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

驱动人 PSC 衍生神经元成熟的细胞内在和外在因素

• 批准号: 10736603
• 负责人: LORENZ P. STUDER
• 金额: $ 65.53万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736603
• 关键词: 3-Dimensional; Acceleration; Address; Adopted; Adult; Astrocytes; Automobile Driving; Basic Science; Behavior; Biological Assay; Biology; Birth; Brain; Cell Maturation; Cells; Characteristics; Chemicals; Chromatin; Complex; Conceptions; Data; Development; Developmental Process; Disease; Disease model; Embryo; Epigenetic Process; Exhibits; Fingerprint; Future; Genes; Genetic; Goals; Human; Human Characteristics; Human Development; In Vitro; Intrinsic factor; Link; Measures; Microglia; Modeling; Molecular; Molecular Profiling; Mus; Nervous System; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neuroglia; Neuronal Differentiation; Neurons; Organoids; Process; Property; Protocols documentation; Regenerative Medicine; Reporter; Research Personnel; Role; Study models; System; Testing; Transplantation; Work; Xenograft procedure; cell motility; cell type; differentiation protocol; directed differentiation; excitatory neuron; fetal; genetic approach; human disease; human model; human pluripotent stem cell; human stem cells; improved; in utero transplantation; in vivo; induced pluripotent stem cell; inhibitory neuron; nerve stem cell; neural; neuron development; neuropsychiatric disorder; novel; pharmacologic; predictive marker; prevent; programs; stem; stem cell differentiation; stem cells; three dimensional cell culture; timeline; tool

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.

项目摘要 人类胚胎（HESC）和人类诱导的多能干细胞（HIPSC）为基础提供了巨大的希望 研究和用于疾病建模中的应用。利用这种潜力的最初挑战是指导 干细胞与特定神经细胞或与疾病相关的神经胶质命运的分化。在过去的几年中，我们 已经开发了许多这样的协议，这些方案现在使研究人员能够生成> 50种不同的人类细胞类型 一道菜。但是，剩下的主要挑战是胎儿而不是像成人一样的特征 产生的细胞，这限制了它们的实用性。他们不成熟的原因尚不清楚，但可能与 控制成熟时机的细胞中心，类似时钟样的机制。虽然人类需要9个月 从概念到出生，婴儿才能在鼠标中仅需20天。那些戏剧性的 在菜肴中概括了时序差异，其中人类细胞的成熟可能需要数月 达到成人的特性。有趣的是，我们观察到2D和3D文化中的这种时间差异 包括在神经器官中。即使将人类细胞移植到小鼠大脑中后，细胞仍在继续 遵循人类特异性的成熟轨迹，目的地被成人宿主微环境所包围。 在这里，我们将通过建立测定法来解决和量化神经元和神经胶质的挑战 成熟并通过制定覆盖固有成熟时钟的策略。达到这些目标，我们 已经建立了一种独特的基于干细胞的评估，以非常高的精度和 暂时同步的方式。然后，所得细胞从胎儿到成人阶段逐渐成熟 在几个月的时间里，我们可以定义预测神经元成熟态的标记。目标 1，我们将基于这些初步数据，并建立神经细胞成熟的特定阶段的“指纹” 以前所未有的精度确定成熟状态。另外，我们将表征神经胶质的成熟 新型三培养系统中的细胞（星形胶质细胞和小胶质细胞），以测试胶质的存在是否可以改善 神经元成熟。在AIM 2中，我们将应用成熟的“指纹”作为识别因素的读数 可以加速成熟时机。在初步研究中，我们确定了化学物质和基因 强大的候选神经元成熟的候选者。我们将进一步验证三文化中的这些发现 确定固有和外在成熟因子的综合作用的系统。最后，我们将表演 机械研究以了解这些因素如何影响人类细胞中更多的成年特征。在AIM3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，以评估是否 诱导的成熟策略会影响其他发展过程，例如细胞迁移和组织。 最后，我们将评估是否可以采用“诱发的成熟”策略来实现加速 将神经元成熟的时间表移植到发育中的鼠大脑中，该策略可以 启用新的人类疾病模型，以研究体内神经发育或神经精神疾病。