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Berber et al. J Transl Genet Genom 2021;5:292-303 Journal of Translational
DOI: 10.20517/jtgg.2021.35
Genetics and Genomics

Original Article Open Access

Retinal organoid differentiation methods determine
organoid cellular composition

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1
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Patricia Berber , Andrea Milenkovic , Lisa Michaelis , Bernhard H. F. Weber 1,2
1
Insitute of Human Genetics, University of Regensburg, Regensburg 93053, Germany.
2
Institute of Clinical Human Genetics, University Hospital Regensburg, Regensburg 93053, Germany.
Correspondence to: Prof. Dr. Bernhard H. F. Weber, Institute of Human Genetics, University of Regensburg, Franz-Josef-Strauss-
Allee 11, Regensburg 93053, Germany. E-mail: bweb@klinik.uni-regensburg.de
How to cite this article: Berber P, Milenkovic A, Michaelis L, Weber BHF. Retinal organoid differentiation methods determine
organoid cellular composition. J Transl Genet Genom 2021;5:292-303. https://dx.doi.org/10.20517/jtgg.2021.35
Received: 8 Jul 2021 First Decision: 3 Aug 2021 Revised: 11 Aug 2021 Accepted: 23 Aug 2021 First online: 25 Aug 2021

Academic Editor: Sanjay Gupta Copy Editor: Yue-Yue Zhang Production Editor: Yue-Yue Zhang

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.

© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0
International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing,
adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as
long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and
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