In vitro and in vivo characterization of iris pigment epithelialcells cultured on amniotic membranes

[摘要] Purpose: To determine whether human amniotic membranes (AMs) caninduce human and rat iris pigment epithelial (IPE) cells grown on themto develop characteristics of RPE cells in situ better than IPE cellsgrown on plastic plates, and to determine whether subretinaltransplantation of IPE cell sheets grown on AMs can protectphotoreceptor cells in dystrophic Royal College of Surgeons (RCS) rats.Methods: IPE cells from humans and Long-Evans rats were cultured onthe basement membrane side of dispase-treated AMs. Two weeks afterseeding, ultrastructural changes were evaluated by transmission electronmicroscopy, and the level of expression of several genes present indifferentiated retinal pigment epithelial (RPE) cells was determined byreal time PCR and western blotting. IPE cell sheets cultured on AMs weretransplanted into the subretinal space of 4-week-old RCS rats, and eyeswere analyzed histologically 12 weeks after grafting.Results: IPE cells cultured on AMs showed ultrastructural featureslike intercellular junctions, similar to RPE cells in situ. IPE cellsgrown on AMs had a greater upregulation in the expression of genesimportant for the function of differentiated RPE cells (e.g., pigmentepithelium-derived factor [PEDF], RPE65, bestrophin, VEGF, and BDNF)than IPE cells grown on plastic plates. The number of photoreceptorspresent in RCS rats after subretinal transplantation of IPE cell sheetsgrown on AMs was significantly higher than that of sham injected ratsand rats receiving transplantation of AMs without IPE cells.Conclusions: The more advanced degree of differentiation of IPEcells grown on AMs indicates that AMs are a better substrate to cultureIPE cells than plastic plates. This was supported by the greaterprotection of photoreceptors of RCS rats when IPE cell sheets culturedon AMs were transplanted in the subretinal space.

[学科分类] 生物化学/生物物理