Primed bovine embryonic stem cell lines can be derived at diverse stages of blastocyst development with similar efficiency and molecular characteristics
In this study, we established bovine embryonic stem cell (bESC) lines from early (eBL) and full (BL) blastocysts to determine the efficiency of bESC derivation from an earlier embryonic stage and compare the characteristics of the resulting lines. Using established medium and protocols for derivation of primed bESCs from expanded blastocysts, we derived bESC lines from eBLs and BLs with the same efficiency (4/12 each, 33%). Regardless of original blastocyst stage, bESC lines had a similar phenotype, including differentiation capacity, stable karyotype, and pluripotency marker expression over feeder-free transition and long-term culture. Transcriptome and functional analyses indicated that eBL- and BL-derived lines were in primed pluripotency. We additionally compared RNA-sequencing data from our lines to bovine embryos and stem cells from other recent reports, finding that base medium was the predominant source of variation among cell lines. In conclusion, our results show that indistinguishable bESC lines can be readily derived from eBL and BL, widening the pool of embryos available for bESC establishment. Finally, our investigation points to sources of variation in cell phenotype among recently reported bESC conditions, opening the door to future studies investigating the impact of factors aside from signaling molecules on ESC derivation, maintenance, and performance. Overall design: Bovine embryonic stem cells were derived from early and full blastocysts using the same methods and medium. RNA sequencing was performed on two cell lines/embryo stage at passage 9 (cells maintained on MEF feeders) and 23-36 (after transtion to feeder-free conditons and long-term culture). Pure MEF were also sequenced and any aligned counts to the bovine genome were subtracted from MEF-cultured bESC sample data.