3 Derivation of bovine trophoblast stem cells

Placental trophoblasts play an essential role in communication between the fetus and mother. In the bovine, inadequate placental trophoblast development and subsequent dysfunction results in a range of adverse outcomes in conceptus/offspring; for example, the abnormalities seen in IVF or somatic cell nuclear transfer embryos. The most significant barrier to progress in this field in the bovine is the lesser feasibility of an in vivo experimental system or the lack of manipulatable in vitro cell culture models that recapitulate placental cell differentiation. To date, trophoblast stem cells (TSC) have been established from various species, including mouse, human, and nonhuman primates, but the generation of self-renewal and stable bovine TSC lines remains unexplored. Building upon signalling required for mouse and human TSC pluripotency, we screened 11 culture conditions and found that a culture condition containing a chemical cocktail of human leukemia inhibitory factor (hLIF), CHIR99021 (an inhibitor of glycogen synthase kinease-3 (GSK-3)), DiM (antagonist of muscarinic M2 and histamine H1 receptors), and MiH (inhibitor of matrix metalloproteinase (MMP)) enables the long-term culture (over 55 passages) of bovine TSC without altered morphology and differentiation from bovine IVF embryos. Three stable cell lines were maintained and used for downstream characterisations (n = 3). Real-time quantitative (qRT)-PCR and immunostaining assays showed that resulting cells highly express bovine trophectoderm markers including CDX2, GATA3, and KRT8. These cells had the capacity to differentiate into multinuclear trophoblast cells in vitro that secreted IFN-τ and expressed high levels of binuclear trophoblast cell marker genes: PAG2, PAG11, PAG12, and uninucleate trophoblast cell marker genes: CYP17A1, HSD3B1, and HAND1. To investigate the pluripotency state of bovine TSCs and to map bovine trophoblast differentiation, we generated transcriptomes and accessible chromatin of in vitro trophoblast cell cultures including TSCs, multinuclear cells from in vitro differentiation at Day 2 and Day 6, and trophoblast lineages including trophectoderm (TE) of Day 7 IVF blastocysts, and trophoblast cells of Day 14 elongating embryos by RNA-seq and assay for transposase-accessible chromatin (ATAC)-seq, respectively. Sequencing analysis were performed with at least two replicates per development stage (n ≥ 2). Transcriptomes of bovine pluripotent stem cell models that represent epiblasts, prime bovine embryonic stem cells (ESC) and expanded potential stem cells (EPSC) were also compared to identify the bovine trophoblast stem niche. Our analysis revealed important transcriptional factors (e.g. GATA3, ELF3, TFAP2A, KLF5, KRT8, SFN, DNMT1 and DNMT3A; adjusted P < 0.05) and signalling pathways (Wnt, LIF, HIF-1 and AMPK signalling pathway; adjusted P < 0.05) required for capturing bovine TSCs state, and reconstructed molecular trajectories of bovine placental trophoblast development. The bovine TSC we established in this study will provide a powerful model to study bovine early placental establishment and early pregnancy failure.