Investigating alterations in bovine embryonic pluripotency through scRNA-seq and establishing robust Formative stem cell lines

Bovine, as one of the most significant domestic animals providing humans with milk and meat, while also serving as bioreactors for producing valuable proteins, pose challenges in establishing embryo-derived stable pluripotent stem cells (PSCs) due to the unclear relationship between embryonic epiblast development and maintenance of stem cell pluripotency and self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, E14) to track pluripotency changes and their signal pathway dependence using modified single-cell transcription sequencing technology. Based on the remarkable similarity of the dependence of WNT/β-catenin, LIF/STAT3, TGFβ/Smads, and FGF/ERK signaling pathways between bovine and porcine during early embryonic lineages development, we have successfully established bovine embryonic epiblast stem cells (bEpiSCs) using porcine pre-gastrulation epiblast stem cell culture system 3i/LAF. The generated bEpiSCs exhibited consistent pluripotency gene expression levels and maintained clonal morphology, normal karyotype, and proliferative capacity for over 110 passages. Moreover, their high-efficiency teratoma formation potential as well as their ability to differentiate into various cell lineages were demonstrated. At the transcriptome level, bEpiSCs displayed similarities with bovine Formative epiblast cells. We also assessed the application potential of bEpiSCs in targeted differentiation towards muscle cells and as donor cells for somatic cell nuclear transfer (SCNT). In summary, this study establish stable Formative bEpiSCs which hold promise for advancements in cell-cultured meat production, gene-edited cloned cattle generation, and animal breeding. To investigate the dynamic changes of early bovine embryo development and the characteristics of bovine embryonic stem cells, we constructed the bovine early embryonic single cell dataset, the bovine embryonic stem cell single cell dataset and the transcriptome dataset The STRT-seq samples are multplexed so each library contains cells of different developmental stages. Please see the file cell_meta.xlsx available at the foot of this record to find the developmental stage for each cell ID and cell barcode.