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Berber et al. J Transl Genet Genom 2021;5:292-303 https://dx.doi.org/10.20517/jtgg.2021.35 Page 300

Method 3 only increased the proportion of BRN3A- and CRX-positive cells and not the proportion of AP-
2-alpha-, SNCG-, or RCVRN-positive cells. Each of these markers is expressed by a different subset of
retinal cells, and some markers are not exclusive to one cell type. For example, although BRN3A [44-46] and
SNCG [36,37] are both expressed in ganglion cells, there is evidence that some amacrine cells also express
SNCG . The AP-2-alpha amacrine cell marker which we investigated in this study was not significantly
[38]
altered as a consequence of the differentiation method. Taken together, these results indicate that Method 3
increases the proportion of ganglion cells but not amacrine cells in the retinal organoids. This underscores
that all three methods successfully generated retinal organoids, while the differentiation method
substantially alters their cellular composition. This is especially interesting when comparing Methods 2 and
3, since only a single extrinsic inductive stimulus (BMP4) significantly altered the cellular composition of
the ensuing retinal organoids.

BMP4 belongs to a family of signaling molecules which were initially discovered for their ability to induce
[49]
bone formation but are now known to play a number of crucial roles in other organ systems . Germline
[50]
deletion of BMP4 is embryologically lethal in mice between 6.5 and 9.5 days post conception . Conditional
[51]
deletion of BMP4 from the optic vesicle resulted in an optic cup devoid of neural retinal markers, indicating
that BMP4 is required for retinal specification . In vitro, BMP4 does not appear to be required for retinal
[52]
differentiation, since the protocols without BMP4 also produce organoids. Instead, it is possible that BMP4
exposure reinforces or amplifies retinal differentiation in vitro, while the timepoint of BMP4 exposure is
thought to be critical . An effect of the differentiation method on the ensuing organoid has previously
[53]
[54]
been observed in the study of cortical organoids. Tanaka et al. performed a synthetic comparative analysis
of single-cell transcriptome profiling of cortical organoids from eight different protocols and observed that
the cellular composition and differentiation routes varied across the individual differentiation protocols. In
line with these findings, Chichagova et al. compared two retinal organoid differentiation methods and
[55]
found that, with one of their hiPSC lines, one method was skewed towards an interneuron fate, whereas
another method skewed the retinal organoids towards a rod photoreceptor fate.

Ours and similar studies which compare retinal organoid differentiation protocols are vital for the scientific
community, especially since there are still some open questions. Based on our study, we should not surmise
that 3D-2D-3D techniques (such as Methods 2 and 3) are inherently superior to 3D techniques (such as
Method 1). In our hands, the organoids from Method 1 showed insufficient lamination despite staining
positive for several retinal cell types. Notably, the lamination in the original publication is above reproach. It
is rather speculative to question why Method 1 did not work better in our hands, especially because the
method was described very well in the original publication. Furthermore, retinal organoids are plagued by a
certain degree of variability . In our study, the quantification of the retinal domains showed some
[33]
variability, which may have been caused by small numbers and differences in the counting methodology. It
is plausible that weaker effects may have been masked by the variability.

To date, many retinal organoid differentiation protocols [18,42,56,57] have not been tested in side-to-side
comparisons by independent groups, most likely due to the considerable time commitment associated with
these protocols. To facilitate the comparability of experimental applications of retinal organoids across
studies, standardized retinal organoid differentiation protocols are needed, which should focus on the type
of differentiation technique and the extrinsic differentiation cues.

DECLARATIONS
Acknowledgments
We sincerely thank Prof. M. Valeria Canto-Soler (Department of Ophthalmology, University of Colorado