Retinal organoid differentiation methods determine organoid cellular composition

Abstract

Aim:We aimed to compare the quantity and quality of aging retinal organoids generated by applying three distinct differentiation protocols for human-derived induced pluripotent stem cells (hiPSC).Methods: hiPSC were differentiated to retinal organoids using a 3D technique (Method 1) and a 3D-2D-3D technique (Method 2), the latter modified by the addition of BMP4 (Method 3). To investigate the retinal organoid quantity, we counted the number of retinal domains, precursors to organoids during differentiation. The retinal organoid quality was evaluated by immunostaining for markers of different retinal cell types in whole cryosections after days 85, 120, and 200 in culture.Results: Method 3 produced strikingly more retinal domains per differentiation (65 ± 27) than Methods 1 (12.3 ± 11.2) and 2 (6.3 ± 6.7). Furthermore, retinal organoids differentiated with Method 3 contained significantly more CRX-positive photoreceptors and BRN3A-positive ganglion cells after 85 days in culture, compared to Methods 1 and 2. After 200 days in culture, the retinal organoids differentiated with Method 3 showed proper maturation, as demonstrated by the expression of mature rod and cone photoreceptor markers. Conclusion:This study demonstrates that the retinal organoid differentiation method can significantly impact the cellular composition of retinal organoids at various time points of development.

Transient Retention of Photoreceptor Outer Segments in Matrigel-Embedded Retinal Organoids

“…A technique was developed to simultaneously differentiate ROs and byproduct RPE (bRPE) ( Figure 2 ). Following existing protocols, ROs are excised from a 2D culture and further cultivated, while the non-RO cells are discarded [ 10 , 20 ]. In our modified protocol, the non-RO cells were treated with nicotinamide for several weeks to reinforce RPE differentiation [ 26 ].…”

“…ROs were differentiated according to Method 3 (M3) in Ref. [ 20 ], modified after [ 10 , 25 ]. Upon excision of the retinal domains on day 21–25, the remaining cells were used for bRPE differentiation.…”

“…ROs regularly develop ectopic clumps of RPE. ROs generally recapitulate their native retinal spatial organization, and form a ganglion, inner nuclear, and outer nuclear layer [ 10 , 19 , 20 , 21 , 22 ]. Still, there is an important incongruency between ROs and native tissue.…”

Bioengineering Human Pluripotent Stem Cell-Derived Retinal Organoids and Optic Vesicle-Containing Brain Organoids for Ocular Diseases

“…One limitation of retinal organoid engineering is the variability in organoid formation due to different formation methods. The differentiation procedure for retinal organoids can have a considerable impact on the cellular composition at different developmental stages [ 228 ]. The quantity and quality of mature retinal organoids produced by three different procedures hiPSCs were compared [ 228 ].…”

“…The differentiation procedure for retinal organoids can have a considerable impact on the cellular composition at different developmental stages [ 228 ]. The quantity and quality of mature retinal organoids produced by three different procedures hiPSCs were compared [ 228 ]. Standardized retinal organoid development methods are required, with an emphasis on the differentiation technique and the extrinsic differentiation cues, to enable the comparison of experimental applications of retinal organoids across research.…”