Russell Ryan P, Fu Yu, Liu Yaling, Maye Peter
Department of Reconstructive Sciences, School of Dental Medicine, University of Connecticut Health Center, United States.
Department of Reconstructive Sciences, School of Dental Medicine, University of Connecticut Health Center, United States.
Stem Cell Res. 2018 Jul;30:85-95. doi: 10.1016/j.scr.2018.05.016. Epub 2018 May 22.
We have investigated the differentiation of paraxial mesoderm from mouse embryonic stem cells utilizing a Tbx6-EYFP/Brachyury (T)-Cherry dual reporter system. Differentiation from the mouse ESC state directly into mesoderm via Wnt pathway activation was low, but augmented by treatment with AGN193109, a pan-retinoic acid receptor inverse agonist. After five days of differentiation, T cells increased from 12.2% to 18.8%, Tbx6 cells increased from 5.8% to 12.7%, and T/Tbx6 cells increased from 2.4% to 14.1%. The synergism of AGN193109 with Wnt3a/CHIR99021 was further substantiated by the increased expression of paraxial mesoderm gene markers Tbx6, Msgn1, Meox1, and Hoxb1. Separate to inverse agonist treatment, when mouse ESCs were indirectly differentiated into mesoderm via a transient epiblast step the efficiency of paraxial mesoderm formation markedly increased. Tbx6 cells represented 65-75% of the total cell population after just 3 days of differentiation and the expression of paraxial mesoderm marker genes Tbx6 and Msgn increased over 100-fold and 300-fold, respectively. Further evaluation of AGN193109 treatment on the indirect differentiation protocol suggested that RARs have two distinct roles. First, AGN193109 treatment at the epiblast step and mesoderm step promoted paraxial mesoderm formation over other mesoderm and endoderm lineage types. Second, continued treatment during mesoderm formation revealed its ability to repress the maturation of presomitic mesoderm into somitic paraxial mesoderm. Thus, the continuous treatment of AGN193109 during epiblast and mesoderm differentiation steps yielded a culture where ~90% of the cells were Tbx6. The surprisingly early effect of inverse agonist treatment at the epiblast step of differentiation led us to further examine the effect of AGN193109 treatment during an extended epiblast differentiation protocol. Interestingly, while inverse agonist treatment had no impact on the conversion of ESCs into epiblast cells based on the expression of Rex1, Fgf5, and pluripotency marker genes Oct4, Nanog, and Sox2, after three days of differentiation in the presence of AGN193109 caudal epiblast and early paraxial mesoderm marker genes, T, Cyp26a1, Fgf8, Tbx6 and Msgn were all highly up-regulated. Collectively, our studies reveal an earlier than appreciated role for RARs in epiblast cells and the modulation of their function via inverse agonist treatment can promote their differentiation into the paraxial mesoderm lineage.
我们利用Tbx6-EYFP/短尾（T）-Cherry双报告系统研究了小鼠胚胎干细胞中轴旁中胚层的分化。通过Wnt信号通路激活从小鼠胚胎干细胞状态直接分化为中胚层的效率较低，但用全反式维甲酸受体反向激动剂AGN193109处理可增强这种分化。分化五天后，T细胞从12.2%增加到18.8%，Tbx6细胞从5.8%增加到12.7%，T/Tbx6细胞从2.4%增加到14.1%。轴旁中胚层基因标志物Tbx6、Msgn1、Meox1和Hoxb1表达的增加进一步证实了AGN193109与Wnt3a/CHIR99021的协同作用。与反向激动剂处理不同，当小鼠胚胎干细胞通过短暂的上胚层步骤间接分化为中胚层时，轴旁中胚层形成的效率显著提高。仅分化3天后，Tbx6细胞就占总细胞群的65-75%，轴旁中胚层标志物基因Tbx6和Msgn的表达分别增加了100倍和300倍以上。对AGN193109处理间接分化方案的进一步评估表明，视黄酸受体有两个不同的作用。第一，在上胚层步骤和中胚层步骤用AGN193109处理可促进轴旁中胚层形成，而不是其他中胚层和内胚层谱系类型。第二，在中胚层形成过程中持续处理显示其能够抑制前体中胚层成熟为体节轴旁中胚层。因此，在上胚层和中胚层分化步骤中持续用AGN193109处理产生了一种培养物，其中约90%的细胞为Tbx6。反向激动剂在分化的上胚层步骤中令人惊讶的早期作用促使我们进一步研究在延长的上胚层分化方案中AGN193109处理的效果。有趣的是，虽然基于Rex1、Fgf5以及多能性标志物基因Oct4、Nanog和Sox2的表达，反向激动剂处理对胚胎干细胞转化为上胚层细胞没有影响，但在AGN193109存在下分化三天后，尾侧上胚层和早期轴旁中胚层标志物基因T、Cyp26a1、Fgf8、Tbx6和Msgn均高度上调。总的来说，我们的研究揭示了视黄酸受体在上胚层细胞中的作用比之前认为的更早，并且通过反向激动剂处理调节其功能可以促进它们分化为轴旁中胚层谱系。
