Abstract:
Animal models have provided many insights into ocular development and disease, but they remain suboptimal for understanding human oculogenesis. Eye development requires spatiotemporal gene expression patterns and disease phenotypes can differ significantly between humans and animal models, with patient-associated mutations causing embryonic lethality reported in some animal models. The emergence of human induced pluripotent stem cell (hiPSC) technology has provided a new resource for dissecting the complex nature of early eye morphogenesis through the generation of three-dimensional (3D) cellular models. By using patient-specific hiPSCs to generate in vitro optic vesicle-like models, we can enhance the understanding of early developmental eye disorders and provide a pre-clinical platform for disease modelling and therapeutics testing. A major challenge of in vitro optic vesicle generation is the low efficiency of differentiation in 3D cultures. To address this, we adapted a previously published protocol of retinal organoid differentiation to improve embryoid body formation using a microwell plate. Established morphology, upregulated transcript levels of known early eye-field transcription factors and protein expression of standard retinal progenitor markers confirmed the optic vesicle/presumptive optic cup identity of in vitro models between day 20 and 50 of culture. This adapted protocol is relevant to researchers seeking a physiologically relevant model of early human ocular development and disease with a view to replacing animal models.
摘要:
动物模型为眼部发育和疾病提供了许多见解,但是它们对于理解人类的眼发育仍然不够理想。眼睛发育需要时空基因表达模式,人类和动物模型之间的疾病表型可能存在显着差异。在一些动物模型中报道了导致胚胎致死率的患者相关突变。人类诱导多能干细胞(hiPSC)技术的出现为通过生成三维(3D)细胞模型来解剖早期眼睛形态发生的复杂性质提供了新的资源。通过使用患者特异性hiPSCs来产生体外视神经泡样模型,我们可以加强对早期发育性眼疾的了解,并为疾病建模和治疗测试提供临床前平台。体外光学囊泡生成的主要挑战是3D培养中分化的低效率。为了解决这个问题,我们使用微孔板调整了以前发表的视网膜类器官分化方案,以改善胚状体的形成.已建立的形态学,已知的早期眼场转录因子的转录水平上调和标准视网膜祖细胞标志物的蛋白质表达证实了在培养的第20天至第50天之间的体外模型的视囊泡/假定视杯身份。这种适应的方案与寻求早期人类眼部发育和疾病的生理相关模型以取代动物模型的研究人员有关。