The retinal pigment epithelium (RPE)-Bruch?s Membrane (BrM)-choriocapillaris (CC) complex is a highly selective diffusional barrier for nutrients and metabolic wastes in the eye. Dysfunctions in the RPE-BrM-CC complex underlies eye pathologies such as age-related macular degeneration (AMD), the leading cause of adult blindness in the US. Within the RPE-BrM-CC, BrM is a multilayered tissue that divides RPE and CC and is the first structure to show anomalies in AMD, yet it is not clear whether RPE and/or CC dysfunction initiates BrM alterations. Murine models fail to recapitulate AMD since mice lack macula. The inability to use animal models or recreate a functional RPE-BrM-CC tissue complex limits our ability to investigate crucial aspects of eye diseases, including AMD, where the integrity of the entire tissue is compromised. Therefore, we hypothesize that by using developmentally-inspired cues, a functional tissue mimetic can be developed for ex vivo study. Specifically, we will exploit human induced pluripotent stem cell (hiPSC) technology and tissue engineering to establish functional tissue mimetics. Because of their modularity, hiPSC-derived in vitro models allow the flexibility to study the role individual cell type(s) and intercellular interaction in disease pathophysiology. Our strong preliminary data demonstrate that hiPSC-RPE seeded onto RGDS-functionalized poly(ethylene glycol) (PEG) hydrogels are stable and become pigmented over 2-3 weeks. RPE layers deposit basement membrane, composed of some BrM components. Furthermore, CC-like vasculature, complete with Col6-postiive basement membrane, can be developed from hiPSC-endothelial cells (EC) and mesenchymal stem cells (MSC) entrapped within the PEG hydrogels underlying RPE. However, our current tissue mimetic lacks structurally complete BrM, which limits overall tissue mimetic structure and function. During development, RPE cells differentiate and become pigmented with support from underlying mesenchymal matrix and soluble cues. Then the CC layer develops while the mature BrM is deposited by coordinated interplay of the RPE and CC. Thus, to improve the mimetic, our aims are to 1) exploit developmentally-inspired cues to further enhance BrM structural development. In Aim 1a, the temporal introduction of the CC tissue mimetics after hydrogel- seeded RPE layer maturation/pigmentation and in Aim 1b, soluble mesenchymal cues via co-culture, will be explored to further promote BrM development. Aim 2 will assess the RPE-BrM-CC mimetic function as compared to in vivo levels and gold standard RPE culture models. Our expertise with engineering biomaterials to regulate the cell environment (Benoit) and eye physiology and hiPSC-derived RPE, MSC, and EC cell types is crucial to developing RPE-BrM-CC tissue mimetics using patient-derived cells. Successful completion of these aims would be a significant step towards ocular disease modeling and subsequent development of novel drug therapies for several retinal degenerative diseases, including age-related macular degeneration (AMD). !