Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines

TLDR

Numerous protocols generate neural retina from human pluripotent stem cells as 3D organoids, yet variability within and between organoids over time and across different hPSC lines complicates their use. The study aimed to evaluate the capabilities and limitations of retinal organoid cultures by generating organoids from 16 hPSC lines and tracking their structural development. Organoids were monitored over time using light microscopy, immunocytochemistry, metabolic imaging, electron microscopy, optical coherence tomography, and 3D imaging to assess whole or broad regions and reduce selection bias. The results enabled the creation of a practical staging system that reduces inconsistencies in retinal organoid cultures and enhances rigor for developmental studies, disease modeling, and transplantation.

Abstract

Numerous protocols have been described that produce neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. While nearly all such methods yield at least partial segments of highly mature-appearing retinal structure, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to better understand the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging, and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.