Characterization of Bruch's Membrane Formation in Human Fetal Retina and De Novo Membrane Synthesis by hPSC-Derived Retinal Pigment Epithelium

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Purpose: Little is known about the development of Bruch's membrane (BrM), the structure separating and supporting the retina and choroid, nor whether differentiation of human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) accurately replicates BrM. This has relevance for tissue engineering strategies, both in the development of accurate in vitro models, and effective RPE transplant strategies. Here, we investigated BrM-associated protein production in human fetal tissue and hPSC-derived RPE. Methods: The presence of laminin, elastin, fibronectin, and types I/III/IV collagen was examined in human fetal eyes at 6 to 21 post-conception weeks (PCWs) and hPSC-derived RPE cultures at 1 to 6 weeks in culture using immunohistochemistry/immunocytochemistry and quantitative PCR (qPCR). Results: In human fetal retina, laminin and fibronectin were present from 6 PCW, type IV collagen from 8 PCW, elastin from 12 PCW, type I collagen by 17 PCW, and type III collagen from 21 PCW. BrM layering was discernible from 12 PCW, becoming distinct by 17 PCW. In hPSC-derived RPE cultures, basement membranes containing laminin and fibronectin were present from week 1, type IV collagen from week 2, and type I collagen from week 4. Type III collagen was present at all timepoints, although not localized as a basement membrane. Elastin was absent at all timepoints. Conclusions: BrM-like membrane synthesis in hPSC-derived RPE largely recapitulates the temporal sequence seen in human development, excluding elastin. These support the utility of hPSC-derived RPE in in vitro systems to model RPE/retina interactions in health and disease, and inform cell therapy approaches, as de novo BrM-like membrane has the potential to support transplanted donor RPE.

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