PDF(Pubmed)
Abstract：
Human retinal organoids have become indispensable tools for retinal disease modeling and drug screening. Despite its versatile applications, the long timeframe for their differentiation and maturation limits the throughput of such research. Here, we successfully shortened this timeframe by accelerating human retinal organoid development using unique pharmacological approaches. Our method comprised three key steps: 1) a modified self-formed ectodermal autonomous multizone (SEAM) method, including dual SMAD inhibition and bone morphogenetic protein 4 treatment, for initial neural retinal induction; 2) the concurrent use of a Sonic hedgehog agonist SAG, activin A, and all-trans retinoic acid for rapid retinal cell specification; and 3) switching to SAG treatment alone for robust retinal maturation and lamination. The generated retinal organoids preserved typical morphological features of mature retinal organoids, including hair-like surface structures and well-organized outer layers. These features were substantiated by the spatial immunostaining patterns of several retinal cell markers, including rhodopsin and L/M opsin expression in the outermost layer, which was accompanied by reduced ectopic cone photoreceptor generation. Importantly, our method required only 90 days for retinal organoid maturation, which is approximately two-thirds the time necessary for other conventional methods. These results indicate that thoroughly optimized pharmacological interventions play a pivotal role in rapid and precise photoreceptor development during human retinal organoid differentiation and maturation. Thus, our present method may expedite human retinal organoid research, eventually contributing to the development of better treatment options for various degenerative retinal diseases.
摘要：
人类视网膜类器官已成为视网膜疾病建模和药物筛选不可或缺的工具。尽管其用途广泛，它们分化和成熟的漫长时间限制了此类研究的吞吐量。这里,我们通过使用独特的药理学方法加速人类视网膜类器官的发育，成功地缩短了这一时间范围。我们的方法包括三个关键步骤:1)改进的自形成外胚层自主多区(SEAM)方法，包括双重SMAD抑制和骨形态发生蛋白4治疗，用于初始神经视网膜诱导；2)同时使用Sonichedgehog激动剂SAG，激活素A,和全反式维甲酸用于快速视网膜细胞规格；3）转换为单独的SAG治疗以实现强大的视网膜成熟和分层。生成的视网膜类器官保留了成熟视网膜类器官的典型形态特征，包括毛发状的表面结构和组织良好的外层。这些特征通过几种视网膜细胞标志物的空间免疫染色模式得到证实，包括最外层的视紫红质和L/M视蛋白表达，伴随着异位圆锥光感受器生成的减少。重要的是,我们的方法只需要90天的视网膜器官成熟，这大约是其他传统方法所需时间的三分之二。这些结果表明，彻底优化的药理学干预措施在人类视网膜器官分化和成熟过程中的快速和精确的光感受器发育中起着关键作用。因此，我们目前的方法可能会加快人类视网膜类器官的研究，最终有助于开发各种退行性视网膜疾病的更好治疗方案。
