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