86 Effects of growth factors in N2B27 basal medium on porcine embryonic development and lineage segregation

N2B27 is a chemically defined medium used for stem cell culture and neural differentiation. Recently, N2B27 has been applied to embryo culture. Previous studies have investigated the effects of adding growth factors to N2B27 in bovine and ovine models; however, studies in porcine models are lacking. Therefore, we aimed to determine whether culturing porcine embryos in N2B27 supplemented with growth factors enhances embryonic development. Parthenogenetic embryos were cultured in Porcine Zygote Medium 3 (PZM3) until Day 4. From Day 4 to Day 7, we compared three conditions: (1) PZM3 alone (P, n = 34), (2) PZM3 supplemented with 10% FBS (P+F, n = 25), and (3) N2B27 medium (N4, n = 41). Statistical analyses were performed using one-way ANOVA with Dunnett’s test for parametric data and the Kruskal-Wallis test with Dunn’s test for nonparametric data. Data are presented as means ± s.e.m. N2B27 demonstrated a significant increase in total cell numbers (P, 65.79 ± 4.191; P+F, 68.08 ± 6.978; N4, 172.6 ± 10.24), SOX17-positive cells (P, 0.7600 ± 0.3070; P+F, 4.650 ± 1.094; N4, 14.72 ± 2.690), and CDX2-positive cells (P, 45.14 ± 5.206; P+F, 53.38 ± 5.207; N4, 127.9 ± 12.60) compared with P and P+F. For SOX2-positive cells, N2B27 exhibited a significant increase compared with P (P, 5.294 ± 0.9991; P+F, 10.38 ± 1.781; N4, 12.91 ± 2.074). Next, we investigated the role of growth factors in regulating embryonic development in N2B27. Among the nontreated group (N, n = 32), FGF2-treated group (F, n = 31), and BMP4-treated group (B, n = 27), SOX17-positive cells were significantly higher in the F and B groups than in the N group (N, 17.33 ± 2.829; F, 30.33 ± 2.321; B, 37.25 ± 6.458). Taken together, the data show that transferring embryos to N2B27 on Day 4 resulted in increased total cell numbers, SOX2-positive cells (epiblast), SOX17-positive cells (hypoblast), and CDX2-positive cells (trophoblast), compared with PZM3 or supplemented with FBS. Furthermore, supplementation with FGF2 and BMP4 increased SOX17-positive cells. These results suggest that N2B27 supports embryonic development effectively, while FGF2 and BMP4 can induce hypoblast differentiation. Our findings provide insights into early embryonic development and lineage segregation.

This work was supported by the BK21 Four program, the Korea Evaluation Institute of Industrial Technology (KEIT) through the Alchemist project funded by the Ministry of Trade, Industry and Energy (MOTIE; 20012411), and the National Research Foundation of Korea (NRF) grant funded by the government of Republic of Korea (NRF-2023R1A2C1005026).