LIN28 and NANOG. Both transposon systems resulted in the successful isolation of murine iPS cell lines. The reduction of the core reprogramming factors to omit the proto-oncogene c-MYC was compatible with iPS cell line derivation, though with reduced reprogramming efficiencies. For targeted differentiation a transgenic mouse model with expression of a vital fluorophore reporter, tdTomato, in the eye lens was exploited. IPS cells from the transgenic mice were generated by SB reprogramming and these iPS cells were differentiated into lentoid bodies in-vitro. The lens-specific reporter allowed fluorescence detection of lens cell differentiation in-vitro. Finally, the optimized conditions for transposon reprogramming were used to derive bovine iPS (biPS) cells. Fetal fibroblasts were electroporated with SB and PB systems, respectively. Different culture media conditions were tested for maintaining the pluripotent status of biPS cells. By using bFGF (8 ng/ml) and hLIF (1000 U/ml) supplementation a stable bovine iPS culture, biPS-1, could be established by PB reprogramming. The derived biPS line expressed typical endogenous markers of embryonic stem cells, proliferated rapidly, showed long term proliferation and readily formed teratomas. Importantly, additive gene transfer was possible in the biPS-1 line. This study is the first demonstration that biPS cells can be generated by a non-viral transposon system, it suggests that ectopic NANOG and LIN 28 are necessary for reprogramming of bovine cells. These results are a major step towards the routine derivation of biPS cells and will facilitate the genetic modifications of the bovine genome.