Abstract
Purpose::
Human Embryonic Stem Cell transplantation is a promising therapeutic approach for the replacement of degenerated retinal cells in patients with age-related macular degeneration, retinitis pigmentosa, Stargardt’s disease, and other peripheral retinal degenerations. Prior to subretinal transplantation of hESC in these individuals it is important to induce the stem cells to differentiate into RPE or retinal neural progenitor cells (NPCs) prior to transplantation into animal models of human retinal degeneration.
Methods::
hESC were cultured on human Bruch`s membrane explant or mouse PA6 stromal cells in differentiation medium (SDIA stromal-derived inducing activity method; Kawasaki H, Neuron 2000). Cells were examined morphologically and stained with different RPE markers (RPE65, Bestrophin, CRALBP) or neuron markers (ß-tubullin III, Neurofilaments etc.) and examined by fluorescence light microscopy.
Results::
hESC cultured on human BM developed a RPE morphology and stained for RPE markers, including bestrophin and CRALBP .These cells could then be expanded on after expansion onto matrigel-coated dishes, hESC became pigmented and showed epithelium morphology, expressing RPE cell markers. Alternately, neural progenitor cells could also be obtained after culturing on by PA6 cells which expressed neuroectoderm marker / retinal progenitor marker Pax-6, neuron markers ß-tubullin III, MAP-2, NF200 and astrocyte marker GFAP.
Conclusions::
hESCs have the potential to differentiate into RPE or retinal progenitor cells. Extracellular matrix may play an important role in hESC differentiation. Additional studies are required to assess hESC-derived RPE function and characterize them with the primary RPE cell lines prior to intraocular transplantation into animal models of retinal degeneration.
Keywords: retinal development • retinal pigment epithelium • Bruch's membrane