Generation of Rat Embryonic Germ Cells Via Inhibition of Tgfb and Mek Pathways

Objective: embryonic germ (eg) cells are the results of reprogramming primordialgerm cells (pgc) in vitro. studying these cells can be of benefit in determining themechanism by which specialized cells acquire pluripotency. therefore in the currentstudy we have tried to derive rat eg cells with inhibition of transforming growthfactor-b (tgfb) and mitogen-activated protein kinase kinase (mek) signaling pathways.materials and methods: in this experimental study, rat pgcs were cultured underfeeder free condition with two small molecules that inhibited the above mentionedpathways. under this condition only two-day presence of stem cell factor (scf) as asurvival factor was applied for pgc reprogramming. pluripotency of the resultant egcells were further confirmed by immunofluorescent staining, directed differentiationability to neural and cardiac cells, and their contribution to teratoma formation as well.moreover, chromosomal stability of two different eg cells were assessed through gbandingtechnique.results: formerly, derivation of rat eg cells were observed solely in the presence of glycogensynthase kinase-3 (gsk3b) and mek pathway inhibitors. due to some drawbacksof inhibiting gsk3b molecules such as increases in chromosomal aberrations, in the presentstudy we have attempted to assess a feeder-free protocol that derives eg cells bythe simultaneous suppression of tgfb signaling and the mek pathway. we have shownthat rat eg cells could be generated in the presence of two inhibitors that suppressed theabove mentioned pathways. of note, inhibition of tgfb instead of gsk3b significantlymaintained chromosomal integrity. the resultant eg cells demonstrated the hallmarks ofpluripotency in protein expression level and also showed in vivo and in vitro differentiationcapacities.conclusion: rat eg cells with higher karyotype stability establish from pgcs by inhibitingtgfb and mek signaling pathways.