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

项目摘要 怀孕期间饮酒会导致各种发育缺陷，包括认知障碍。 赤字胎儿酒精谱系障碍（FASD）患者的中枢神经系统损害， 通过非侵入性成像和行为评估进行了集中表征。虽然离体研究和 动物模型提供了酒精引起的分子反应的快照，模型系统不 完全模仿人类大脑的长时间发育。该提案旨在开发3D体外模型 使用诱导多能细胞（iPSC）衍生的皮质球状体来研究乙醇对皮质细胞的影响。 发育，特别是在外侧脑室下区（oSVZ）。oSVZ被认为是一种进化的 人类皮质发生的适应性变化，以增加祖细胞群体的多样性，神经元 生产和大脑大小。我们提出在限定的， 一种促进分化的培养基，用于测试酒精和其他化学物质， 反应性氧化应激在我们的实验中产生的球状体中层状结构的发展 新的方案模拟了人类皮质生成的早期过程，包括oSVZ样的形成。 结构我们将首先通过以下方式匹配iPSC衍生的皮质球状体的发育时间轴： 免疫组织化学和RNA测序，以明确定义体外培养的窗口 FASD研究（目标1）。然后我们将iPSC衍生的皮质球状体在含乙醇的培养基中孵育 不同的时间段来模拟产前暴露。我们将评估乙醇对大脑皮层的影响， 通过监测细胞死亡、形态学变化、细胞增殖和祖细胞的发育 强调oSVZ的人群（目标2）。利用CRISPR/Cas9基因组编辑技术， 将证明我们的新的皮质球体系统的适用性，研究分子 人类大脑发育过程中酒精介导的细胞病理学机制。我们将选择 RBM 39是本实验室筛选的靶基因，在小鼠大脑皮层中具有一定的功能。 这项研究不仅将表征一个强大的体外平台，用于研究人类皮质发育， 应力暴露，而且还提供了关键的洞察机制FASD对皮质发育， 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.