Isolation and characterization of human retinal progenitor cells isolated from eyes with advanced retinopathy of prematurity

Abstract

Abstract: : Purpose: To characterize retinal progenitor cells isolated from eyes with advanced retinopathy of prematurity. To study their survival and differentiation both in vitro and after subretinal transplantation. Methods: Neovascular membranes collected from patients with stages 4 and 5 ROP were subjected to collagenase digestion for cell cultures. Double–fluorescence immunohistochemistry and RT–PCR were performed on both epiretinal membranes and cultured cells to determine the expression of progenitor marker (nestin), mature neuronal markers (MAP 5, NF–200, B–tubulin, Neu N), glial marker (GFAP) and markers for bipolar cells (PKC) and photoreceptors (opsin). Retinal progenitor cells were tagged with either red fluorescent linker or GFP using adeno/GFP virus delivery and transplanted into subretinal space of SCID mice. Mice were sacrificed between 2–8 days and studied by serial section. Results: cellular components of epiretinal membranes from advanced ROP contain nestin–positive cells which form neurospheres in vitro, supporting their identity as retinal progenitor cells. Retinal progenitor cells could be induced to differentiate into mature neuronal/retinal lineage in preference to glial lineage, including expression of bipolar and photoreceptor markers. Fluorescent–tagged or GFP+ retinal progenitors injected into the subretinal space of SCID mice survived and differentiated into morphologically mature neuronal and retinal–like elements. Conclusions: Epiretinal membranes from advanced stage ROP contain retinal pregenitor cells which can be expanded and induced to differentiate into mature neuronal and retinal–like lineages. These cells survive transplantation into the subretinal space as xenografts and differentiate into mature neuronal and retinal cells. Cultured retinal precursors may be a future source of transplantation for retinal repair especially in cases of advanced ROP.

Keywords: retinopathy of prematurity • transplantation • plasticity