Exploring a new model to study developmental eye diseases

项目摘要 人眼发育缺陷可导致广泛的眼部疾病，其中许多导致 部分或完全视力丧失。人眼由许多特化组织组成， 不同的原始细胞谱系，它们的形成随着时间的推移是高度协调的， 发展是一个非常复杂的过程。常用于疾病的人眼细胞培养系统 模型未能概括这一逐步发展过程的早期阶段。因此，它仍然是一个 有效测试患者变异如何影响眼内发育结构的挑战， 与所观察到的复杂眼部表型有关。为了克服这一障碍，我们的目标是探索最近 提出了细胞培养模型，其中人iPSC被诱导以形成组织化的多种眼部细胞类型。 在四个可识别的同心区域内-称为自形成的外胚层自主多区域， 眼细胞（SEAMs）。独特的是，每个区域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、角膜和透镜，并且区域形成逐渐发生， 时间，模仿人眼发育过程中观察到的时间和细胞-细胞协调。主 这个建议的目的是描绘可量化的形态基准和基因表达 签名与SEAM生成在正常条件下，然后测试这个模型，通过引入 已知眼部基因中的疾病相关遗传变异。具体来说，我们的目标是：（1）描述 SEAM的形态、变异性及其形成过程中的基因/蛋白表达;（2）探讨 致病性人类变异的关键眼部基因对SEAM形成。在我们的方法中，我们将测量 SEAM在沿着的四个时间点的大小、形状和其他物理特征和外观的变化 分化过程。同时进行的scRNA-seq分析将定义细胞内细胞类型的身份。 多区域，并确定其与发育中的人类眼组织的等效性。这些分析将 首先在正常条件下建立，然后扩展到评估源自hiPSC的SEAM 携带已知的眼部基因（PAX 6和FOXE 3）的致病变体。完成这些研究将 确定用于研究人类全眼创新模型系统的优势和可能的局限性 发展因此，这项研究将可能提供一个测试广泛的遗传变异的平台。 在先天性眼部表型中鉴定，包括鉴定那些 各种眼组织协调发育的变体。

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.