Integration, Survival and Function of Transplanted RPE Stem Cells into Mouse Models of Geographic Atrophy

Abstract

Purpose: : Replacement of the degenerating retinal pigment epithelium (RPE) is a promising therapeutic strategy for atrophic age-related macular degeneration(AMD). Here we transplant human embryonic stem cell H9-derived RPE cells (H9-ES-RPE) and human fetal RPE (hf-RPE) into mouse models of retinal degeneration.

Methods: : We employed a high-definition camera to visualize the fundus during trans-scleral injection of RPE cells into the subretinal space of Rag1^-/- and Rpe65^-/- mice. The cells were labeled by lipophilic fluorescent tracers followed by in vivo visualization with an endoscope camera. RPE-cell survival was quantitated by quantitative genomic PCR(qPCR) of the human-specific Alu sequence. RPE integration is demonstrated by light and electron microscopy. RPE function was assessed by electroretinography (ERG) and HPLC retinoid analysis. Immunocytochemical analyses were performed on retina whole mounts and cryosections using established protocols.

Results: : RPE cells transplanted into the retina of normal albino animals demonstrate punctate islands of incorporated H9-ES-RPE on fundus photography 75 days post-transplant. Electron microscopy demonstrates the H9-ES-RPE cells have integrated into the native RPE monolayer and now reside on Bruch’s membrane. Electroretinography of RPE-injected Rpe65^-/- mice demonstrates a peak increase in post-transplant b-wave amplitudes 2 weeks after transplantation. HPLC analysis confirms a 4.2 and 3.3-fold increase in the visual chromophore 9-cis-RAL in hf-RPE and H9-ES-RPE transplanted Rpe65^-/- mice, respectively. Genomic-qPCR demonstrates the 2 week transient survival of transplanted RPE in immune competent mice and a prolonged survival in the immune deficient Rag1^-/- mouse. Fluorescence microscopy revealed that the transplanted cells were present as a continuous layer of cells in the subretinal space with the characteristic cobblestone morphology of mature RPE. The transplanted cells also expressed Rpe65 protein, which was not observed in the host Rpe65^-/- mouse tissue.

Conclusions: : Subretinal injections of fetal and stem cell-derived RPE are an excellent prospect for cell therapies for age-related macular degeneration and other RPE disorders. Transplantation of RPE cell suspensions into the Rpe65^-/-mouse results in cell integration and restoration of a functional visual cycle.

Keywords: retinal pigment epithelium • age-related macular degeneration • transplantation