160 Derivation and evaluation of bovine embryonic stem cells from early and full blastocyst-stage embryos

Embryonic stem cells (ESCs) are pluripotent cells derived from blastocyst-stage embryos that can be maintained indefinitely while keeping their pluripotency and self-renewal capabilities. The derivation of ESC lines can capture distinct cellular states of pluripotency, which resemble developmental checkpoints of in vivo cells as they progress from being less restricted (i.e. naïve) to more lineage committed (i.e. primed). The recent derivation of primed bovine ESCs (bESCs) from full blastocysts represents a major step in the field of stem cell biology. However, these cells have limited applications because of their primed pluripotent state. In this experiment, we derived bESC lines from early (eBL) and full (BL) blastocysts with the goals of determining derivation efficiency of bESC lines from an earlier embryonic stage and comparing the global transcriptomic profiles and performance characteristics of the resulting cell lines. Bovine embryos were produced in vitro using standard procedures. While eBLs were characterised as embryos where the blastocele had just become visible, BLs had a fully formed cavity and clearly delineated inner cell mass and trophectoderm cells. Upon harvest, embryos were treated with Pronase to remove the zona pellucida and plated in wells that were preseeded with mouse embryonic fibroblasts (MEFs) in NBFR medium (Soto et al. 2021 Sci. Rep. 11, 11045). Outgrowths were cultured at 37°C and 5% CO₂ with daily medium changes and passaged following single-cell dissociation using TrypLE onto fresh MEFs at a 1:1–2 split ratio every 7 days. When ESC derivation was successful, colonies were observed by passage 4. Once established, bESCs were passaged at a 1:5–10 split ratio every 3–4 days. We successfully derived bESC lines from eBLs and BLs at the same efficiency (4/12 and 4/12, 33% each). All cell lines homogeneously expressed the pluripotency markers OCT4 and SOX2 at passage 8 ± 1. One line derived from an eBL was tested and exhibited a normal karyotype at passage 9. RNA-sequencing analysis of 2 eBL- and 2 BL-derived lines did not indicate differential expression of the main pluripotency genes after culture for nine passages in NBFR medium on MEFs. Using a false discovery rate < 0.05 and fold change ≥2, we found that 74 genes were down-regulated and 256 genes were up-regulated in eBL compared with BL cell lines. Preliminary gene ontology analysis indicated enrichment of biological processes related to cell migration, adhesion, and proliferation in BL-derived lines, whereas cell metabolism and signal transduction were enriched in eBL-derived lines. In conclusion, our results show that bESCs can be readily established from eBLs and propagated using methods previously described for BL-derived lines. The resulting cell lines are morphologically similar to bESC lines from BLs and appear to be in a similar state of pluripotency after culture for nine passages under identical conditions. However, the transcriptomic changes observed may indicate underlying functional differences, which we will further investigate. These findings will reveal whether the embryonic stage from which cell lines are derived alters the developmental potential of bESCs.

This research was funded by Genus plc, UC Davis Jastro Shields Award, and USDA GEEAP National Needs Fellowship.