June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Advanced vascularized human stem cell-derived retinal organoids
Author Affiliations & Notes
  • Volker Busskamp
    Dep. of Ophthalmology, Rheinische Friedrich-Wilhelms-Universitat Bonn, Bonn, Nordrhein-Westfalen, Germany
  • Kritika Sharma
    Dep. of Ophthalmology, Rheinische Friedrich-Wilhelms-Universitat Bonn, Bonn, Nordrhein-Westfalen, Germany
  • Footnotes
    Commercial Relationships   Volker Busskamp None; Kritika Sharma None
  • Footnotes
    Support  Volkswagen Foundation (Freigeist—A110720), the Paul Ehrlich Foundation (Frankfurt, Germany), and the Deutsche Forschungsgemeinschaft (BU 2974/4-1, EXC-2151-390873048-Cluster of Excellence—ImmunoSensation2 at the University of Bonn.
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 1894. doi:
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    • Get Citation

      Volker Busskamp, Kritika Sharma; Advanced vascularized human stem cell-derived retinal organoids. Invest. Ophthalmol. Vis. Sci. 2023;64(8):1894.

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

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Purpose : Human stem cell-derived retinal organoids (ROs) are an important tool to study retinal development and pathology in a dish. ROs also serve as a human photoreceptor source for cell transplantation therapies. However, human ROs struggle to prolong the survival of functional ganglion cells that are in the innermost part. Ganglion cells suffer from insufficient nutrient and oxygen supply leading to their early degeneration. ROs consist of only neural cell types; a vascular system does not form. We aimed to engineer a vasculature using differentiated endothelial cells to be incorporated into growing ROs to boost maturation, cell survival, and to obtain functional ganglion cell outputs.

Methods : We use the AMASS protocol to generate ROs taking the F49B7 human induced pluripotent stem cell line (hiPSC) that we further genomically modified with an endothelial differentiation and a fluorescent reporter cassette. Here, an isoform of the ETS Variant Transcription Factor 2 (ETV2) was driven from the inducible TetOn promoter system. The fluorescent reporter was under the constitutive EF1a promoter. Both cassettes were stably integrated in F49B7 hiPSCs named ETV2-cells. Live cell imaging, immunohistochemistry, qRT-PCR and flow cytometry were used to validate the formation and to investigate the impact of vasculature within RO. Non-vascularized ROs served as controls. Our data was reproduced in more than three independent RO batches.

Results : Upon induction, ETV2-cells underwent rapid and highly efficient differentiation into endothelial cells within five days. Fluorescent reporter expression was visible using live cell imaging. ETV2-cells were mixed with plain F49B7 hiPSCs to generate ROs. We performed live cell imaging over 30 weeks and detected fluorescent cells forming a vascular system in the inner core of the growing ROs. This led to a significant increase of tissue size compared to non-vascularized organoids. Additionally, the percentage of apoptotic cells was significantly reduced, indicating the beneficial impact of a vascular system on cell survival in ROs. An in-depth analysis on ganglion cell survival and function is ongoing.

Conclusions : Vascularized ROs have the potential to overcome current limitations such as apoptotic cores eliminating ganglion cells from these in vitro tissues. More advanced and physiological ROs are key to exploit these tissues for basic and biomedical vision research.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.


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