Production Improvement of Derived Haploid Mouse Embryonic Stem Cells from Parthenogenetic Mouse Blastocysts by Simultaneous Suppression of TGF-Β and ERK Signaling Pathways

Aim: In this project, production of haploid parthenogenetic embryonic stem cells has been studied in presence of the ERK and TGF-β signaling pathway inhibitors.
Material and First the parthenogenetic blastocyst- produced with chemical activation of oocytes- was cultured in R2i culture condition (containing PD0325901 and SB431542 which inhibit ERK and TGF-β signaling pathways respectively). Then parthenogenetic embryonic stem cell lines was subsequently expanded and preserved in the same culture medium. Using immunofluorescent staining and qRT-PCR, expression of the pluripotent markers were evaluated. Moreover, differentiation potential of these cells was assessed by spontaneous differentiation and teratoma formation assay. Degree of haploid in this cell lines was determined by flowcytometry methods.
In R2i culture condition, about 50% of parthenogenetic embryos produce cell lines, out of which 10-13% are haploid. Established parthenogenetic stem cell lines demonstrate pluripotent stemness characteristics including morphology, passagability as well as expression of pluripotent specific genes in mRNA and protein levels. In addition, these cells were able to differentiate spontaneously to embryonic germ layers and form teratoma.
Inhibition of ERK and TGF-β signaling pathways can provide an appropriate condition for producing haploid stem cells from parthenogenetic embryos.