Graduate Student Chungbuk National University, Republic of Korea
Abstract: Blastoids are three-dimensional (3D) structures resembling blastocysts, serving as ideal models for ethically challenging embryo studies. Unlike rodents, pigs exhibit greater developmental similarity to humans, making porcine blastoids a valuable model for studying early developmental mechanisms. In this study, we aimed to establish the optimal culture medium for porcine blastoid formation using primed-state porcine embryonic stem cells (pESCs) and to generate blastocyst-like structures through 3D culture. pESCs were cultured in media containing different combinations of factors to identify the optimal medium composition. pESCs cultured in FAB5i/X medium supplemented with FGF2, Activin A, BMP4, and small-molecule inhibitors targeting SRC, GSK3, TGFβ, ROCK, HDAC, and WNT signaling differentiated into the three distinct lineages of the blastocyst, namely epiblast, hypoblast, and trophoblast, as demonstrated by qPCR and immunofluorescence. This finding established FAB5i/X as the optimal medium for porcine blastoid formation. Utilizing the FAB5i/X medium, pESCs were cultured in AggreWell for approximately five days, resulting in the generation of a 3D structure closely resembling natural embryos, as confirmed through immunostaining. Future studies will aim to evaluate the efficiency of blastoid formation across various embryonic stem cell types and develop strategies for long-term culture. This research offers valuable insights into porcine embryo development, expands the possibilities for embryo-related experimental approaches, and contributes to advancing the field of early developmental biology.
Funding Source: funded by the Korean Government (2020R1A2C2008276, NRF-2021R1C1C2007132), funded by the MOTIE, Korea (20023068), funded by MAFRA (RS-2024-00398561, RS-2024-00399475)
