背景:培养的小鼠滋养层干细胞(mTSC)在FGF4下保持增殖/正常干性(NS),导致正常分化(ND)。缺氧,或高渗压力迫使滋养细胞巨细胞(TGC)分化。缺氧,高渗,和基因毒性苯并(a)芘(BaP),在烟草烟雾中发现,强制下调分化抑制剂(Id)2,使TGC分化。低氧和高渗胁迫通过SAPK依赖性HAND1增加诱导TGC。在这里,我们测试BaP是否在诱导SAPK和HAND1的同时强制mTSC到TGC。
方法:用免疫印迹法检测Hand1和SAPK活性,在接触BaP72小时后,在Tfinal测定mTSC到TGC的生长和分化,NS,ND,维甲酸(RA),或山梨糖醇。通过实时成像仪自动对细胞核染色的细胞进行显微成像,并由ImageJ/FIJI分析,BiotekGen5,AIVIA专有人工智能(AI)软件或开源,CellPose人工智能/AI软件。
结果:BaP(0.05-1μM)激活了SAPK和HAND1,而不减少生长。对于2-4N循环核和>4N分化的TGC核,TSC到TGC的分化越来越准确,使用ImageJ/FIJI,第五代,AIVIA,或CellPoseAI软件。AIVIA和CellposeAI软件与人类的准确性相匹配。BaP对SAPK激活/HAND1增加的最低影响比mESC的类似影响敏感>10倍。RA诱导44-47%的第一谱系TGC分化,但相同的RA剂量仅诱导1%的第一谱系mESC分化。
结论:首先,这些试验数据表明mTSC可用于高通量筛选(HTS),以预测迫使TGC分化的毒物暴露.第二,对于类似的应激暴露,mTSC比mESC分化更多的细胞,第三,开源AI可以取代人类显微照片量化,并实现预测流产的HTS。
Cultured mouse trophoblast stem cells (mTSC) maintain proliferation/normal stemness (NS) under FGF4, which when removed, causes normal differentiation (ND). Hypoxic, or hyperosmotic stress forces trophoblast giant cells (TGC) differentiate. Hypoxic, hyperosmotic, and genotoxic benzo(a)pyrene (BaP), which is found in tobacco smoke, force down-regulation of inhibitor of differentiation (Id)2, enabling TGC differentiation. Hypoxic and hyperosmotic stress induce TGC by SAPK-dependent HAND1 increase. Here we test whether BaP forces mTSC-to-TGC while inducing SAPK and HAND1.
Hand1 and SAPK activity were assayed by immunoblot, mTSC-to-TGC growth and differentiation were assayed at Tfinal after 72hr exposure of BaP, NS, ND, Retinoic acid (RA), or sorbitol. Nuclear-stained cells were micrographed automatically by a live imager, and assayed by ImageJ/FIJI, Biotek Gen 5, AIVIA proprietary artificial intelligence (AI) software or open source, CellPose artificial intelligence/AI software.
BaP (0.05-1μM) activated SAPK and HAND1 without diminishing growth. TSC-to-TGC differentiation was assayed with increasingly accuracy for 2-4 N cycling nuclei and >4 N differentiating TGC nuclei, using ImageJ/FIJI, Gen 5, AIVIA, or CellPose AI software. The AIVIA and Cellpose AI software matches human accuracy. The lowest BaP effects on SAPK activation/HAND1 increase are >10-fold more sensitive than similar effects for mESC. RA induces 44-47% 1st lineage TGC differentiation, but the same RA dose induces only 1% 1st lineage mESC differentiation.
First, these pilot data suggest that mTSC can be used in high throughput screens (HTS) to predict toxicant exposures that force TGC differentiation. Second, mTSC differentiated more cells than mESC for similar stress exposures, Third, open source AI can replace human micrograph quantitation and enable a miscarriage-predicting HTS.