Exogenous Yamanaka factors (h-oksm) maintain pluripotency of bovine and porcine ipsc-like cells obtained with stemcca delivery system

KLISCH D; GUBERMAN A; CANIZO J; ALLER ATUCHA J; ALBERIO RH; VAZQUEZ ECHEGARAY C; PAZ D; ALBERIO R

CABA

Congreso; Reunión Conjunta de Biociencias 2017; 2017

The use of induced reprogramming of somatic cells to produce transgenic animals requires the development of a biotechnological platform. In our study, different strategies for the generation of induced pluripotent stem cells (iPSC) of bovine and porcine cells were evaluated. Episomal and lentiviral vectors were used for delivering reprogramming human factors OCT4, SOX2, KLF4 and c-MYC (h-OKSM) into bovine(BEF) and porcine (PEF) embryonic fibroblasts. Cells were cultured in different media containing different supplements: DMEM/F12 with KSR + FGF, E8 medium for culturing on feeder and serum free conditions and finally SB43 medium in which MAPK/ERK, Wnt and TGF pathways are modulated. Episomal vectors (1x, 2x and 4x runs of transfections) failed to generate iPSCs in both species. In these cultures, we detected a gradual loss of expression of exogenous factors. On the other hand, protocols based on the integrative lentiviral vector, STEMCCA, produced porcine iPSC-like cells more efficiently than from bovine cells. The iPSC generated maintained stem cells features and expression of exogenous factors. Inactivation of STEMCCA is still a bottleneck for establishing bonafide iPSC, since culture media tested did not probably maintain endogenous OKSM expression.