Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA.
Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02142, USA.
Biomaterials. 2019 Sep;216:119244. doi: 10.1016/j.biomaterials.2019.119244. Epub 2019 Jun 7.
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 mechanotransductive 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 peri-implantation development, processes that have been previously inaccessible to study.
在人类胚胎着床后的早期，囊胚中的上胚层（EPI）极化形成一个具有中央腔的囊。然后，EPI 囊的子宫极细胞经历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 EPI 的腔形成或羊膜发生不当可能是导致失败的因素之一。在这里，我们报告了一种新型的 AM 微组织阵列平台，该平台允许定量表型分析 EPI 的腔形成和羊膜发生，并展示了其在胚胎毒性分析中的潜在应用。具体来说，我们使用两步微图案化技术开发了一种基于人多能干细胞（hPSC）的羊膜发生分化方案，以生成具有定义组织大小的规则 AM 微组织阵列。开发了一个计算机辅助分析管道，用于自动处理成像数据并定量分析 AM 微组织的形态和生物学特征。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 的羊膜发生之间存在明显的联系。使用该平台，我们证明了 ROCK 信号通路的药理学抑制，一种重要的机械转导途径，抑制了腔形成，但不会干扰 hPSC 的羊膜发生分化，这表明 AM 形态发生与细胞分化的调控机制是分离的。该 AM 微组织阵列进一步应用于筛选一组临床相关药物，成功检测到它们对早期人类着床后发育过程中腔形成和羊膜发生的差异致畸性。这项工作为毒理学筛选具有潜在临床意义的药物提供了技术平台，以研究它们对早期人类着床后发育过程中腔形成和羊膜发生的影响，这些过程以前是无法研究的。
