MicroBRAINS: Bioengineered Human Neural Circuits for Aging Research

项目摘要 我们的目标是生成和验证一个创新的3D细胞模型的神经系统从人类 多能干细胞作为衰老研究的新工具。体外建模的现有技术是 这是因为在培养条件下的多细胞人类神经回路仍处于不成熟状态， 长时间的。为了克服这一障碍，我们建议生产一种 生物工程，快速，全诱导神经系统（µBRAINS）。我们将利用我们最近优化的协议 为了产生一致的3D共培养物，所述3D共培养物由具有转录激活因子样蛋白的新型转基因细胞系产生， 效应子核酸酶技术，我们已经产生的初步数据。在Aim 1神经元、星形胶质细胞和 少突胶质细胞将通过神经生成素2的诱导从人多能干细胞单独产生， Sox 9分别与NFIA和Sox 10组合。我们将这些细胞类型组装在一起， 3D共培养球，并评估神经回路随时间的成熟。成熟度将由增加的 有髓纤维的存在和同步网络活动的开始。活动将通过 多电极阵列记录以及活体钙指示剂成像。这些研究将确定最佳的 产生功能性µBRAINS的条件。在目标2中，我们将测试受调节的神经元活动是否会 以快速和一致的方式促进成熟。我们将利用一种新的光遗传学细胞系， 初步研究，以确定光介导的神经元刺激对回路成熟的影响， 的大脑。这些研究的结果将揭示，如果诱发的神经元活动加速了 成熟的电路在目标3中，我们将测试星形胶质细胞中转录因子NFIX的强制表达是否会 通过进行细胞老化测定和测量对髓鞘形成的影响来诱导老化表型 和共培养细胞的神经网络活性。总而言之，我们希望提供新的细胞系和优化的 本发明涉及一种用于系统地生成具有老化表型的人类神经回路模型系统的协议。微脑 可以成为整个研究界用于疾病建模和筛选药物的突破性工具 在与年龄有关的情况下，如阿尔茨海默病的干预措施。

PROJECT SUMMARY-ABSTRACT Our goal is to generate and validate an innovative 3D cellular model of the nervous system from human pluripotent stem cells as a new tool for aging research. The current state of the art for in vitro modeling is insufficient because multicellular human neural circuits in culture conditions remain in immature states for prolonged periods of time. To overcome this barrier, we are proposing to produce a Microassembly of Bioengineered, Rapid, All-Inducible Neural System (µBRAINS). We will utilize our recently optimized protocols to produce consistent 3D cocultures generated from novel transgenic cell lines with transcription activator-like effector nuclease technology that we have generated in preliminary data. In Aim 1 neurons, astrocytes and oligodendrocytes will be individually produced from human pluripotent stem cells by induction of neurogenin 2, Sox9 in combination with NFIA, and Sox10, respectively. We will assemble these cell types together in defined 3D coculture spheres and assess neural circuit maturation over time. Maturation will be determined by increased presence of myelinated fibers and onset of synchronous network activity. Activity will be measured by multielectrode array recordings as well as live calcium indicator imaging. These studies will determine the optimal conditions for producing the functional µBRAINS. In Aim 2, we will test whether regulated neuronal activity will enforce maturation in a rapid and consistent manner. We will utilize a novel optogenetic cell line generated in preliminary studies to determine the effect of light-mediated stimulation of neurons upon circuit maturation within the µBRAINS. The results from these studies will reveal if evoked neuronal activity accelerates production of mature circuits. In Aim 3, we will test whether forced expression of the transcription factor NFIX in astrocytes will induce aging phenotypes by performing assays for cellular aging and by measuring the effect upon myelination and neural network activity of cocultured cells. Altogether, we expect to deliver novel cell lines and an optimized protocol to systematically generate a human neural circuit model system with aging phenotypes. The µBRAINS can be a breakthrough tool for use throughout the research community for disease modeling and screening drug interventions in the context of age-related conditions such as Alzheimer’s disease.