effects of klf4 and c-myc knockdown on pluripotency maintenance in porcine induced pluripotent stem cell

Objective: the importance of oct4 and sox2 in maintaining pluripotency and self-renewal is well-understood, but the functions of klf4 and c-myc has not been fully investigated. in the present study, we attempted to determine the roles of klf4 and c-myc on pluripotency maintenance of porcine induced pluripotent stem (pips) cells. materials and methods: in this experimental study, we performed short hairpin rna (shrna) to knock down the klf4 and c-myc functions of pips cells and examined pluripotency markers and teratoma formation to evaluate pips cell pluripotency. the shrna-klf4 and shrna-c-myc vectors containing a reporter gene, tagfp635, were transfected into pips cells by lentivirus infection. the pips cells fully expressing infrared fluorescence were selected to confirm gene knockdown of klf4 and c-myc reverse transcription-polymerase chain reaction (rt-pcr). next, for pluripotency evaluation, expression of pluripotency markers was detected by immunocytochemical staining, and capability of teratoma formation was investigated by pips cell transplantation into nonobese diabetic-severe combined immunodeficiency (nod-scid) mice. results: our findings indicated that klf4 and c-myc functions of pips cells were knocked down by shrna transfection, and knockdown of klf4 and c-myc functions impaired expression of pluripotency markers such as oct4, ap, ssea-3, ssea-4, tra-1-6, and tra-1-81. furthermore, pips cells without klf4 and c-myc expression failed to form teratomas. conclusion: the pluripotency of pips cells are crucially dependent upon klf4 and c-myc expression. these findings, suggesting potential mechanisms of klf4 and c-myc contribution to pips cell formation, have important implications for application, regulation, and tumorigenesis of pips cells.