SummaryPluripotent stem cells can be differentiated into 3D retinal organoids, with major cell types self-patterning into a polarized, laminated architecture. In static cultures, organoid development may be hindered by limitations in diffusion of oxygen and nutrients. Herein, we report a bioprocess using rotating-wall vessel (RWV) bioreactors to culture retinal organoids derived from mouse pluripotent stem cells. Organoids in RWV demonstrate enhanced proliferation, with well-defined morphology and improved differentiation of neurons including ganglion cells and S-cone photoreceptors. Furthermore, RWV organoids at day 25 (D25) reveal similar maturation and transcriptome profile as those at D32 in static culture, closely recapitulating spatiotemporal development of postnatal day 6 mouse retina in vivo. Interestingly, however, retinal organoids do not differentiate further under any in vitro condition tested here, suggesting additional requirements for functional maturation. Our studies demonstrate that bioreactors can accelerate and improve organoid growth and differentiation for modeling retinal disease and evaluation of therapies.

SUMMARY Mutations in CEP290 cause ciliogenesis defects, leading to diverse clinical phenotypes, including Leber congenital amaurosis (LCA). Gene therapy for CEP290-associated diseases is hindered by the 7.4 kb CEP290 coding sequence, which is difficult to deliver in vivo. The multi-domain structure of the CEP290 protein suggests that a specific CEP290 domain may complement disease phenotypes. Thus, we constructed AAV vectors with overlapping CEP290 regions and evaluated their impact on photoreceptor degeneration in Cep290rd16/rd16 and Cep290rd16/rd16;Nrl—/— mice, two models of CEP290- LCA. One CEP290 fragment (the C-terminal 989 residues, including the domain deleted in mutant mice) reconstituted CEP290 function and resulted in cone preservation and delayed rod death. The CEP290 C-terminal domain also improved cilia phenotypes in mouse embryonic fibroblasts and iPSC-derived retinal organoids carrying the Cep290rd16 mutation. Our study strongly argues for in trans complementation of CEP290 mutations by a cognate fragment and suggests therapeutic avenues.

Numerous degenerative diseases affecting visual function, including glaucoma and retinitis pigmentosa, are produced by the loss of different types of retinal cells. Cell replacement therapy has emerged as a promising strategy for treating these and other retinal diseases. The retinal margin or ciliary body (CB) of mammals has been proposed as a potential source of cells to be used in degenerative conditions affecting the retina because it has been reported it might hold neurogenic potential beyond embryonic development. However, many aspects of the origin and biology of the CB are unknown and more recent experiments have challenged the capacity of CB cells to generate different types of retinal neurons. Here we review the most recent findings about the development of the marginal zone of the retina in different vertebrates and some of the mechanisms underlying the proliferative and neurogenic capacity of this fascinating region of the vertebrates eye. In addition, we performed experiments to isolate CB cells from the mouse retina, generated neurospheres and observed that they can be expanded with a proliferative ratio similar to neural stem cells. When induced to differentiate, cells derived from the CB neurospheres start to express early neural markers but, unlike embryonic stem cells, they are not able to fully differentiate in vitro or generate retinal organoids. Together with previous reports on the neurogenic capacity of CB cells, also reviewed here, our results contribute to the current knowledge about the potentiality of this peripheral region of the eye as a therapeutic source of functional retinal neurons in degenerative diseases.