Cell Intrinsic and Extrinsic Factors Driving Maturation in Human PSC-derived Neurons

项目摘要 人类胚胎干细胞(HESC)和人类诱导多能干细胞(HiPSC)为基础 研究和在疾病建模中的应用。开发这一潜力的最初挑战是指导 干细胞向与疾病相关的特定神经细胞或神经胶质细胞分化。在过去的几年里，我们 已经开发了许多这样的协议，现在使研究人员能够在体内生成50种不同的人类细胞类型 一道菜。然而，剩下的一个主要挑战是胎儿而不是成人的特征 生成的单元格，这限制了它们的用途。他们不成熟的原因尚不清楚，但可能与 细胞固有的时钟状机制，控制成熟的时间。而人类需要9个月的时间 婴儿发育，从受孕到出生，同样的过程在老鼠身上只需要20天。那些戏剧性的 时间差在培养皿中重现，人类细胞的成熟可能需要数月时间。 才能达到成人般的特性。有趣的是，我们在2D和3D文化中都观察到了这样的时间差异 包括在神经器官中。即使在将人类细胞移植到小鼠大脑后，细胞仍在继续 遵循人类特有的成熟轨迹，尽管周围是成年宿主的微环境。 在这里，我们将通过建立检测方法来测量和量化神经元和神经胶质细胞来应对这一挑战 通过制定策略来覆盖内在的成熟时钟。为了实现这些目标，我们 已经建立了一种独特的基于干细胞的分析方法，以非常高的精度和在 时间同步的方式。由此产生的细胞然后从胎儿阶段逐渐成熟到成人期。 几个月的时间，使我们能够定义预测神经元成熟状态的标记。在AIM 1，我们将在这些初步数据的基础上，建立神经细胞成熟的特定阶段“指纹”，以 以前所未有的精度确定成熟状态。此外，我们还将描述神经胶质细胞的成熟 细胞(星形胶质细胞和小胶质细胞)在一种新的三种培养体系中，以测试胶质细胞的存在是否可以改善 神经元成熟。在目标2中，我们将应用成熟“指纹”作为读数，以确定哪些因素 可以加快成熟时间。在初步研究中，我们已经识别出 是推动神经元成熟的有力候选者。我们将在三种文化中进一步验证这些发现。 系统，以确定内在和外在成熟因素的综合作用。最后，我们将表演 机制研究，以了解这些因素是如何在人类细胞中诱导更多成人型特征的。在Aim3， 我们将在更复杂的3D培养系统中测试我们优化的成熟策略，以评估 诱导成熟策略影响其他发育过程，如细胞迁移和组织。 最后，我们将评估是否可以采用“诱导成熟”策略来实现加速。 移植到发育中的小鼠大脑中的神经元成熟的时间表，这一策略可能 使新的人类疾病模型能够在体内研究神经发育或神经精神障碍。

Project Summary Human embryonic (hESC) and human induced pluripotent stem cells (hiPSC) offer great promise for basic research and for applications in disease modeling. The initial challenge for exploiting this potential was to direct stem cell differentiation towards specific nerve cell or glial fates relevant to disease. Over the last few years, we have developed many such protocols that now enable researchers to generate > 50 distinct human cell types in a dish. However, a major remaining challenge is the fetal rather than adult-like features exhibited by the resulting cells, which limits their usefulness. The reason for their immaturity is unclear but may be linked to a cell-intrinsic, clock-like mechanism that controls the timing of maturation. While it takes 9 months for a human baby to develop, from conception to birth, the same process takes only 20 days in a mouse. Those dramatic timing differences are recapitulated in a dish, where the maturation of human cells may require many months to reach adult-like properties. Interestingly, we observe such timing differences in both 2D and 3D culture including in neural organoids. Even after transplanting human cells into the mouse brain, cells continue to follow a human-specific maturation trajectory, despite being surrounded by an adult host microenvironment. Here we will address this challenge by building assays to measure and quantify neuronal and glial maturation and by developing strategies to override the intrinsic maturation clock. Towards these goals, we have established a unique stem cell-based assay to produce nerve cells at very high precision and in a temporally synchronized manner. The resulting cells then progressively mature from fetal to adult-like stages over a period of several months allowing us to define markers that predict the neuronal maturation state. In Aim 1, we will build on these preliminary data and establish stage-specific “fingerprints” of nerve cell maturation to determine maturation states at unprecedented precision. In addition, we will characterize the maturation of glial cells (astrocytes and microglia) in a novel tri-culture system to test whether the presence of glia can improve neuronal maturation. In Aim 2, we will apply maturation “fingerprints” as a readout for identifying factors that can accelerate maturation timing. In preliminary studies, we have identified chemicals and genes that are strong candidates for driving neuronal maturation. We will further validate those findings in the tri-culture system to determine the combined effect of intrinsic and extrinsic maturation factors. Finally, we will perform mechanistic studies to understand how those factors induce more adult-like features in human cells. In Aim3, we will test our optimized maturation strategies in more complex 3D culture systems to assess whether induced maturation strategies impact other developmental processes such as cell migration and organization. Finally, we will assess whether “induced maturation” strategies can be adopted to achieve accelerated timelines of neuronal maturation upon transplantation into the developing murine brain, a strategy that could enable new human disease models for the study of neurodevelopmental or neuropsychiatric disorders in vivo.