Exploring a new model to study developmental eye diseases

项目总结 人类眼睛发育的缺陷会导致多种眼部疾病，其中许多会导致 部分或完全失明。人类的眼睛由许多特殊的组织组成，这些组织来自 不同的原始细胞谱系，它们的形成随着时间的推移高度协调，使眼睛 发展是一个非常复杂的过程。疾病常用的人眼细胞培养系统 建模未能概括这一逐步发展过程的早期阶段。因此，它仍然是一种 有效测试患者变异如何影响眼睛内发育的结构的挑战，从而有助于 观察到的复杂的眼部表型。为了克服这个障碍，我们的目标是探索最近 提出了一种诱导人IPSCs形成多种眼组织细胞类型的细胞培养模型 在四个可识别的同心带内-称为自形成的外胚层自治多带 眼细胞(缝隙)。独一无二的是，每个区域都由眼睛内发现的不同细胞类型组成，包括 神经视网膜、视网膜色素上皮、角膜和晶状体，以及区域形成逐渐发生。 时间，模仿人眼发育过程中观察到的时间和细胞间的协调。初级阶段 这项提案的目的是描绘可量化的形态基准和基因表达 在正常情况下与接缝生成相关联的签名，然后通过引入 众所周知的眼部基因中与疾病相关的遗传变异。具体地说，我们的目标是：(1)表征 菌丝形成过程中的形态、变异性和基因/蛋白表达；(2)探讨 影响缝隙形成的关键眼部基因中的致病人类变异。在我们的方法中，我们将测量 沿四个时间点接缝的大小、形状和其他物理特征和外观的变化 分化的过程。并发scRNA-seq分析将定义 并确定它们与发育中的人类眼组织的等效性。这些分析将是 首先在正常条件下建立，然后扩展到评估来自HiPSC的接缝 携带众所周知的眼部基因(PAX6和FOXE3)的致病变异。完成这些研究后， 确定这一用于研究人类全眼的创新模型系统的优点和可能的局限性 发展。因此，这项研究可能会为测试广泛的遗传变异提供一个平台 先天性眼部表型的识别，包括对这些表型的直接和次要影响的识别 各种眼组织协调发展的变种。

PROJECT SUMMARY Defects in human eye development can result in a broad spectrum of ocular disorders, many of which lead to partial or complete vision loss. The human eye is comprised of many specialized tissues derived from different primordial cell lineages, and their formation is highly coordinated over time, making ocular development a very complex process. Human ocular cell culture systems commonly used for disease modeling fail to recapitulate earlier stages of this stepwise developmental process. Consequently, it remains a challenge to efficiently test how patient variants impact developing structures within the eye, thus contributing to the observed complex ocular phenotypes. To overcome this barrier, we aim to explore the recently presented cell culture model where human iPSCs are induced to form multiple ocular cell types organized within four identifiable, concentric zones - referred to as self-formed ectodermal autonomous multi-zone of ocular cells (SEAMs). Uniquely, each zone is comprised of different cell types found within the eye, including neuroretina, retinal pigmented epithelium, cornea and lens, and zone formation occurs progressively over time, mimicking the timing and cell-cell coordination observed during human eye development. The primary objective of this proposal is to delineate quantifiable morphological benchmarks and gene expression signatures associated with SEAM generation under normal conditions and then test this model by introducing disease-associated genetic variants in well-known ocular genes. Specifically, we aim: (1) To characterize SEAM morphology, variability and gene/protein expression during their formation; (2) To explore the effects of pathogenic human variants in key ocular genes on SEAM formation. In our approach we will measure changes in size, shape, and other physical characteristics and appearance of SEAMs at four timepoints along the differentiation process. Concurrent scRNA-seq analysis will define the identity of the cell types within the multi-zones and determine their equivalence to developing human ocular tissues. These analyses will be established first under normal conditions and then extended to assessing SEAMs derived from hiPSCs carrying pathogenic variants in well-known ocular genes (PAX6 and FOXE3). Completion of these studies will identify strengths and possible limitations of this innovative model system for studying human whole eye development. Thus, this study will likely provide a platform for testing a wide range of genetic variants identified in congenital ocular phenotypes including identification of the direct and secondary effects of those variants on coordinated development of various ocular tissues.