Characterisation of Bruch’s membrane formation in human fetal retina and de novo membrane synthesis by hPSC-derived retinal pigment epithelium

Purpose. Little is known about the development of Bruch’s membrane (BrM), the structure that separates and supports the retina and choroid, nor whether this is accurately replicated during differentiation of human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE). This has relevance for tissue engineering strategies, both in the generation of accurate in vitro models, as well as the development of effective RPE transplant strategies. Here, we investigated BrM-associated protein production in human fetal tissue and hPSC-derived RPE.<br/>Methods. The presence of Laminin, Elastin, Fibronectin and Types I/III/IV Collagen was examined in human fetal eyes at 6-21 post-conception weeks (PCW) and hPSC-derived RPE cultures at 1-6 weeks in culture using immunohistochemistry/immunocytochemistry and qPCR.<br/>Results. In human fetal retina, Laminin and Fibronectin were present from 6PCW, the earliest time point examined, Type IV Collagen from 8PCW, Elastin from 12PCW, Type I Collagen by 17PCW and Type III Collagen from 21PCW. BrM layering was discernible from 12PCW, at the start of Elastin deposition, becoming distinct by 17PCW. 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, though not localised as a basement membrane. Elastin was absent at all timepoints. <br/>Conclusion. BrM-like membrane synthesis in hPSC-derived RPE cultures largely recapitulates the temporal sequence seen in human fetal development, except for deposition of Elastin, and some lamination is apparent. 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.<br/>