Background: Age-related macular degeneration (AMD) is a disease that affects the outer retina, causing the irreversible atrophy of the retinal pigment epithelium (RPE). Current therapies aim to delay the progression of the disease but fail to promote regeneration or offer a definitive cure. Regenerative medicine strategies have been tested, in which the damaged RPE was effectively replaced by autologous transplantation. However, this approach is likely applicable only to a limited number of patients.  
We have previously investigated the native expression of certain marker genes expressed by RPE in human amniotic epithelial cells (hAECs) derived from term placentas. Therefore, we examined the effect of specific factors known to play a key role during retinal embryogenesis in inducing full differentiation of hAECs into mature RPE. 

Methods: Term placentas were collected from uncomplicated cesarean sections, with informed consent from the mothers. hAECs were obtained from these placentas and subjected to an RPE differentiation protocol for up to 8 days. The protocol employed various doses and combinations of specific factors, including Sonic Hedgehog (a key morphogen involved in eye development and RPE specification), Noggin (a BMP antagonist), and CHIR99021 (a Wnt/β-catenin agonist). Samples were collected at different time points for gene expression analysis using qRT-PCR and for protein expression analysis using immunofluorescence. 

Results: Some of the selected factors, particularly when used in combination, promoted increased expression of typical RPE markers at both gene and protein level. In particular, Zonulin-1 (a tight junction protein) was detected at intercellular junctions at the end of treatment; the expression of transcription factor PAX6 was upregulated at day 8, although it only showed partial nuclear localization, suggesting the cells were not fully competent. On the other hand, transcription factor OTX2 was found to be functionally expressed at the nuclear level, but only in rare cells. We also found that the expression of certain RPE markers varied among different placental samples, suggesting that biological variability in the starting material may significantly influence the outcome of the differentiation protocol. 

Conclusion: The preliminary results of this study suggest that hAECs can acquire some phenotypical and functional features of RPE in vitro. These findings support the potential of hAECs as an accessible and ethically non-contentious source for generating RPE-like cells. However, the observed variability among samples highlights the need for further optimization of the differentiation protocol and standardization of starting material. Future studies should also aim to assess the functional integration and long-term stability of these cells in in vivo models of AMD.