Stress Forces First Lineage Differentiation of Mouse Embryonic Stem Cells; Validation of a High-Throughput Screen for Toxicant Stress.
Stress Forces First Lineage Differentiation of Mouse Embryonic Stem Cells; Validation of a High-Throughput Screen for Toxicant Stress.
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DOI：
10.1089/scd.2018.0157
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发表时间：
2019-01
影响因子：
4
通讯作者：
Quanwen Li;E. Louden;Jordan Z Zhou;S. Drewlo;Jing Dai;E. Puscheck;Kang Chen;D. Rappolee
中科院分区：
文献类型：
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作者：
Quanwen Li;E. Louden;Jordan Z Zhou;S. Drewlo;Jing Dai;E. Puscheck;Kang Chen;D. Rappolee
小鼠胚胎干细胞（mESC）具有独特的自我更新和多能性。假设，mESC模型妊娠应激效应或体外受精/辅助生殖技术或药物/环境暴露的压力，危及胚胎。测试mESCs应激反应应该减少和加快体内胚胎筛选。用于分化的绿色荧光蛋白（GFP）报告基因的转基因mESC使用血小板衍生生长因子受体（Pdgfr）的启动子（一种驱动GFP表达的启动子）来监测高渗应激迫使的mESC增殖减少（发育迟缓）和分化增加（进一步阻碍mESC群体生长）。在分化mESC中，Pdgfra标记第一谱系胚外原始内胚层（ExEndo）。高渗胁迫迫使mESC分化增益（Pdgfra-GFP）在单层或三维胚状体。尽管与维持效力的白血病抑制因子（LIF）一起培养，但应力迫使ExEndo，如使用酶标仪测定的，并通过免疫荧光共表达Pdgfra-GFP、Disabled 2（Dab 2）和层粘连蛋白以及通过免疫印迹共表达GFP蛋白和Dab 2来验证。与以前的报道一致，尽管LIF，但用高渗山梨醇处理后，Rex 1和Oct 4的损失与Pdgfra-GFP mESC的增加成反比。Pdgfra-GFP+细胞亚群>背景的增加在0.23%与先前报道的在匹配的高山梨醇剂量下Rex 1-红色荧光蛋白（RFP）阴性亚群增加0.25%相似。通过酶标仪，与单独的LIF相比，在高非病态剂量和病态剂量下，尽管有LIF，GFP仍增加了107 -11倍。通过流式细胞术（FACS），Pdgfra-GFP+细胞>背景的亚群在这些剂量下增加了18 -16倍。两者合计，微孔板，流式细胞仪，免疫印迹，免疫荧光数据表明，视黄酸或高渗应力的力量剂量依赖性分化是否LIF存在，这是负相关的，并可能补偿应力迫使减少ESC人口扩张和效力损失。
Mouse Embryonic Stem Cells (mESCs) are unique in their self-renewal and pluripotency. Hypothetically, mESCs model gestational stress effects or stresses of in vitro fertilization/assisted reproductive technologies or drug/environmental exposures that endanger embryos. Testing mESCs stress responses should diminish and expedite in vivo embryo screening. Transgenic mESCs for green fluorescent protein (GFP) reporters of differentiation use the promoter for platelet-derived growth factor receptor (Pdgfr)a driving GFP expression to monitor hyperosmotic stress-forced mESC proliferation decrease (stunting), and differentiation increase that further stunts mESC population growth. In differentiating mESCs Pdgfra marks the first-lineage extraembryonic primitive endoderm (ExEndo). Hyperosmotic stress forces mESC differentiation gain (Pdgfra-GFP) in monolayer or three-dimensional embryoid bodies. Despite culture with potency-maintaining leukemia inhibitory factor (LIF), stress forces ExEndo as assayed using microplate readers and validated by coexpression of Pdgfra-GFP, Disabled 2 (Dab2), and laminin by immunofluorescence and GFP protein and Dab2 by immunoblot. In agreement with previous reports, Rex1 and Oct4 loss was inversely proportional to increased Pdgfra-GFP mESC after treatment with high hyperosmotic sorbitol despite LIF. The increase in subpopulations of Pdgfra-GFP+ cells>background at ∼23% was similar to the previously reported ∼25% increase in Rex1-red fluorescent protein (RFP)-negative subpopulation at matched high sorbitol doses. By microplate reader, there is a ∼7-11-fold increase in GFP at a high nonmorbid and a morbid dose despite LIF, compared with LIF alone. By flow cytometry (FACS), the subpopulation of Pdgfra-GFP+ cells>background increases ∼8-16-fold at these doses. Taken together, the microplate, FACS, immunoblot, and immunofluorescence data suggest that retinoic acid or hyperosmotic stress forces dose-dependent differentiation whether LIF is present or not and this is negatively correlated with and possibly compensates for stress-forced diminished ESC population expansion and potency loss.