Generation of Rat Embryonic Germ Cells via Inhibition of TGFß and MEK Pathways

Abstract

Objective: Embryonic germ (EG) cells are the results of reprogramming primordial germ cells (PGC) in vitro. Studying these cells can be of benefit in determining the mechanism by which specialized cells acquire pluripotency. Therefore in the current study we have tried to derive rat EG cells with inhibition of transforming growth factor-β (TGFβ) and mitogen-activated protein kinase kinase (MEK) signaling pathways. Materials and Methods: In this experimental study, rat PGCs were cultured under feeder free condition with two small molecules that inhibited the above mentioned pathways. Under this condition only two-day presence of stem cell factor (SCF) as a survival factor was applied for PGC reprogramming. Pluripotency of the resultant EG cells were further confirmed by immunofluorescent staining, directed differentiation ability to neural and cardiac cells, and their contribution to teratoma formation as well. Moreover, chromosomal stability of two different EG cells were assessed through Gbanding technique. Results: Formerly, derivation of rat EG cells were observed solely in the presence of glycogen synthase kinase-3 (GSK3β) and MEK pathway inhibitors. Due to some drawbacks of inhibiting GSK3β molecules such as increases in chromosomal aberrations, in the present study we have attempted to assess a feeder-free protocol that derives EG cells by the simultaneous suppression of TGFβ signaling and the MEK pathway. We have shown that rat EG cells could be generated in the presence of two inhibitors that suppressed the above mentioned pathways. Of note, inhibition of TGFβ instead of GSK3β significantly maintained chromosomal integrity. The resultant EG cells demonstrated the hallmarks of pluripotency in protein expression level and also showed in vivo and in vitro differentiation capacities. Conclusion: Rat EG cells with higher karyotype stability establish from PGCs by inhibiting TGFβ and MEK signaling pathways