Enhanced development of mouse single blastomeres into blastocysts via the simultaneous inhibition of TGF-β and ERK pathways in microdroplet culture.

Optimization of an in vitro culture that supports blastocyst (BL) development from single blastomeres (SBs) is essential to generate additional embryos for farm animals and humans and unravel the mechanisms that underlie totipotency. In this study, we have examined BL development from SBs that were derived from 2-cell and 4-cell mouse embryos in different media. Moreover, BLs were assessed for inner cell mass (ICM) by staining with Oct4. We found that BL development was improved in a lower volume of medium (1 µL) compared with a higher volume (5 µL). Furthermore, the supplementation of medium with the inhibitors of ERK1/2 and TGFβ (R2i) signaling pathways in 1 µL droplets of T6 medium improved BL development. The co-culture of SBs with intact embryos in the presence of R2i showed more BL development and ICM to trophectoderm cell number ratio in comparison with SB culture and SB group culture. We also observed reduced total cell number, ICM, and trophectoderm cell numbers in all of the SB culture conditions versus intact embryo development. These findings might facilitate the successful generation of additional embryos for biomedical applications and elucidate the mechanisms that underlie totipotency.