Abstract
Purpose :
Human retinal organoids (hRO) take a long time to develop. It typically takes more than 200 days for photoreceptors to become light sensitive. Meanwhile, early born retinal ganglion cells (RGCs) within the organoid core begin to degenerate, likely due to limited nutrient and oxygen supply as well as lack of axon guidance. We wondered whether adding a vascular system to growing organoids would improve RGC survival and whether providing axon projections for RGCs might result in functionally active hROs.
Methods :
We generated inducible human stem cell-derived endothelial cells (ECs) that we added to growing hROs. We genomically integrated an optogenetic tool (ChRimson fused to yellow fluorescent protein) driven by the BRN3A promoter into the starting human induced pluripotent stem cell line to specifically target RGCs. The hROs were generated using the AMASS protocol. After eight weeks, hROs were placed within multicompartment microfluidic devices aligned with electrodes of multielectrode arrays (MEA). Microfluidic microchannels guided RGCs axonal outgrowth on MEA electrodes. Spontaneous and light-induced RGC activity was recorded and live cell fluorescence microscopy was performed. At least three independent batches for vascularized and non-vascularized hRO were analyzed.
Results :
The hRO and EC co-cultures resulted in the formation of thin capillaries inside the hRO layers and larger lumens with openings at the hRO surface. Vascularized hRO were enlarged and showed less apoptosis. After 8 weeks of hRO age, we detected fluorescent RGCs within the hRO and subsequently axons entering the microchannels. At 12 weeks, we recorded robust spontaneous and light-evoked neuronal activity up to 60 Hz from RGC axons traversing the MEA sensing area.
Conclusions :
The addition of a vascular system is extremely beneficial for the physiology of hROs. Microfluidic devices combined with MEAs provide a stable microenvironment and facilitate channelling RGC axons to the electrode surface for functional recordings. Delivery of optogenetic tools to RGCs provides robust induction of RGC function. Overall, our work demonstrates that advanced vascularized and optogenetically modified hROs can be used for functional studies, overcoming some limitations of these human retinal model systems.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.