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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 296

Statistical analysis
The Kolmogorov-Smirnov test was used to test for normality. Significance was investigated as per the
retinal organoid yield quantification. To compare organoids from two timepoints, a Student’s t-test was
performed, and a P-value < 0.05 was considered significant.

RESULTS
To investigate the efficacy of retinal organoid differentiation protocols, we differentiated the same hiPSC
[15]
line to retinal organoids using three differentiation protocols termed Method 1 (based on Wahlin et al. ),
[14]
[30]
Method 2 (based on Zhong et al. ), and Method 3 (based on Capowski et al. ) [Figure 1A]. First, we
investigated whether each differentiation method could successfully generate retinal organoids. In our
hands, each differentiation method successfully produced retinal organoids showing a phase bright outer
rim, which corresponds to the developing neuroepithelium [14,15,30] , known as a characteristic morphological
feature of high-quality retinal organoids [Figure 1B]. To further confirm effective retinal differentiation, the
expression of a ganglion [36,37] and amacrine cell marker synuclein gamma (SNCG) and two early
[38]
photoreceptor markers recoverin (RCVRN) and cone-rod homeobox (CRX) [40,41] were confirmed in
[39]
retinal organoids from each differentiation method [Figure 1B]. We used these stains to evaluate the
histoarchitecture of the retinal organoids and observed an optimal lamination in the organoids from
Method 3.

The primary objective of this study was to identify the differentiation method which produced the highest
yield and best quality of retinal organoids. To determine the retinal organoid quantity, we counted the
retinal domains produced using Method 1 on day 10 and using Methods 2 and 3 on day 23 [Figure 2A and
B]. These timepoints were chosen based on recommendations given by Wahlin et al. and Zhong et al.
[14]
[15]
concerning the optimal timepoint for retinal domain excision. Method 3 produced strikingly more retinal
domains per differentiation (65 ± 27) than Method 1 (12.3 ± 11.2) and Method 2 (6.3 ± 6.7), despite some
variability. Furthermore, the retinal domains from Method 3 were more clearly defined, and therefore easier
to excise [Figure 2A]. The number of retinal domains which matured to retinal organoids was counted on
day 63. Again, Method 3 produced more retinal organoids than Method 2 (24.7 ± 17.2 vs. 1.7 ± 0.6)
[Supplementary Figure 1].

To investigate the quality of retinal organoids produced by the three different methods, we quantified the
expression of several retinal markers in 85-day-old retinal organoids. This timepoint was chosen, since the
cellular composition of retinal organoids at this timepoint has been well documented by previous
groups [14,30,42] . To control for regional variability, we analyzed whole cryosections. First, we investigated the
expression of CRX, a transcription factor which is one of the earliest genes expressed in photoreceptor
precursors, as well as mature photoreceptors [40,41,43] . Retinal organoid cryosections were immunostained for
CRX [Figure 2C], and the CRX-positive area was quantified relative to the DAPI-positive area (blue
staining). We found the highest CRX expression in the retinal organoids differentiated using Method 3 (25%
± 5.3%), while the CRX expressions for Methods 1 (5.7% ± 4.8%) and 2 (8.6% ± 2%) were significantly lower
than that of Method 3 but comparable to each other [Figure 2D]. Next, we investigated the expression of
BRN3A, a transcription factor and an early retinal ganglion cell marker [44-46] [Figure 2E]. Again, the retinal
organoids produced by Method 3 showed the highest BRN3A expression (Method 1: 0.3% ± 0.5%; Method
2: 0.3% ± 0.4%; Method 3: 1.8% ± 0.3%) [Figure 2F]. In contrast, the different methods did not have a
significant effect on the expression of the amacrine cell marker transcription factor AP-2-alpha, the
ganglion cell marker SNCG, and the photoreceptor marker RCVRN [Supplementary Figure 2].