July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
RNAseq analysis of in vitro retinal organoids shows comparable pathway expression to developing eye in vivo
Author Affiliations & Notes
  • Philip Wagstaff
    Department of Clinical Genetics, Amsterdam University Medical Center, Amsterdam, Netherlands
  • Anneloor ten Asbroek
    Department of Clinical Genetics, Amsterdam University Medical Center, Amsterdam, Netherlands
  • Valeria Lo Faro
    Department of Ophthalmology, University Medical Center Groningen, Groningen, Netherlands
  • Aldo Jongejan
    Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Amsterdam University Medical Center, Amsterdam, Netherlands
  • Camiel Boon
    Department of Ophthalmology, Amsterdam University Medical Center, Amsterdam, Netherlands
    Department of Ophthalmology, Leiden University Medical Center, Leiden, Netherlands
  • Clara van Karnebeek
    Department of Pediatric Metabolic Diseases, Amsterdam University Medical Center, Amsterdam, Netherlands
  • Frederik Wijburg
    Department of Pediatric Metabolic Diseases, Amsterdam University Medical Center, Amsterdam, Netherlands
  • Ronaldus Wanders
    Department of Genetic Metabolic Diseases, Amsterdam University Medical Center, Amsterdam, Netherlands
  • Elizabeth Meijers-Heijboer
    Department of Clinical Genetics, Amsterdam University Medical Center, Amsterdam, Netherlands
  • Nomdo M Jansonius
    Department of Ophthalmology, University Medical Center Groningen, Groningen, Netherlands
  • Arthur A Bergen
    Department of Clinical Genetics, Amsterdam University Medical Center, Amsterdam, Netherlands
    Department of Ophthalmology, Amsterdam University Medical Center, Amsterdam, Netherlands
  • Footnotes
    Commercial Relationships   Philip Wagstaff, None; Anneloor ten Asbroek, None; Valeria Lo Faro, None; Aldo Jongejan, None; Camiel Boon, None; Clara van Karnebeek, None; Frederik Wijburg, None; Ronaldus Wanders, None; Elizabeth Meijers-Heijboer, None; Nomdo Jansonius, None; Arthur Bergen, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 3334. doi:https://doi.org/
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      Philip Wagstaff, Anneloor ten Asbroek, Valeria Lo Faro, Aldo Jongejan, Camiel Boon, Clara van Karnebeek, Frederik Wijburg, Ronaldus Wanders, Elizabeth Meijers-Heijboer, Nomdo M Jansonius, Arthur A Bergen; RNAseq analysis of in vitro retinal organoids shows comparable pathway expression to developing eye in vivo. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3334. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Retinal diseases, like glaucoma and age-related macular degeneration, form a major global health problem. There is no effective cure for these diseases, largely due to the lack of suitable models for these disorders. Lately, a lot of research is focused on the development of a new type of in vitro model, representing all retinal cell types: retinal organoids. It is important when considering the use of these organoids for disease models and treatments that they are comparable to the in vivo situation, both in the expression of important retinal genes, and the developmental timeline. To confirm this, we performed an in-depth RNAseq analysis to look at the developmental pathways compared to the developing eye.

Methods : Human-derived stem cells (hESCs or iPSCs) were encased in 3D Matrigel drops for 4 days in a mixture of iPSC medium and neural induction medium, before being plated on matrigel coated 6 well plates for 7 days, and lifted off to form retinal organoids. These organoids were grown for a total of 160 days, being dissociated and grown as single cells at specific intervals. Organoid samples were taken at important stages during the differentiation process, up to the formation of ganglion cells, and taken forward for RNAseq analysis.

Results : We found that the initial encasement of embryoid bodies in matrigel boosted the growth of our organoids. We could generate retinal ganglion cells within 28 days without the addition of other external factors to direct cells towards this fate. We have also been able to generate all other retinal cell types, confirmed by sq-PCR and immunohistochemistry using both developmental and cell specific markers. These cells include retinal pigmented epithelium and photoreceptors. The RNAseq showed similar developmental pathways were upregulated at certain timepoints, comparable to the in vivo developing eye.

Conclusions : Our results present a developmentally similar method of generating retinal ganglion cells in a shorter timeframe compared to already published protocols using RNAseq analysis. This will not only allow us to study the development and morphology of the emerging eye with different degenerative disease models, but also gives us the opportunity to generate multiple different cell types that can be used for transplantations in a variety of animal disease models and ultimately, human transplantations in the future.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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