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Page 293 Berber et al. J Transl Genet Genom 2021;5:292-303 https://dx.doi.org/10.20517/jtgg.2021.35

Keywords: Retina, retinal organoid, BMP4, photoreceptor

INTRODUCTION
Organoids are three-dimensional (3D) in vitro miniature organs, which contain multiple organ-specific cell
[1]
types and a comparable spatial organization to the native tissue . To date, differentiation protocols for
multiple organoid types exist, including cerebral organoids , intestinal organoids , lung organoids ,
[8,9]
[6,7]
[2-5]
and kidney organoids , among many others. Organoid research was pioneered in 2005 when the Sasai
[10]
group developed a protocol to selectively differentiate murine embryonic stem cells to neurons [2,11] . In the
following years, the same group developed methods to differentiate murine and later human embryonic
stem cells to optic cups [12,13] . These advances served as a cornerstone for other groups to develop human
induced pluripotent stem cell (hiPSC) derived retinal organoid differentiation protocols [14-16] .

Retinal organoids provide many exciting new avenues of research, in part filling a void created by the
incongruencies between animal models and human diseases. hiPSC derived retinal organoids contain the
main cell types native to the retina: rod and cone photoreceptors, bipolar cells, horizontal cells, amacrine
cells, ganglion cells, and Müller cells. The development and molecular profile of the organoid cells appear to
be fairly similar to their native counterparts [17,18] . Recently, retinal organoids have also been used to model
inherited retinal dystrophies [19-23] , although it seems that organoids may be best suited to model severe
phenotypes with an early disease onset.

To date, a variety of retinal organoid differentiation protocols have been established. Broadly, the
procedures can be classified into two main categories: 3D protocols and 3D-2D-3D protocols . The 3D
[24]
differentiation protocols are based on the serum-free embryoid body-like quick aggregation culture
technique developed by Eiraku et al. [2,11] and Watanabe et al. . In this technique, the cells are cultured in
[25]
suspension for the entire differentiation process (referred to as “3D” culture). Later, other groups added a
temporary adherent phase (the “2D” phase) after the aggregation step, which improved the retinal domain
[26]
development . Retinal domains are organoid precursors, which are excised and cultured in suspension
until they are harvested (the second “3D” phase) . Protocols from both main categories can be modified by
[26]
including a variety of possible extrinsic chemical signals to promote retinal differentiation or enhance cell
survival.

In this study, the efficacy of differentiation protocols from both main categories was investigated including a
[15]
protocol by Wahlin et al. which operates according to the 3D technique (Method 1). This differentiation
protocol utilizes a Wnt inhibitor (Inhibitor of Wnt response compound-1-endo, IWR-1e) to induce retinal
differentiation , a Hedgehog agonist [Smoothened agonist (SAG)] to enhance the survival of neural
[27]
cells , and a Notch signaling inhibitor {N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl
[28]
ester, DAPT} to increase photoreceptor yield . The differentiation protocol by Zhong et al. is based on
[14]
[29]
the 3D-2D-3D technique (Method 2) and uses minimal extrinsic inductive cues to facilitate autonomous
differentiation. We also tested an adaptation of Method 2 which includes a brief early exposure to bone
morphogenic protein 4 (BMP4), as developed by Kuwahara et al. and more recently demonstrated by
[16]
Capowski et al. (Method 3). The overall aim of this study was to compare quantity and quality of retinal
[30]
organoids produced by the three methods and to identify any discrepancies in cellular composition upon
long-term development.