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  • Title: Comparison of human amniotic membrane decellularisation approaches for hESC-derived RPE cells culture.
    Author: Daniele E, Ferrari B, Rassu N, Ben-Nun J, Bosio L, Barbaro V, Ferrari S, Ponzin D.
    Journal: BMJ Open Ophthalmol; 2022 Sep; 7(1):. PubMed ID: 36161850.
    Abstract:
    OBJECTIVE: Recent clinical studies have shown that the transplantation of functional retinal pigment epithelium (RPE) cells can prevent the onset of RPE degeneration in age-related macular degeneration. This study aimed to investigate the potential of human amniotic membrane (hAM) as a viable scaffold for the growth and proliferation of pluripotent-derived RPE cells. METHODS AND ANALYSIS: Three enzymatic hAM de-epithelialisation methods (thermolysin, trypsin-EDTA and dispase II) were assessed by histological analysis and optical coherence tomography (OCT). We generated RPE cells from a human embryonic stem cell (hESC) line subjected to spontaneous differentiation in feeder-free conditions. The hESC-derived RPE cells were seeded over denuded hAM at a density of 2.0×105 cells/cm2 and maintained in culture for up to 4 weeks. Immnofluorescence was carried out to evaluate the development of a confluent monolayer of RPE cells on the top of the hAM. Conditioned medium was collected to measure pigment epithelium-derived factor (PEDF) concentration by ELISA. RESULTS: Laminin α5 and collagen IV staining confirmed the efficiency of the de-epithelialisation process. In particular, thermolysin showed good retention of tissue integrity on OCT images and greater preservation of the hAM basement membrane. The hESC-derived RPE cells formed patches of pigmented cells interspersed along the denuded hAM, but failed to form a regular sheet of RPE cells. These cells expressed typical RPE markers, such as PMEL17 and RPE65, but they secreted low levels of PEDF. CONCLUSION: The biological variability of the hAM could influence the adhesion and the expansion of hESC-derived RPE cells. Further studies are required to verify whether a non-confluent monolayer might represent a limit to transplantation.
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