Unlocking the postnatal human brain using activity augmented organoids

项目总结 理解支配极其复杂的人脑发育的原理是一个 生物学的根本目标。人类多能干细胞已被证明是这一努力的强大工具，作为 活跃领域的研究人员利用这项技术来定义触发内源性的因素 基因程序最终会产生不同的特殊脑细胞类型。其中最激动人心的一个 这项技术的最新应用是产生三维(3D)脑细胞培养物或“有机体”， 使神经前体细胞能够增殖、分化和自我组织成复杂的微生理学 系统。脑有机化合物已被证明在结构、转录和功能上具有相似性。 对于妊娠中晚期的人脑来说，外部输入非常少。目前，它们表示 最接近天然人脑组织的细胞模型。虽然一个非常强大的探测系统 出生前发育的机制，通过脑器官进入出生后阶段的努力一直是 令人沮丧。许多改善脑器官成熟状态的尝试已被证明是 递增的、不确定的或复制得很差的因此，大脑器官系统在很大程度上仍然 不适合对发育的高级阶段(例如，出生后大脑)进行建模。同时，这些 先前的研究强调了改进人脑模型的动力和未得到满足的需求。给定 基础和临床进展的潜力，打破这种发育障碍应该被认为是最重要的 神经科学研究的优先方向。我的核心假设是大脑有机体不能发育的一个主要原因 过去的妊娠晚期时间点是因为他们缺乏来自重要来源的发育指导 投入。这项提议的主要目标是解开人类大脑器官的高级阶段。 应用多管齐下的方法替代大脑中缺失的外源性神经输入的发展 具有合成“虚拟”输入的有机化合物，并确定这种操作对发育的影响， 重点是不同神经细胞类型的规范和高级状态的获得 活动。我们将尝试几种并行的方法，在较长的时间内激活电池和电路，并 以不同的生物相关方式。为了达到这些目标，我们还将开发新的方法，使 重复测量在人脑过程中发生显著变化的不同神经元活动 发展。最后，我们将利用我们的洞察力在一个疾病模型中研究致病机制 严重的发育迟缓和神经传递受损的癫痫。工具和方法 所建立的模型可以很容易地适应于其他器官系统的细胞培养模型 投入很重要。如果成功，这项新的创新者提案将导致以下方面的开创性发现 依赖活动的成熟，并将利用这些洞察力来解锁人类大脑更高级的阶段 开发使临床前研究能够讯问神经疾病的新途径。

PROJECT SUMMARY Understanding the principles that govern the development of the enormously complex human brain is a fundamental goal in biology. Human pluripotent stem cells have proven to be a powerful tool in this effort as an active field of researchers have leveraged this technology to define the factors required to trigger endogenous genetic programs that ultimately give rise to diverse specialized brain cell types. One of the most exciting recent applications of this technology is the generation of tri-dimensional (3D) brain cell cultures or "organoids," which enable neural progenitors to proliferate, differentiate, and self-organize into complex microphysiological systems. Brain organoids have been shown to develop structural, transcriptional, and functional similarities up to the mid-to-late gestation human brain with remarkably little external input. Currently they represent the closest cellular model to native human brain tissue available. While an enormously powerful system for probing mechanisms of prenatal development, efforts to access postnatal stages with brain organoids have been frustrating. Numerous attempts to improve upon the maturation state of brain organoids have proved to be incremental, inconclusive, or poorly reproduced. As a result, brain organoid systems remain largely inappropriate for modeling advanced stages of development (e.g., postnatal brain). At the same time, these prior studies underscore the momentum and unmet need to improve models of the human brain. Given the potential for fundamental and clinical advances, breaking this developmental wall should be considered a top priority in neuroscience research. My core hypothesis is that a major reason brain organoids fail to develop past late gestational time points is because they lack developmental guidance from significant sources of inputs. The primary objective of this proposal is to unlock advanced stages of human brain organoid development by applying a multipronged approach to replace missing exogenous neural inputs in brain organoids with synthetic "virtual" inputs and to determine the effects such manipulations have on development, with an emphasis on the specification of diverse neuronal cell types and acquisition of advanced states of activity. We will try several methods in parallel that activate cells and circuits over extended periods of time and in different biologically-relevant ways. To achieve these goals, we will also develop new methods to enable the repeated measurement of diverse neuronal activities that change dramatically over the course of human brain development. Finally, we will leverage our insights to study pathogenic mechanisms in a disease model of severe developmental delay and epilepsy in which neurotransmission is impaired. The tools and approaches established here may be readily adapted to cell culture models of other organ systems for which neuronal inputs are important. If successful, this New Innovator proposal will lead to ground breaking discoveries about activity-dependent maturation and will leverage those insights to unlock more advanced stages of human brain development to enable a new avenue of preclinical research to interrogate neurological disease.