234 Bovine blastocyst-like structures derived from pluripotent stem cell cultures

Understanding the molecular mechanisms that underline blastocyst formation and implantation is critical for improving the efficiency of assisted reproductive technologies. Recent advancements in in vitro embryo models derived from human and mouse stem cell cultures have opened new avenues for understanding the mechanistic insights into early lineage segregation and implantation. Here, we have developed two strategies for the generation of blastocyst-like structures (blastoids) from an ungulate species, Bos taurus: (1) 3D differentiation and self-organisation (SO) directly from naïve-like bovine embryonic stem cells (bESCs), and (2) 3D assembly (AS) of bovine trophoblast stem cells (bTSCs) and naïve-like bovine ESCs. These naïve-like cells are cultured with activators of the AKT and WNT pathways to promote proliferation and inhibitors of the Ras-Raf-Mek-ERK/MAPK, SRC, and PARP pathways to inhibit differentiation. These blastoids can be generated with high efficiency (AS ∼98%, SO ∼80% blastocele expansion) and resemble bovine blastocysts in terms of morphology, size, cell number, lineage composition, and allocation, as revealed by immunostaining of epiblast (SOX2), hypoblast (SOX17), and trophectoderm (CDX2, AP-2α, AP-2γ, KRT18, ZOI) markers. The SO blastoids were significantly smaller and more heterogenous on day 8 after assembly than AS blastoids, indicating a growth delay in SO over AS protocol. In addition, the extended culture of blastoids in in vitro growth media resulted in trophectoderm and blastocele expansion up to ∼5 mm in diameter, comparable to IVF blastocyst controls. To evaluate the developmental competency, we transferred IVF blastocysts or blastoids into synchronised surrogates. We were able to detect the maternal recognition of pregnancy hormone Interferon-Tau (IFN-τ) in the serum of surrogate cows via ELISA assay in IVF embryos (78.36 ± 21.54 pm/mL, n = 2), assembled (56.53 ± 25.13 pm/mL, n = 2) and self-organised (74.47 pm/mL, n = 1) blastoids 8 days after transfer. By testing different signalling pathways using agonists or inhibitors, we have shown that WNT, Hippo, Ras-Raf-Mek-ERK/MAPK, and Activin/TGF-β/SMAD pathways play essential roles in the differentiation and segregation of the bovine trophectoderm, epiblast, and hypoblast lineages from naïve-like bovine ESCs. Overall, our findings show that bovine ESCs and TSCS can utilise external cues and inductive signals to self-organise into blastocyst-like structures in a scalable and high-throughput manner, creating an in vitro platform to study early bovine embryogenesis.

We thank Dr Joel Carter from J A Carter, CETS, LLC, for his assistance with embryo transfer. We also thank our funding sources, the Hamon Center for Regenerative Science and Medicine, the New York Stem Cell Foundation, the Cancer Prevention & Research Institute of Texas, the Welch Foundation, the United States Department of Agriculture (2019-67016-29863), the National Institutes of Health (R01HD102533), and UT Southwestern & Texas A&M clinical translation and translational award program.