Title: Retinal Organoids Derived from Human Pluripotent Stem Cells Recapitulate in Vivo Outer Retina
Legend: Stage 3 organoids possess an outer plexiform-like layer (OPL), ribbon synapses, and an inner nuclear layer (INL) containing rod and cone bipolar cells (BPCs). (A-D) ICC images of a stage 3 organoid (d220) showing two distinct nuclear layers (asterisks) separated by a thin nuclei-free zone (arrow) (A). Expression of ARR3 in cone pedicles (B) overlaps with expression of the PR synapse marker VGLUT (C; merge in D) in the OPL (scale bar = 50 mm). (E, E’) Immunostaining in a d225 stage 3 organoid for the ribbon synapse marker CTBP2 (also known as RIBEYE), which co-localizes with ARR3+ cone pedicles in the OPL. E’ is a magnified image of the region within the hatched square in panel E (scale bars = 50 mm). (F, G) ICC of ARR3+ cones and RHO+ rods demonstrating pedicle and spherule morphology, respectively (asterisks; scale bar = 10 mm). (H) Electron micrograph demonstrating ribbon synapses in a d212 stage 3 organoid. (scale bar = 500 nm).
Citation: Capowski EE, Samimi K, Mayerl SJ, Phillips MJ, Pinilla I, Howden SE, Saha J, Jansen AD, Edwards KL, Jager LD, Barlow K, Valiauga R, Erlichman Z, Hagstrom A, Sinha D, Sluch VM, Chamling X, Zack DJ, Skala MC, Gamm DM. (2018). Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines. Development. 2018 Dec 19. pii: dev.171686. doi: 10.1242/dev.171686.
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.
About the Lab: David Gamm’s laboratory at the Waisman Center uses stem cell technology to investigate the cellular and molecular events that occur during human retinal differentiation and to generate cells for use in human retinal disease modeling and cell-based rescue or replacement strategies. To meet these goals, Gamm utilizes a variety of human cell types, including ES and iPS cells, which have the capacity to mimic retinal development and disease, as well as to delineate the genetic “checkpoints” necessary to produce particular retinal cell types. By understanding the behavior of these cell types in vitro and in vivo, Gamm hopes to optimize strategies to delay or reverse the effects of blinding disorders such as retinitis pigmentosa and age–related macular degeneration.