Generation of vascularised immunocompetent retinal assembloids from human pluripotent stem cells

The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. Most drug studies are performed in vivo in experimental animals, but this approach is far from optimal because there are fundamental, structural and functional differences between the rodent and human retina. The advent of organoid technology has made possible the generation of three dimensional (3D) self-organised laminated and light-responsive retinal organoids from pluripotent stem cells (PSCs), a powerful tool for disease modelling and drug discovery. However the lack of microglia and vasculature currently limits their utility in two ways: firstly, it is known that microglia and vasculature play an important role in retinal development and disease, and secondly, the vascular system is necessary to prevent formation of necrosis in tissues  that grow  beyond 500µm diameter because of impaired diffusion of oxygen and nutrients. The vascular cells in the retina interact closely with the retinal neurons and glial cells, forming the retinal neurovascular unit (NVU), which together with the retinal pigment epithelium (RPE) form the core of the blood retina barrier (BRB). The BRB mediates highly selective diffusion of molecules from the blood to the retina and maintains retinal homeostasis. Alterations of the BRB play a crucial role in the development of retinal diseases, thus dynamic modelling of human BRB in highly desirable.

Current conventional co-culture models of retinal organoids with endothelial and microglia cells bypass the developmental processes that lead to formation of spatially organised retinal NVU and BRB. Furthermore, the mesodermal origin of microglia and vascular cells, and ectodermal origin of neural retina, provide a significant barrier for the generation of vascularised immunocompetent retinal organoids incorporating NVU/BRB. Our aim is to reach beyond the state of the art to combine multiple organoid types in 3D culture to generate vascularised immunocompetent retinal assembloids that combine the neural and mesodermal cells and provide all the components of retinal NVU/BRB. To achieve this goal, we will generate PSC-vascular organoids that contain endothelial cells, pericytes, smooth muscle, astrocytes and microglia, and assemble those with novel blood-generating heart-forming and retinal organoids to make vascular immune competent functional retinal assembloids. Combining cardiac contractility with blood-like flow can provide significant improvements to the development and function of retinal assembloids in a tractable fashion with the potential to reduce animal usage and increase their use in drug discovery and disease modelling.