Generation of embryonic stem cell lines from IVF embryos and parthenotes

Abstract

Efficient isolation and maintenance of pluripotent cell lines in livestock species have proven to be a major hurdle despite tremendous efforts over 3 decades. Little is known about the signalling pathways controlling pluripotency in mammals other than rodents and primates. To understand these mechanisms in the bovine due to a biomedical model for human diseases, biotechnology applications and commercial use, embryonic stem cells (ESCs) were isolated from IVF embryos in a conventional medium and in a medium with 3 small-molecule inhibitors (3i) which permit efficient self-renewal of ESCs in mice and rats. SU5402 inhibits the FGF receptor tyrosine kinases and PD184352 inhibits the ERK cascade. CHIR99021 inhibits GSK3 to increase ES-cell proliferation. The effect of three inhibitors “3i” on 1) the efficiency of ESC isolation and 2) maintenance of established bovine ESC lines (bESCs) was examined in the different media. Colony formation and attachment to the feeder layer differed among media and media containing the 3i components resulted in better growth and maintenance of putative ESCs compared with the others. Expression of 6 pluripotency related genes (Oct4, rex1, sox2, ssea1, alp and nanog) and four proteins (Oct4, SSEA1, SSEA4, Alkaline Phosphatase) were assessed. The results indicate that 3i can improve bovine ESC isolation and increase the pluripotent component of putative bovine ES cells. The second series of experiments investigated the generation of autologous cell lines. ES cells from parthenogenetically activated oocytes can provide autologous transplantable cells, which are immuno-compatible for the oocyte donors as well as an invaluable tool for genetic engineering for transplantation and epigenetic studies. We report the efficient isolation of 8 putative bovine parthenogenetic ESC lines in 3i medium from 15 in vitro produced parthenotes. The cells displayed pluripotency similar to IVF ESC lines and differentiated in suspension culture to form embryoid bodies (EBs) expressing markers of the three embryonic germ layers. The third series of experiments investigated the long-term maintenance of pluripotent bESCs during in vitro culture further and developed a robust method of cryopreservation which is essential requirements to maximize the use of ESCs in research and for biotechnology applications. 1) Eight putative bESC lines established in N2B27-3i medium and 1 established in conventional medium were cultured over 30 passages (> 240 days) and characterized at P23-30. All cell lines expressed markers of pluripotency. The cells formed EBs containing cells of the three embryonic germ layers. For cryopreservation, vitrification was compared with conventional slow-freezing. Survival rate and colony morphology of vitrified cell lines after thawing was significantly higher than after slow-freezing, (97% v/s 56% respectively; p<0.01). Cell lines survived and proliferated, with good morphology, following vitrification in both N2B27-3i, 95% (104/110) and conventional medium, 97% (93/96). Post-thawed vitrified cell lines were cultured for an additional 12 passages (P¬18+12, P19+12 and P23+12) and shown to retain pluripotency. The cells formed EBs in suspension culture and expressed genes representative of the three embryonic germ layers. Vitrification did not change the karyotype of cells. Finally, three cell lines isolated in conventional medium were maintained for 80-95 passages and vitrified. These cell lines also retained expression of pluripotent markers. In summary, the results in this thesis present 1) efficient derivation and characterization of pluripotent putative bESCs from IVF and parthenotes in 3i medium and 2) maintenance of cell lines in both conventional and 3i media which express pluripotent markers and retain the ability to differentiation in vitro after long-term culture, and 3) successful cryopreservation of cell lines, especially by vitrification. This thesis adds significantly to the knowledge on isolation, characterization and cryopreservation of pluripotent cell lines in livestock, especially the bovine