Structural and Functional Characterization of Human Stem-Cell-Derived Retinal Organoids by Live Imaging.
Structural and Functional Characterization of Human Stem-Cell-Derived Retinal Organoids by Live Imaging.
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DOI：
10.1167/iovs.16-20796
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发表时间：
2017-07-01
影响因子：
4.4
通讯作者：
Cobrinik D
中科院分区：
文献类型：
--
作者：
Browne AW;Arnesano C;Harutyunyan N;Khuu T;Martinez JC;Pollack HA;Koos DS;Lee TC;Fraser SE;Moats RA;Aparicio JG;Cobrinik D
人类多能干细胞 (hPSC) 衍生的视网膜类器官是研究视网膜发育、病理生理学和细胞疗法的平台。与使用在不同时间固定的多个标本来重建发育过程的组织学分析相反，对相同活体类器官的重复分析提供了一种更直接的方法来表征变化。新的实时成像方式可以深入了解体外生长过程中的视网膜类器官结构和代谢功能。这项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) 检查不同发育阶段的活 hPSC 来源的视网膜类器官的显微解剖组织和代谢功能。将特征与固定类器官组织的组织学染色、免疫染色和微计算机断层扫描 (micro-CT) 所揭示的特征进行比较。我们使用 FLIM 和 HSpec 来检测类器官分化为有组织的片层时代谢活动的变化。 FLIM 检测到糖酵解活性增加，HSpec 检测到类器官外层中视黄醇和视黄酸的积累，这与光感受器的发生一致。 OCT 能够对类器官成熟过程中形成的片层进行成像。显微CT显示了三维结构，但未能检测到片层。当视网膜类器官组织成层状结构时，实时成像方式有助于对它们进行实时和无损成像。 FLIM 和 HSpec 能够快速检测层状结构和光感受器代谢。实时成像技术可能有助于在不同的实验和细胞治疗环境中持续评估视网膜类器官的发育。
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.