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）为基础研究提供了巨大的希望。 研究和疾病建模中的应用。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命运分化。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产生超过50种不同的人类细胞类型， 一个盘子。然而，一个主要的剩余挑战是胎儿而不是成人一样的功能表现出的胎儿， 这限制了它们的有用性。他们不成熟的原因尚不清楚，但可能与 细胞内在的，时钟样的机制，控制成熟的时间。人类需要9个月的时间 婴儿从受孕到出生，同样的过程在老鼠身上只需要20天。这些戏剧性 时间差异在培养皿中重现，人类细胞的成熟可能需要数月 达到成人的特性。有趣的是，我们在2D和3D培养中都观察到了这种时间差异 包括神经类器官。即使在将人类细胞移植到小鼠大脑中后，细胞仍继续 遵循人类特定的成熟轨迹，尽管被成年宿主微环境所包围。 在这里，我们将通过建立测定和定量神经元和神经胶质细胞的方法来解决这一挑战。 成熟和发展战略，以推翻内在的成熟时钟。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检测方法，以非常高的精度和 时间同步的方式。由此产生的细胞然后逐渐成熟，从胎儿到成人样阶段 在几个月的时间里，我们可以确定预测神经元成熟状态的标志物。在Aim中 我们将在这些初步数据的基础上建立神经细胞成熟的阶段特异性“指纹”， 以前所未有的精确度确定成熟状态。此外，我们将描述神经胶质细胞的成熟过程。 细胞（星形胶质细胞和小胶质细胞）在一个新的三培养系统，以测试是否存在胶质细胞可以改善 神经元成熟在目标2中，我们将应用成熟“指纹”作为识别因素的读数， 可以加快成熟时间。在初步研究中，我们已经确定了化学物质和基因， 驱动神经元成熟的强有力的候选者。我们将在三文化中进一步验证这些发现 系统来确定内在和外在成熟因子的组合效应。最后，我们将表演 机制研究，以了解这些因素如何诱导人类细胞中更多的成人样特征。在目标3中， 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，例如细胞迁移和组织。 最后，我们将评估是否可以采用“诱导成熟”策略来实现加速成熟。 移植到发育中的小鼠脑中后神经元成熟的时间表，这一策略可以 使新的人类疾病模型能够用于体内神经发育或神经精神障碍的研究。

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.