Microengineered human amniotic ectoderm tissue array for high-content developmental phenotyping
Microengineered human amniotic ectoderm tissue array for high-content developmental phenotyping
复制标题
DOI：
10.1016/j.biomaterials.2019.119244
复制
发表时间：
2019-09-01
期刊：
影响因子：
14
通讯作者：
Fu, Jianping
中科院分区：
文献类型：
--
作者：
Esfahani, Sajedeh Nasr;Shao, Yue;Fu, Jianping
在植入后人类胚胎发生的早期，胚泡内的外胚层(EPI)极化，产生一个带有中央管腔的囊。然后，外膜包囊子宫极的细胞经过分化形成羊膜外胚层(AM)，这是进一步胚胎发育所必需的组织。虽然早期妊娠失败的原因很复杂，但外周血腔发生或羊水发生不当是可能的致病因素。在这里，我们报告了一种新的AM微组织阵列平台，该平台允许对外膜蛋白的管腔发生和羊膜形成进行定量表型，并展示了其在胚胎毒性分析中的潜在应用。具体地说，开发了一种基于人类多能干细胞(HPSC)的羊膜分化方案，该方案使用两步微图案化技术来产生具有限定组织尺寸的规则AM微组织阵列。开发了一种计算机辅助分析流水线，用于自动处理AM显微组织的成像数据并对其形态和生物学特征进行量化。对细胞密度、囊泡大小和培养条件的影响分析表明，囊泡大小与hPSC的羊水发生有明显的联系。利用这一平台，我们证明了药物抑制ROCK信号，这是一种重要的机制转导途径，抑制了管腔发生，但不干扰hPSC的羊膜分化，提示AM形态发生与细胞分化的调节机制不耦合。进一步应用AM微组织芯片筛选了一组临床相关药物，成功地检测了它们的差异致畸作用。这项工作为临床相关药物在人类植入后早期发育过程中对管腔形成和羊膜形成的影响进行毒理学筛选提供了一个技术平台，这些过程以前一直无法研究。
During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechan-otransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human pert-implantation development, processes that have been previously inaccessible to study.