Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno-Free and Feeder-Free Conditions

Abstract

Human induced pluripotent stem cells (hiPSCs) are potentially useful in regenerative therapies for retinal disease. For medical applications, therapeutic retinal cells, such as retinal pigmented epithelial (RPE) cells or photoreceptor precursors, must be generated under completely defined conditions. To this purpose, we have developed a two-step xeno-free/feeder-free (XF/FF) culture system to efficiently differentiate hiPSCs into retinal cells. This simple method, relies only on adherent hiPSCs cultured in chemically defined media, bypassing embryoid body formation. In less than 1 month, adherent hiPSCs are able to generate self-forming neuroretinal-like structures containing retinal progenitor cells (RPCs). Floating cultures of isolated structures enabled the differentiation of RPCs into all types of retinal cells in a sequential overlapping order, with the generation of transplantation-compatible CD73 photoreceptor precursors in less than 100 days. Our XF/FF culture conditions allow the maintenance of both mature cones and rods in retinal organoids until 280 days with specific photoreceptor ultrastructures. Moreover, both hiPSC-derived retinal organoids and dissociated retinal cells can be easily cryopreserved while retaining their phenotypic characteristics and the preservation of CD73 photoreceptor precursors. Concomitantly to neural retina, this process allows the generation of RPE cells that can be effortlessly amplified, passaged, and frozen while retaining a proper RPE phenotype. These results demonstrate that simple and efficient retinal differentiation of adherent hiPSCs can be accomplished in XF/FF conditions. This new method is amenable to the development of an in vitro GMP-compliant retinal cell manufacturing protocol allowing large-scale production and banking of hiPSC-derived retinal cells and tissues. Stem Cells 2017;35:1176-1188.

Dual SMAD inhibition and Wnt inhibition enable efficient and reproducible differentiations of induced pluripotent stem cells into retinal ganglion cells

“…[62] The long-term cultures of retinal organoids (RO) when supplemented with B27 allowed the differentiation of all retinal cell types including RGCs. [63] The RGC survival in these ROs derived retinal cultures decreased in vitro as the culture time was extended as floating cultures were a hinderance to the normal RGC growth and induced apoptosis. Adherent cultures obtained by enzymatic dissociation of ROs greatly improved the survival of these RGC [64] Several protocols following this study, differentiated iPSCs to neural rosettes and embryoid bodies that were further differentiated into RGCs using chemically defined media.…”

CRISPR-Cas Genome Surgery in Ophthalmology

“…Further development of our ability to differentiate CRISPR-Cas modified iPSCs into retinal, corneal and other cell types, as well as methods for autologous transplantation, could make sight-saving advances in numerous genetic diseases of the eye possible. 99–101 Overall, the future is bright for the use of CRISPR-Cas systems in ophthalmology, and it is likely that the important experiments discussed in this review represent a small sampling of what is to come.…”

Improved Ocular Tissue Models and Eye-On-A-Chip Technologies Will Facilitate Ophthalmic Drug Development

“…Increased automation Generation of storable retinal organoids from human-induced pluripotent stem cells in xeno-free and feeder-free conditions 33 reproducibility and validation. These barriers are not trivial and will require efforts from both biological and technical fields to address.…”