Exploring a new model to study developmental eye diseases

探索研究发育性眼病的新模型

• 批准号: 10678123
• 负责人: Elena V Semina
• 金额: $ 22.8万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678123
• 关键词: Affect; Alleles; Animal Model; Aniridia; Anophthalmos; Anterior; Anterior segment dysgenesis; Aphakia; Appearance; Benchmarking; Biological Models; Blindness; Cataract; Cell Culture System; Cell Culture Techniques; Cell Lineage; Cells; Cellular Morphology; Characteristics; Code; Coloboma; Complex; Cornea; Defect; Development; Developmental Gene; Developmental Process; Disease; Disease model; Drainage procedure; Ectoderm; Evaluation; Eye; Eye Development; Eye diseases; FOXC1 gene; FOXE3 gene; Fostering; Gene Expression Profile; Generations; Genes; Genetic; Genetic Predisposition to Disease; Hip region structure; Human; In Situ Hybridization; Individual; Iris; Lens development; Measures; Mesoderm; Microphthalmos; Modeling; Morphology; Neural Crest Cell; Neural tube; Normal Range; Optic vesicle; Organoids; Pathogenicity; Pathway interactions; Patients; Phenotype; Process; Proteins; Protocols documentation; Publishing; Reproducibility; Retina; Role; Self Assessment; Shapes; Structure; Structure of retinal pigment epithelium; Surface Ectoderm; System; Testing; Time; Tissues; Transcript; Untranslated RNA; Variant; Vertebrates; Vesicle; Visual Fields; cell motility; cell type; diencephalon; genetic disorder diagnosis; genetic variant; human pluripotent stem cell; induced pluripotent stem cell; innovation; insight; lens; model development; neural; novel; optic cup; protein expression; single-cell RNA sequencing; stem cells; transcription factor; usability

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.

项目概要 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。人眼由许多专门的组织组成，这些组织来源于 不同的原始细胞谱系，并且它们的形成随着时间的推移高度协调，使得眼睛 开发是一个非常复杂的过程。常用于疾病治疗的人眼细胞培养系统 建模无法概括这个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，从而有助于 观察到的复杂的眼部表型。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，其中人类 iPSC 被诱导形成多种有组织的眼部细胞类型 在四个可识别的同心区域内 - 称为自我形成的外胚层自主多区域 眼细胞（SEAM）。独特的是，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安排和细胞间协调。初级 该提案的目标是描绘可量化的形态学基准和基因表达 正常条件下与 SEAM 生成相关的签名，然后通过引入来测试该模型 众所周知的眼部基因中与疾病相关的遗传变异。具体来说，我们的目标是：（1）表征 SEAM 形态、变异性和形成过程中的基因/蛋白质表达； (2) 探讨效果 SEAM 形成的关键眼部基因的致病性人类变异。在我们的方法中，我们将测量 SEAM 在四个时间点的大小、形状和其他物理特征和外观的变化 分化过程。并行的 scRNA-seq 分析将定义细胞类型的身份 多区域并确定它们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 携带众所周知的眼部基因（PAX6 和 FOXE3）的致病变异。完成这些研究将 确定这种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，这项研究可能会提供一个测试各种遗传变异的平台 在先天性眼表型中鉴定，包括鉴定这些表型的直接和继发影响 各种眼组织协调发育的变异。