我们最近开发了重新分化的马输卵管上皮细胞(REOEC)单层,证明了各种体内形态特征,但缺乏次要的调解。在这项研究中,我们评估了胎牛血清的效果,生殖类固醇激素,Wnt-和Notch配体和抑制剂,和不同的EOEC播种密度,在常规井和微孔膜上,关于EOEC形态和,特别是,次要调解。在合并细胞核染色后,通过共聚焦显微镜评估REOEC单层,纤毛和细胞骨架。只有Wnt配体,Notch抑制剂和输卵管外植体细胞浓度影响EOEC形态。在暴露于含有Wnt3a的培养基和Wnt配体CHIR99021的REOEC单层中观察到不期望的上皮-间质转化。关于次要调解,只有输卵管外植体细胞浓度和Notch抑制作用的综合作用将REOEC单层引导到体内样的分化模式。去分化的EOEC,输卵管外植体细胞接种后10天形成,重新接种在插入物上;仅在最初的输卵管外植体细胞浓度为每孔1和5×106个细胞时,才形成具有高扩散率支持的REOEC单层。气液接口引入后1个月内,>40%和>20%的REOECs显示继发性纤毛,分别。在较高的输卵管外植体细胞接种密度下,再分化后不支持二次分裂。此外,在REOEC单层中,Notch抑制有助于将次级分化率提高到>60%,仅具有弥漫性分化。这些单层在卵泡期条件下显示出较高的网格蛋白表达。总的来说,纤毛REOEC单层在体内比以前的模型更像输卵管上皮细胞。
We recently developed re-differentiated equine oviduct epithelial cell (REOEC) monolayers demonstrating various in vivo morphological characteristics, but lacking secondary
ciliation. In this study, we evaluated the effects of fetal bovine serum, reproductive steroid hormones, Wnt- and Notch ligands and inhibitors, and different EOEC seeding densities, in both conventional wells and on microporous membranes, on EOEC morphology and, in particular, secondary ciliation. REOEC monolayers were assessed by confocal microscopy after combined staining of nuclei, cilia and the cytoskeleton. Only Wnt ligands, Notch inhibitors and oviduct explant cell concentration affected EOEC morphology. Undesirable epithelial-mesenchymal transition was observed in REOEC monolayers exposed to Wnt3a containing medium and Wnt ligand CHIR 99021. With respect to secondary
ciliation, only the combined effect of oviduct explant cell concentration and Notch inhibition steered REOEC monolayers to in vivo-like
ciliation patterns. De-differentiated EOECs, formed 10 days after oviduct explant cell seeding, were reseeded on inserts; only at initial oviduct explant cell concentrations of 1 and 5 x106 cells per well was the formation of REOEC monolayers with a high rate of diffuse ciliation supported. Within 1 month after air-liquid interface introduction, >40% and > 20% of the REOECs showed secondary cilia, respectively. At higher oviduct explant cell seeding densities secondary ciliation was not supported after re-differentiation. Additionally, Notch inhibition helped boost secondary
ciliation rates to >60% in REOEC monolayers with diffuse
ciliation only. These monolayers demonstrated higher clathrin expression under follicular phase conditions. Overall, the ciliated REOEC monolayers better resemble in vivo oviduct epithelial cells than previous models.