Understanding Down Syndrome Brain Development Using Human iPSC-Based Mouse Chimeras

唐氏综合征（Down syndrome，DS）是由人类21号染色体（human chromosome 21，HSA 21）三倍体引起的一种常见的遗传性疾病， 智力残疾的起源。研究DS发病机制具有挑战性，因为功能性人类DS 脑组织几乎不可用，DS的转基因小鼠模型显示不完整/不准确 HSA 21基因的表达。人类诱导多能干细胞（hiPSC）技术的出现已经导致 DS患者来源的hiPSC的产生，这为研究 DS的发病机制与无限的人脑细胞在体外。在使用基于hiPSC的体外模型时，基本 可以检查疾病表型的各个方面，发育中的大脑神经回路的中断 在疾病条件下仍有待于用hiPSC研究。最终，特定的发育和疾病 机制只能在活体动物中建模，以确定细胞表型和行为之间的联系。 性能因此，我们建议采用基于hiPSC的嵌合小鼠脑模型来研究 DS的神经病理生理学。小胶质细胞在大脑发育中起着关键作用，也是一种活跃的 参与学习和记忆过程。令人惊讶的是，很少有信息是关于如何三体性的， HSA 21改变小胶质细胞的发育和功能以及小胶质细胞在异常大脑中发挥的作用 发育和认知缺陷。此外，越来越多的证据表明，啮齿类动物的小胶质细胞不是 能够完全反映人类小胶质细胞在正常和疾病条件下的特性。在这项研究中，我们将使用 我们最近创建了hiPSC小胶质细胞嵌合小鼠模型，以阐明小胶质细胞在DS发病机制中的作用 在一个具有完整神经网络的体内系统中。我们假设与移植的正常人类小胶质细胞不同， 移植的患病DS人小胶质细胞将显示异常的生物学特性和功能，如突触 修剪功能。DS小胶质细胞的这些异常特性将导致它们对神经元的负调节。 海马神经网络的突触活动和可塑性，对认知缺陷有重要作用 在DS中看到这一假设将在三个具体目标中得到检验。目标1：我们将确定 DS和对照hiPSC衍生的小胶质细胞在人小胶质细胞嵌合小鼠脑中的体内表达。目标2：使用 小胶质细胞嵌合小鼠模型，我们将进一步研究DS小胶质细胞整合对突触的影响。 海马的可塑性以及动物的学习和记忆行为。目标3：我们将标准化 通过CRISPR/Cas9表达HSA 21基因，以检查这将如何改变DS小胶质细胞的性质。 此外，将进行hiPSC小胶质细胞嵌合小鼠脑的单细胞RNA测序分析，以确定hiPSC小胶质细胞嵌合小鼠脑的基因表达。 比较对照和DS小胶质细胞的基因表达谱。我们的研究结果使用了一种强大的，新的 hiPSC小胶质细胞嵌合小鼠模型将为人类小胶质细胞的病理作用提供新的见解 在DS。鉴定出能够靶向改善小胶质细胞功能的潜在分子可能会提供一种新的 治疗DS的治疗途径。

Down syndrome (DS), caused by triplication of human chromosome 21 (HSA21), is the most common genetic origin of intellectual disability. Studying DS disease mechanism is challenging because functional human DS brain tissues are scarcely available and transgenic mouse models of DS demonstrate incomplete/inaccurate expression of HSA21 genes. The advent of human induced pluripotent stem cell (hiPSC) technology has led to the generation of DS patient-derived hiPSCs, which presents an unprecedented opportunity for studying the pathogenesis of DS with unlimited human brain cells in vitro. While using the hiPSC-based in vitro models, basic aspects of the disease phenotypes can be examined, the disruption of neural circuits in the developing brain under disease conditions remains to be studied with hiPSCs. Ultimately, specific developmental and disease mechanisms can only be modeled in live animals to identify links between cellular phenotypes and behavioral performance. Therefore, we propose to employ hiPSC-based chimeric mouse brain models to study the neuropathophysiology of DS in vivo. Microglia play critical roles in brain development and are also an active player in learning and memory processes. Surprisingly, very little information is available on how trisomy of HSA21 alters the development and functions of microglia and what roles microglia play in the abnormal brain development and cognitive deficits in DS. In addition, mounting evidence indicates that rodent microglia are not able to fully mirror the properties of human microglia in normal and disease conditions. In this study, we will use our recently created hiPSC microglial chimeric mouse model to unravel the role of microglia in DS pathogenesis in an in vivo system with intact neural networks. We hypothesize that unlike engrafted normal human microglia, engrafted diseased DS human microglia will show abnormal biological properties and functions, such as synaptic pruning function in vivo. These abnormal properties of DS microglia will result in their negative regulation of the synaptic activity and plasticity of the hippocampal neural network, critically contributing to the cognitive deficits seen in DS. This hypothesis will be tested in three specific aims. Aim 1: we will determine the differences between DS and control hiPSC-derived microglia in vivo in human microglial chimeric mouse brains. Aim 2: Using the microglial chimeric mouse model, we will further examine the impact of integration of DS microglia on synaptic plasticity of the hippocampus and learning and memory behavior of the animals. Aim 3: We will normalize the expression of the HSA21 genes by CRISPR/Cas9 to examine how this will alter the properties of DS microglia. Moreover, single-cell RNA-sequencing analysis of hiPSC microglial chimeric mouse brains will be performed to compare gene expression profiles of control and DS microglia. Findings from our study using a powerful, new hiPSC microglial chimeric mouse model will provide novel insights into the pathological roles of human microglia in DS. Identifying the potential molecules that can be targeted to improve microglial function may provide a new therapeutic avenue for the treatment of DS.