Primary Bovine Extra-Embryonic Cultured Cells: A New Resource for the Study of In Vivo Peri-Implanting Phenotypes and Mesoderm Formation

In addition to nourishing the embryo, extra-embryonic tissues (EETs) contribute to early embryonic patterning, primitive hematopoiesis, and fetal health. These tissues are of major importance for human medicine, as well as for efforts to improve livestock efficiency, but they remain incompletely understood. In bovines, EETs are accessible easily, in large amounts, and prior to implantation. We took advantage of this system to describe, in vitro and in vivo, the cell types present in bovine EETs at Day 18 of development. Specifically, we characterized the gene expression patterns and phenotypes of bovine extra-embryonic ectoderm (or trophoblast; bTC), endoderm (bXEC), and mesoderm (bXMC) cells in culture and compared them to their respective in vivo micro-dissected cells. After a week of culture, certain characteristics (e.g., gene expression) of the in vitro cells were altered with respect to the in vivo cells, but we were able to identify “cores” of cell-type-specific (and substrate-independent) genes that were shared between in vitro and in vivo samples. In addition, many cellular phenotypes were cell-type-specific with regard to extracellular adhesion. We evaluated the ability of individual bXMCs to migrate and spread on micro-patterns, and observed that they easily adapted to diverse environments, similar to in vivo EE mesoderm cells, which encounter different EE epithelia to form chorion, yolk sac, and allantois. With these tissue interactions, different functions arose that were detected in silico and corroborated in vivo at D21–D25. Moreover, analysis of bXMCs allowed us to identify the EE cell ring surrounding the embryonic disc (ED) at D14-15 as mesoderm cells, which had been hypothesized but not shown prior to this study. We envision these data will serve as a major resource for the future in the analysis of peri-implanting phenotypes in response to the maternal metabolism and contribute to subsequent studies of placental/fetal development in eutherians.胚外组织（extra-embryonic tissues, EETs）除为胚胎提供营养外，还参与早期胚胎模式建成、原始造血及胎儿健康的维持。这类组织在人类医学领域与提升畜禽生产性能的研究中均具有重要价值，但目前人们对其认知仍不够全面。在牛体内，胚外组织可在着床前大量且便捷地获取。本研究借助这一优势，对发育第18天的牛胚外组织中的细胞类型进行了体外与体内的系统表征。具体而言，我们分别对培养状态下的牛胚外外胚层（又称滋养层；bTC）、内胚层（bXEC）及中胚层（bXMC）细胞的基因表达谱与细胞表型进行了表征，并将其与体内显微切割获取的对应细胞进行了对比。培养一周后，体外培养细胞的部分特征（如基因表达谱）与体内细胞出现差异，但我们仍成功鉴定出了在体外与体内样本中均存在的细胞类型特异性（且不依赖培养底物）的核心基因集。此外，诸多细胞表型均呈现出细胞类型特异性的细胞外黏附特性。我们评估了单个bXMC细胞在微图案表面的迁移与铺展能力，发现其可轻松适应多种环境，这与体内胚外中胚层细胞的特性一致——体内胚外中胚层细胞可接触不同的胚外上皮，进而形成绒毛膜、卵黄囊与尿囊。借助这类组织间的相互作用，产生了多种功能，这些功能通过计算机模拟分析得到预测，并在D21至D25的体内样本中得到了验证。此外，通过对bXMC细胞的分析，我们确认了D14至D15时期围绕胚盘（embryonic disc, ED）的胚外细胞环为中胚层细胞——这一结论此前仅为假说，尚未被实验证实。我们认为，本研究数据将为未来响应母体代谢的围植入期表型分析提供重要资源，并为后续真兽类动物的胎盘/胎儿发育研究提供助力。