Abstract
Purpose :
Bruch’s membrane (BrM) is a penta-laminar, acellular substrate separating the retina and choroid that provides structural support for retinal pigment epithelium (RPE), without which the retina degenerates. While age- and disease-related changes to BrM have been comprehensively studied, little is known about human BrM development. In addition, while it has been reported that human pluripotent stem cell (hPSC)-derived RPE can synthesise BrM-associated proteins in vitro, this has yet to be thoroughly characterised. To develop effective RPE transplant strategies, it is important to understand the in vivo development of BrM and its de novo synthesis by hPSC-derived RPE in vitro. Here, we investigated BrM-associated extracellular matrix protein production in human fetal tissue and hPSC-derived RPE.
Methods :
The presence of known BrM markers (Laminin, Elastin, Type I/IV Collagen) was examined in human fetal eyes between 6 and 21 post conception weeks (PCW) and aged adults using immunohistochemistry (n=1/timepoint). RPE was differentiated from hPSCs, passaged, then cultured for up to 6 wks. Presence of BrM markers was assessed weekly with immunocytochemistry (n=3/tp).
Results :
In fetal retina, Laminin was present and localised basally to the RPE in all timepoints, Type IV Collagen from 8PCW, Elastin from 12PCW and Type I Collagen only visible from 21PCW. BrM layering, whereby the central Elastin layer is visible between Laminin+ve layers, was discernible from 12PCW, becoming more defined in 17 and 21PCW. Laminin was present in hPSC-derived RPE cultures from wk1. Deposits of Type IV Collagen were visible at wk1 and visible as fibrous strands from wk2 onwards. Similarly, Type I Collagen was visible at wks 1-3 and became more fibrous from wk4. Elastin was observed in one sample at wk6. Barring Elastin, the sequence in which BrM proteins are synthesised in fetal retina is recapitulated in hPSC-derived RPE cell cultures.
Conclusions :
Here, we provide the first time-course of BrM protein expression in human BrM development and demonstrate that BrM-like synthesis in hPSC-derived RPE cultures largely recapitulates the sequence seen in development. These findings will inform cell therapy approaches for treatment of AMD, as de novo BrM-like membrane may have the potential to support donor RPE in a transplant scenario, with/without additional artificial support.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.