A 3D Model of Human Brain Development for Studying Gene/Environment Interactions

描述（由申请人提供）：将在集体培养中开发3d模型以进行发育神经毒性测试（DNT）。该模型将捕捉星形胶质细胞和神经元在发育过程中发生的复杂形态和生化相互作用。星形胶质细胞通过为轴突生长和树突形成提供基质参与神经元分化。它们参与突触的形成，并通过包膜突触的过程监控突触的活动。星形胶质细胞将谷氨酸从突触中移除，它们通过钙反应对谷氨酸和ATP作出反应，并且它们解毒由活跃的线粒体和神经递质生物合成产生的活性氧。在来源于啮齿动物和人类的脑细胞中，发现聚集培养物可促进神经元分化、突触形成和髓鞘形成。在具体目标中，将比较星形胶质细胞和神经元共培养和单层培养之间的神经元分化、突触形成、突触可塑性和对物质的反应性。星形胶质细胞和神经元将由人类诱导多能干细胞株系（iPSC）发育而来。突触形成将在形态学上进行评估。星形胶质细胞参与突触的形成将通过测量星形胶质细胞过程与突触的并列来确定形态学。星形胶质细胞参与的功能评估将完成基因驱动钙敏感荧光探针。同样，神经元和星形胶质细胞对增加氧化应激和炎症的不同物质的反应将通过生化测定和含有荧光探针的报告基因构建来测量。由于遗传变异性会影响对药物和其他物质的反应，因此还将利用来自不同遗传背景的多能干细胞开发DNT模型。为了证明遗传背景在DNT中起重要作用的可行性，将对唐氏综合症和结节性硬化症患者的iPSC进行测试。在这两种综合征中，基因修饰增加了对产生氧化应激物质的敏感性。几种DNT物质将在来自两种综合征的iPSC诱导的神经元和星形胶质细胞的聚集培养物中进行检查。总的来说，本提案中开发的模型将有可能评估来自不同遗传背景的个体的人类神经元和星形胶质细胞中的DNT。

DESCRIPTION (provided by applicant): A 3 D model will be developed in aggregate cultures to conduct developmental neurotoxicity testing (DNT). The model will capture the complex morphological and biochemical interactions between astrocytes and neurons that take place during development. Astrocytes participate in neuronal differentiation by providing a matrix for axonal growth and dendritic arborization. They participate in synapse formation and monitor synaptic activity through their processes that ensheath synapses. Astrocytes remove glutamate from the synapse, they respond to glutamate and ATP through a calcium response, and they detoxify reactive oxygen species generated by the active mitochondria and by neurotransmitter biosynthesis. Aggregate cultures were found to promote neuronal differentiation, synapse formation, and myelination in brains cells derived from rodents and humans. In the specific aims, neuronal differentiation, synapse formation, synaptic plasticity, and responsiveness to substances will be compared between co-cultures of astrocytes and neurons in aggregate and monolayer cultures. Astrocytes and neurons will be developed from human induced pluripotent stem cell lines (iPSC). Synapse formation will be assessed morphologically. Astrocyte participation in synapse formation will be determined morphologically by measuring the juxtaposition of astrocyte processes with synapses. Functional assessment of astrocyte participation will be accomplished with genetically driven calcium sensitive fluorescent probes. Similarly, the responses of neurons and astrocytes to different substances that increase oxidative stress and inflammation will be measured with biochemical assays and reporter gene constructs containing fluorescent probes. Because genetic variability will influence responses to drugs and other substances, the DNT model will also be developed with iPSC from different genetic backgrounds. To demonstrate the feasibility that genetic background is important in DNT, iPSC from individuals with Down's Syndrome and Tuberous Sclerosis Complex will be tested. In both syndromes, the genetic modification increases sensitivity to substances that generate oxidative stress. Several DNT substances will be examined in aggregate cultures of neurons and astrocytes induced from iPSC that are derived from both syndromes. Overall, the model developed in this proposal will have the potential to assess DNT in human neurons and astrocyte derived from individuals with different genetic backgrounds. PUBLIC HEALTH RELEVANCE: A 3D rat mini brain model successfully used for developmental neurotoxicity testing will be humanized using induced pluripotent stem cells. This allows to use cells from healthy donors and patients and study for the first time gene/environment interactions. The model promises to replace demanding animal testing with a more predictive human model.