Browne Andrew W, Arnesano Cosimo, Harutyunyan Narine, Khuu Thien, Martinez Juan Carlos, Pollack Harvey A, Koos David S, Lee Thomas C, Fraser Scott E, Moats Rex A, Aparicio Jennifer G, Cobrinik David
USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States.
Translational Imaging Center, University of Southern California, Los Angeles, California, United States 3Department of Molecular and Computational Biology, University of Southern California, Los Angeles, California, United States.
Invest Ophthalmol Vis Sci. 2017 Jul 1;58(9):3311-3318. doi: 10.1167/iovs.16-20796.
Human pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating retinal development, pathophysiology, and cellular therapies. In contrast to histologic analysis in which multiple specimens fixed at different times are used to reconstruct developmental processes, repeated analysis of the same living organoids provides a more direct means to characterize changes. New live imaging modalities can provide insights into retinal organoid structure and metabolic function during in vitro growth. This study employed live tissue imaging to characterize retinal organoid development, including metabolic changes accompanying photoreceptor differentiation.
Live hPSC-derived retinal organoids at different developmental stages were examined for microanatomic organization and metabolic function by phase contrast microscopy, optical coherence tomography (OCT), fluorescence lifetime imaging microscopy (FLIM), and hyperspectral imaging (HSpec). Features were compared to those revealed by histologic staining, immunostaining, and microcomputed tomography (micro-CT) of fixed organoid tissue.
We used FLIM and HSpec to detect changes in metabolic activity as organoids differentiated into organized lamellae. FLIM detected increased glycolytic activity and HSpec detected retinol and retinoic acid accumulation in the organoid outer layer, coinciding with photoreceptor genesis. OCT enabled imaging of lamellae formed during organoid maturation. Micro-CT revealed three-dimensional structure, but failed to detect lamellae.
Live imaging modalities facilitate real-time and nondestructive imaging of retinal organoids as they organize into lamellar structures. FLIM and HSpec enable rapid detection of lamellar structure and photoreceptor metabolism. Live imaging techniques may aid in the continuous evaluation of retinal organoid development in diverse experimental and cell therapy settings.
人多能干细胞（hPSC）来源的视网膜类器官是研究视网膜发育、病理生理学和细胞治疗的一个平台。与使用在不同时间固定的多个标本重建发育过程的组织学分析不同，对同一活类器官进行重复分析提供了一种更直接的方法来表征变化。新的活体成像方式可以深入了解体外生长过程中视网膜类器官的结构和代谢功能。本研究采用活体组织成像来表征视网膜类器官的发育，包括伴随光感受器分化的代谢变化。
通过相差显微镜、光学相干断层扫描（OCT）、荧光寿命成像显微镜（FLIM）和高光谱成像（HSpec），对不同发育阶段的活hPSC来源的视网膜类器官进行微观解剖组织和代谢功能检查。将这些特征与固定类器官组织的组织学染色、免疫染色和微型计算机断层扫描（micro-CT）所显示的特征进行比较。
我们使用FLIM和HSpec来检测类器官分化为有组织的板层时代谢活性的变化。FLIM检测到糖酵解活性增加，HSpec检测到类器官外层视黄醇和视黄酸的积累，这与光感受器的发生相吻合。OCT能够对类器官成熟过程中形成的板层进行成像。Micro-CT揭示了三维结构，但未能检测到板层。
活体成像方式有助于对视网膜类器官组织成板层结构的过程进行实时和非破坏性成像。FLIM和HSpec能够快速检测板层结构和光感受器代谢。活体成像技术可能有助于在各种实验和细胞治疗环境中对视网膜类器官的发育进行持续评估。
