HUMAN AMNIOTIC EPITHELIAL CELLS FOR RETINAL REGENERATION: INVESTIGATING THEIR DIFFERENTIATIVE POTENTIAL IN VITRO

R. Pisu¹, S. Saponara², S. Angioni², E. Peiretti², S.G. Vitale², F. Marongiu¹ | ¹Department of Biomedical Sciences, University of Cagliari, Cagliari; ²Department of Surgical Sciences, University of Cagliari, Cagliari, Italy

The human amniotic membrane (hAM) has long been used in ophthalmology for its regenerative and immunomodulatory properties. Its emerging application in retinal repair -particularly for macular holes, retinal tears, and age-related macular degeneration (AMD)- typically focuses on structural or paracrine support, often overlooking its cellular components. Notably, human amniotic epithelial cells (hAECs), which form a natural monolayer on a basement membrane, resemble the architecture of the retinal pigment epithelium (RPE). This structural similarity suggests the potential to engineer RPE-like cell sheets directly on hAM for therapeutic use. Term placentas were obtained from uncomplicated cesarean deliveries with informed maternal consent. hAECs were either maintained on their native hAM scaffold or enzymatically isolated and cultured on plastic. Cell morphology was assessed under maintenance conditions. Expression of key RPE-specific genes and proteins was analyzed over time using qRT-PCR and immunofluorescence, following exposure to differentiation inducing factors involved in retinal development. hAECs cultured on native hAM retained superior epithelial integrity, exhibited reduced stress markers, and showed more stable intercellular organization compared to those cultured on plastic. Several RPE markers were present in freshly isolated hAM and remained more stably expressed in native cultures. Combinatorial use of specific differentiation cues further enhanced expression of characteristic RPE markers at both the transcript and protein levels. However, marker expression varied between placental samples, indicating biological variability that may influence differentiation outcomes. These preliminary findings suggest that hAECs possess the capacity to acquire RPE-like features in vitro, particularly when cultured on their native membrane. This highlights their promise as an ethically acceptable and readily available source for developing RPE-like grafts. Nonetheless, inter-donor variability underscores the need for standardized differentiation protocols and further investigation into in vivo integration and functional stability in AMD models.