Maintaining retinal ganglion cells within human retinal organoids by implementing a vascular system.

• Applicant: Professor Dr. Volker Busskamp, Ph.D.
• Subject Area: Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 531985111

Stem cell technology has boosted the life sciences over the last decades, especially the bottom-up generation of human tissues from stem cells, so-called organoids, are now enabling new experimental approaches. We are interested in exploiting human retinal organoids for our research. However, we face severe limitations and shortcomings of this technology. As in most brain organoids, only neural cell types, glia and neurons are present. The lack of vascularization leads to the development of a necrotic core. Neighboring cells are negatively impacted and degenerate over time. Within retinal organoids, retinal ganglion cells (RGCs) reside inside the tissue and degenerate shortly after development. RGCs normally form a defined retinal layer and their axons are bundled as the optic nerve, relaying visual information to higher brain areas. However, RGC morphology and function is not recapitulated in retinal organoids. It takes about 30 weeks until the photoreceptors become light-sensitive but RGCs are already degenerated by week 16. To overcome these structural and functional limitations, we hypothesize that the implementation of a vascular system within developing retinal organoids will prevent the formation of the necrotic core and thereby inhibit RGC degeneration. The vascularization will enhance oxygen and nutrient supply to the inner part of the organoids. We take advantage of our previously described transgenic stem cell line in which a transcription factor, ETV2, can be induced leading to almost 100% endothelial differentiation in four days. To this end, we will add stem-cell derived endothelial cells to growing organoids or we will forward program stem cells into endothelial cells in situ. Our preliminary data look very promising. Retinal organoids become significantly larger and apoptotic rates around week 16 are significantly reduced. However, individual parameters of our technology require extensive and systematic modifications to obtain a robust protocol for vascularized retinal organoids. We need to perform in-depth quality control of retinal cell types, cellular morphologies and retinal laminations using imaging techniques. Retinal cell types will also be studied at the transcriptome level using single cell RNA sequencing. Importantly, we are also keen on revealing the functional improvements of RGC survival and function. We will perform micro-electrode array (MEA) recordings over time. We will also develop microchannel devices that are placed on MEAs that RGCs from retinal organoids send their axons via microchannels onto the electrode area. We will record light-evoked neuronal activity originating from the retinal organoids from these postsynaptic neurons, providing a functional simplified assembloid model for functional retinal studies using organoids. Overall, implementing a vascular system within neuronal organoids is an essential step to enlarge the applications of this fascinating system in basic and biomedical research.

• DFG Programme: Research Grants