Original research: Comparison of human amniotic membrane decellularisation approaches for hESC-derived RPE cells culture

BMJ Open Ophthalmology

article

Elena Daniele¹  Barbara Ferrari²  Nicolò Rassu³  Joshua Ben-Nun⁴  Lorenzo Bosio²  Vanessa Barbaro²  Stefano Ferrari²  Diego Ponzin² 

[1] Department of Translational Medicine , University of Ferrara;Venice Eye Bank;Ophthalmic Unit , Ospedale dell’Angelo;Medicel AG

关键词: Stem Cells;    Retina;    Experimental & laboratory;    Degeneration;   

DOI  :  10.1136/bmjophth-2022-000981

学科分类：工业工程学

来源: BMJ Publishing Group

【 摘 要 】

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.

【 授权许可】

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