Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 116, Issue 22, Pages 10824-10833Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1901572116
Keywords
retinal organoid; retinal differentiation; RNA-seq; single cell; cone and rod photoreceptor
Categories
Funding
- New York State Stem Cell Science Grant [C029154]
- BrightFocus Grant [M2012044]
- Retina Research Foundation
- National Eye Institue [R01EY022645, R01EY012200, R01EY014237, EY014800, R01EY018571, R01EY022356, R01EY026065, R01EY17353]
- Research to Prevent Blindness
- Carl Marshal AMP
- Mildred Almen Reeves Foundation
- Macular Degeneration Foundation
- National Institute of General Medical Sciences [T32GM007491]
- Yale Vision Science Core [P30EY026878]
- NIH [S10OD018033, S10OD023469, P30EY002520]
- Empire State Stem Cell Fund through New York State Department of Health [C30292GG]
- [P30CA013330]
- NATIONAL EYE INSTITUTE [P30EY002520, P30EY014800] Funding Source: NIH RePORTER
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Rod and cone photoreceptors are light-sensing cells in the human retina. Rods are dominant in the peripheral retina, whereas cones are enriched in the macula, which is responsible for central vision and visual acuity. Macular degenerations affect vision the most and are currently incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids differentiated from hESCs using an improved retinal differentiation system. Induced by extracellular matrix, aggregates of hESCs formed single-lumen cysts composed of epithelial cells with anterior neuroectodermal/ectodermal fates, including retinal cell fate. Then, the cysts were en bloc-passaged, attached to culture surface, and grew, forming colonies in which retinal progenitor cell patches were found. Following gentle cell detachment, retinal progenitor cells self-assembled into retinal epithelium-retinal organoid-that differentiated into stratified cone-rich retinal tissue in agitated cultures. Electron microscopy revealed differentiating outer segments of photoreceptor cells. Bulk RNA-sequencing profiling of time-course retinal organoids demonstrated that retinal differentiation in vitro recapitulated in vivo retinogenesis in temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-sequencing profiling of 8-mo retinal organoids identified cone and rod cell clusters and confirmed the cone enrichment initially revealed by quantitative microscopy. Notably, cones from retinal organoids and human macula had similar single-cell transcriptomes, and so did rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-rich retinal organoids and a reference of transcriptomes that are valuable resources for retinal studies.
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