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

使用 3D 体外模型研究酒精暴露对人类皮质发育的影响

• 批准号: 10231088
• 负责人: Ray Yueh Ku
• 金额: $ 7.5万
• 依托单位: CHILDREN'S RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-12 至 2024-11-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10231088
• 关键词: 3-Dimensional; Acute; Alcohol consumption; Alcohols; Animal Model; Behavioral; Biological Models; Biomedical Research; Bioreactors; Brain; CRISPR/Cas technology; Cell Death; Cell Line; Cell Proliferation; Cells; Cerebrum; Chemicals; Chronic; Cognitive deficits; Data; Defect; Development; Dose; Embryonic Development; Ethanol; Evaluation; Exposure to; Fetal Alcohol Exposure; Fetal Alcohol Spectrum Disorder; Fetal Development; Genes; Human; Hyperoxia; Immunohistochemistry; Impairment; In Vitro; Incubated; Investigation; Knowledge; Laboratories; Mediating; Modeling; Modernization; Molecular; Monitor; Morphology; Mus; Neuraxis; Neuroglia; Neuronal Differentiation; Neurons; Oxidative Stress; Pathology; Patients; Population; Predisposition; Pregnancy; Pregnant Women; Preparation; Primates; Process; Production; Protocols documentation; RNA-Binding Proteins; Radial; Reporting; Research; Role; Stress; Structure; Study models; System; Techniques; Testing; Time; TimeLine; Up-Regulation; alcohol consumption during pregnancy; alcohol effect; alcohol exposure; alcohol response; alcohol testing; base; brain size; cell injury; cell type; cognitive function; falls; genome editing; human data; human fetal brain; in vitro Model; insight; migration; mouse model; nerve stem cell; non-invasive imaging; novel; overexpression; prenatal exposure; prenatal influence; progenitor; relating to nervous system; response; stem cell proliferation; stem cells; subventricular zone; transcriptome sequencing; transcriptomics

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.

项目概要 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) 患者的中枢神经系统受损 通过非侵入性成像和行为评估对其进行了深入的表征。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，但模型系统不提供 完全模仿人类大脑的长期妊娠发育过程。该提案旨在开发3D体外模型 使用诱导多能细胞 (iPSC) 衍生的皮质球体研究乙醇对皮质的影响 发育，特别是外脑室下区（oSVZ）。 oSVZ 被认为是一种进化的 人类皮质发生的适应性变化增加祖细胞群、神经元的多样性 产量和大脑大小。我们提议以定义的方式生成人类 iPSC 衍生的皮质球体， 分化促进培养基，针对酒精和其他产生化学物质的测试进行了优化 反应性氧化应激。我们生成的球体中分层结构的发展 新方案模仿了人类皮质生成的早期过程，其中包括 oSVZ 样的形成 结构。我们首先将通过以下方式匹配 iPSC 衍生的皮质球体的发育时间表： 对人胎儿大脑进行免疫组织化学和 RNA 测序，以明确体外实验的窗口 FASD 研究（目标 1）。然后我们将在含乙醇的培养基中孵育 iPSC 衍生的皮质球体 不同的时间段来模拟产前暴露。我们将评估乙醇对皮质的影响 通过监测细胞死亡、形态变化、细胞增殖和祖细胞来发育 重点关注 oSVZ 的人口（目标 2）。最后，利用 CRISPR/Cas9 基因组编辑技术，我们 将证明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学的潜在机制。我们将选择 RBM39作为我们实验室筛选的靶基因，并在小鼠皮质中定义了功能。 这项研究不仅将成为研究人类皮质发育的强大体外平台 压力暴露，而且还提供了关于 FASD 对皮质发育的机制的重要见解 细胞和分子水平。