Investigating the effect of alcohol exposure on human cortical development using a 3D in vitro model

项目总结 怀孕期间饮酒会导致各种发育缺陷，包括认知缺陷 赤字。胎儿酒精谱系障碍(FASD)患者中枢神经系统损害 通过非侵入性成像和行为评估进行了密集的特征。在体外研究和 动物模型提供了酒精引起的分子反应的快照，而模型系统不提供 完全模仿人脑漫长的孕期发育。这项提案旨在开发一种3D体外模型 用诱导多能细胞(IPSC)来源的皮质球体研究乙醇对皮质的影响 发展，特别是在外室下区(OSVZ)。OSVZ被认为是一个进化的 人类皮质新生的适应性变化以增加祖细胞群体、神经元的多样性 产量和大脑大小。我们建议在定义的， 一种促进分化的培养基经优化，可用于酒精和其他产生 反应性氧化应激。球体中分层结构的发展是由我们的 一种新的方案模拟了人类皮质发生的早期过程，其中包括OSVZ样的形成 结构。我们将首先匹配IPSC衍生的皮质球体的发育时间表 以人胎脑免疫组织化学和RNA测序为窗口，明确体外培养 FASD研究(目标1)。然后，我们将在含乙醇的培养液中培养IPSC衍生的皮质球体 用于模拟不同时间段的产前暴露。我们将评估乙醇对大脑皮层的影响 通过监测细胞死亡、形态变化、细胞增殖和祖细胞的发育 人口，重点是小岛屿发展中国家(目标2)。最后，利用CRISPR/Cas9基因组编辑技术，我们 将展示我们的新型皮质球体系统在分子研究中的适用性 人脑发育过程中酒精介导的细胞病理机制。我们将选择 RBM39作为我们实验室筛选的目的基因，并在小鼠皮质中确定了其功能。 这项研究不仅将为研究人类大脑皮质发育提供一个强大的体外平台 应激暴露，但也为FASD在皮质发育中的机制提供了至关重要的见解 细胞和分子水平。

Project summary Alcohol consumption during pregnancy causes various developmental defects, including cognitive deficit. The impairment of the central nervous system in patients of fetal alcohol spectrum disorder (FASD) has been characterized intensively by non-invasive imaging and behavioral evaluations. While Ex vivo studies and animal models provide snapshots of the molecular responses elicited by alcohol, the model systems do not fully mimic the long gestational development of human brain. This proposal aims to develop a 3D in vitro model using induced pluripotent cells (iPSC)-derived cortical spheroids to study the effect of ethanol on cortical development, specifically on the outer subventricular zone (oSVZ). oSVZ is considered as an evolutionary adaptive changes of human corticogenesis to increase the diversity of progenitor population, neuronal production, and brain size. We are proposing to generate human iPSC-derived cortical spheroids in defined, differentiation-promoting culture medium that is optimized for test of alcohol and other chemicals that produce reactive oxidative stress. The development of stratified structure in the spheroids that were generated by our novel protocol mimics the early process of human corticogenesis, which includes the formation of oSVZ like structure. We will first match the developmental timeline of iPSC-derived cortical spheroids by immunohistochemistry and RNA sequencing to human fetal brain, to clearly define the window for in vitro FASD studies (Aim 1). Then we will incubate the iPSC-derived cortical spheroids in ethanol-containing medium for different periods of time to mimic the prenatal exposure. We will assess the effects of ethanol on cortical development by monitoring cell death, morphological change, cellular proliferation and the progenitor cell population with an emphasis on oSVZ (Aim 2). Lastly, leveraging CRISPR/Cas9 genome editing technique, we will demonstrate the applicability of our novel cortical spheroid system for investigation of molecular mechanisms underlying alcohol-mediated cell pathology during human brain development. We will select RBM39 as a target gene that was screened in our laboratory and functions defined in mouse cortex. This study will not only characterize a powerful in vitro platform for studying human cortical development under stress exposure, but also provide crucial insight into the mechanisms of FASD on cortical development at cellular and molecular levels.