Abstract / Summary
Abstract The retinal pigment epithelium (RPE) plays a central role in maintaining retinal function, and its degeneration contributes to diseases such as age-related macular degeneration. The human amniotic membrane (hAM), an ethically accessible tissue with immune privilege and epithelial organization, has been proposed as a potential source for RPE-like cells, particularly in its epithelial cell component. In this study, we investigated the baseline expression of RPE markers in freshly collected hAM to assess interindividual variability. In addition, we compared the behavior of intact hAM sheets and isolated human amniotic epithelial cells (hAECs) in culture, both under maintenance conditions and after treatment with RPE-inductive factors. Fresh hAM fragments and isolated hAECs were analyzed for RPE-associated markers and cultured either as intact epithelial sheets or isolated monolayers. Morphology, gene expression, and protein localization were assessed over time under standard conditions and following exposure to RPE-inductive factors noggin (NOG), CHIR99021 (CHIR), nicotinamide (NIC), and trichostatin A (TSA). Our results show that fresh hAM and isolated hAECs inherently express several RPE-associated markers, although with marked donor-dependent variability. hAM fragments preserved epithelial morphology and marker expression more effectively than isolated hAECs. Stimulation with NOG and CHIR variably modulated RPE-associated marker expression and localization, with largely consistent treatment-related trends across donors. NIC + TSA produced only limited modulation of RPE-associated features and was associated with a progressive reduction in nuclear density, even at the lower TSA concentration tested. Variability in responsiveness compared to previous studies suggested that inter-placental heterogeneity might also affect the response to RPE-inductive cues. Epithelial cells within hAM display donor-dependent RPE-like priming that is better preserved in the native sheet context. Intact hAM may therefore provide a useful system for refining RPE-induction strategies while accounting for donor variability and preserving a tissue format that may be advantageous for future retinal repair applications.