MicroBRAINS: Bioengineered Human Neural Circuits for Aging Research

项目摘要-摘要 我们的目标是生成并验证人类神经系统的创新3D细胞模型 多能干细胞作为衰老研究的新工具。目前体外建模的最新技术状态是 不足是因为多细胞人类神经回路在培养条件下仍处于未成熟状态 延长的一段时间。为了克服这一障碍，我们提议生产一种微型组装 生物工程、快速、全感应神经系统(µBrains)。我们将利用我们最近优化的协议 从新型转基因细胞系中产生一致的3D共培养物与转录激活剂样物 效应器核酸酶技术，我们已经在初步数据中产生。在目标1中，神经元、星形胶质细胞和 少突胶质细胞将通过神经原蛋白2的诱导从人类多能干细胞中单独产生， SOX9分别与NFIA和SOX10相结合。我们将这些单元格类型组合在定义的 3D共培养球体，并评估随着时间的推移神经回路的成熟。成熟度将由增加 有髓纤维的存在和同步网络活动的开始。活动将通过以下方式来衡量 多电极阵列记录以及实时钙指示器成像。这些研究将确定最佳的 生产功能性脑的条件。在目标2中，我们将测试受调控的神经元活动是否会 以快速和一致的方式推动成熟。我们将利用一种新的光遗传细胞系 光介导刺激神经元对脑内神经回路成熟影响的初步研究 他们的大脑。这些研究的结果将揭示，诱发的神经元活动是否加速了 成熟的电路。在目标3中，我们将测试转录因子NFIX在星形胶质细胞中的强制表达是否会 通过进行细胞老化试验和测量对髓鞘形成的影响来诱导衰老表型 以及共培养细胞的神经网络活性。总之，我们希望提供新的细胞系和优化的 系统地生成具有衰老表型的人类神经电路模型系统的协议。我的大脑 可以成为整个研究社区用于疾病建模和药物筛选的突破性工具 在阿尔茨海默病等与年龄相关的疾病的背景下进行干预。

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.