1 State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestocks, Inner Mongolia University, Hohhot, China.
2 Department of Urology, Inner Mongolia Autonomous Region People's Hospital, Hohhot, China.
DNA Cell Biol. 2019 May;38(5):410-422. doi: 10.1089/dna.2018.4458. Epub 2019 Mar 21.
Trophoblast stem cells (TSCs), the precursors of placental cells, are effective for studying placental formation . Using a dual inhibition (2i) medium and mixed L-Wnt3a/mouse embryonic fibroblast feeder cells, we previously established the bovine trophoblast cell line BTS-1. In this study, we used bovine fetal fibroblasts and added Wnt3a to the 2i medium to establish another bovine TSC line (BTSW). BTSW cells expressed pluripotency markers, including , , , , , , , and , and TSC markers , , and . Methylation sequencing of the promoter regions of , , and revealed no significant differences between BTS-1 and BTSW cells. Removal of Wnt3a from the culture medium resulted in downregulation ( < 0.05) of , , , and TSC marker genes, and upregulation of TSC differentiation markers, including , , and . Western blotting indicated activation of the WNT-YAP/TAZ signaling pathway in BTS-1 and BTSW cells, consequently activating transcription. However, this pathway was not activated in BCFF cells, an established bovine embryonic stem-like cell line that expresses , , and , but not . Thus, Wnt3a may play a critical role in bovine TSC maintenance by activating and regulating expression through the WNT-YAP/TAZ signaling pathway.
滋养层干细胞（TSC）是胎盘细胞的前体细胞，对于研究胎盘形成非常有效。我们之前使用双重抑制（2i）培养基和混合的 L-Wnt3a/小鼠胚胎成纤维细胞饲养细胞，建立了牛滋养层细胞系 BTS-1。在这项研究中，我们使用牛胎儿成纤维细胞，并在 2i 培养基中添加 Wnt3a，建立了另一种牛 TSC 系（BTSW）。BTSW 细胞表达多能性标记物，包括 、 、 、 、 、 、和 ，以及 TSC 标记物 、 、和 。对 BTS-1 和 BTSW 细胞的 、 和 启动子区域的甲基化测序没有发现明显差异。从培养基中去除 Wnt3a 导致 、 、 和 TSC 标记基因的下调（ < 0.05），同时 TSC 分化标记物，包括 、 、和 的上调。Western blot 分析表明，BTS-1 和 BTSW 细胞中的 WNT-YAP/TAZ 信号通路被激活，从而激活 转录。然而，在 BCFF 细胞中，该通路没有被激活，BCFF 细胞是一种已建立的牛胚胎样细胞系，表达 、 、和 ，但不表达 。因此，Wnt3a 可能通过激活和调节 WNT-YAP/TAZ 信号通路中的 表达，在牛 TSC 的维持中发挥关键作用。
