Functional rescue of photoreceptor cells in RCS rats by transplanting retinal pigment epithelial cells differentiated from primate embryonic stem cells

Abstract

Abstract: : Purpose:Embryonic stem (ES) cells retain significant developmental potential and replicative capability and are expected to alleviate the problem of the shortage of donor cells for cell–replacement therapy. Compared to the extensive potential demonstrated by mouse ES cells, there is no reported case showing that primate ES cells can be successfully applied to animal disease models. The purpose of the study was to determine whether primate embryonic stem cell–derived pigment epithelial cells (ESPEs) develop the well–known characteristics of retinal pigment epithelial (RPE) cells, and have functional properties that will be of value in treating diseases when transplanted into an animal model of RPE dysfunction. Methods:Cynomolgus monkey ES cells were induced to differentiate into pigment epithelial cells by co–culturing them with PA6 stromal cells in a differentiating medium. The expanded, single layer ESPEs were examined by light and electron microscopy. The expression of standard RPE markers by the ESPEs was determined by RT–PCR and Western blot, and immunocytochemical analyses. The ESPEs were labeled with CM–DiI, transplanted into the subretinal space of 4–week–old Royal College of Surgeons (RCS) rats, and the eyes were analyzed immunohistochemically at 8 weeks postgrafting. The effect of the ESPE–graft on the visual function of RCS rats was estimated by optokinetic reflex. Results:The expanded ESPEs were hexagonal and contained significant amounts of pigment. The ESPEs expressed typical RPE markers: viz., ZO–1, RPE65, CRALBP, and Mertk. They had extensive microvilli and were able to phagocytose latex beads. When transplanted into the subretinal space of RCS rats, the grafted ESPEs enhanced the survival of the host photoreceptors. The effects of the transplanted ESPEs were confirmed by histological analyses and behavioral tests. Conclusions:The ESPEs had morphological and physiological properties of normal RPE cells, and these findings suggest that these cells may provide an unlimited source of primate cells to be used for the study of pathogenesis, drug development, and cell–replacement therapy in eyes with retinal degenerative diseases due to primary RPE dysfunction. To the best of our knowledge, this is the first study to show detailed functions of specific cells differentiated from primate ES cells both in vitro and in vivo. In addition, this is also the first study to demonstrate the successful therapeutic application of primate ES cells in an animal disease model.

Keywords: retinal culture • retinal pigment epithelium • transplantation