Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202.
Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202.
Proc Natl Acad Sci U S A. 2024 Jun 18;121(25):e2317285121. doi: 10.1073/pnas.2317285121. Epub 2024 Jun 13.
Human pluripotent stem cell (hPSC)-derived retinal organoids are three-dimensional cellular aggregates that differentiate and self-organize to closely mimic the spatial and temporal patterning of the developing human retina. Retinal organoid models serve as reliable tools for studying human retinogenesis, yet limitations in the efficiency and reproducibility of current retinal organoid differentiation protocols have reduced the use of these models for more high-throughput applications such as disease modeling and drug screening. To address these shortcomings, the current study aimed to standardize prior differentiation protocols to yield a highly reproducible and efficient method for generating retinal organoids. Results demonstrated that through regulation of organoid size and shape using quick reaggregation methods, retinal organoids were highly reproducible compared to more traditional methods. Additionally, the timed activation of BMP signaling within developing cells generated pure populations of retinal organoids at 100% efficiency from multiple widely used cell lines, with the default forebrain fate resulting from the inhibition of BMP signaling. Furthermore, given the ability to direct retinal or forebrain fates at complete purity, mRNA-seq analyses were then utilized to identify some of the earliest transcriptional changes that occur during the specification of these two lineages from a common progenitor. These improved methods also yielded retinal organoids with expedited differentiation timelines when compared to traditional methods. Taken together, the results of this study demonstrate the development of a highly reproducible and minimally variable method for generating retinal organoids suitable for analyzing the earliest stages of human retinal cell fate specification.
人多能干细胞（hPSC）衍生的视网膜类器官是三维细胞聚集体，可分化并自我组织，以紧密模拟人类视网膜的时空模式。视网膜类器官模型是研究人类视网膜发生的可靠工具，但目前视网膜类器官分化方案的效率和可重复性存在局限性，限制了这些模型在高通量应用（如疾病建模和药物筛选）中的使用。为了解决这些缺点，本研究旨在对先前的分化方案进行标准化，以产生一种高度可重复且高效的生成视网膜类器官的方法。结果表明，通过使用快速再聚集方法调节类器官的大小和形状，与更传统的方法相比，视网膜类器官具有高度的可重复性。此外，在发育细胞中定时激活 BMP 信号，可从多种广泛使用的细胞系中以 100%的效率生成纯视网膜类器官群体，通过抑制 BMP 信号产生默认的前脑命运。此外，鉴于能够以完全纯净的方式指导视网膜或前脑命运，然后利用 mRNA-seq 分析来鉴定在这两个谱系从共同祖细胞特化过程中发生的一些最早的转录变化。与传统方法相比，这些改进的方法还使视网膜类器官具有更快的分化时间线。总之，这项研究的结果表明，开发了一种高度可重复且最小变化的生成视网膜类器官的方法，适用于分析人类视网膜细胞命运特化的最早阶段。
