Towards an experimental stem cell-based therapy for Age-related Macular Degeneration

Abstract

Purpose : Age-related macular degeneration is a major cause of severe, progressive visual impairment among elderly. There is no treatment to restore vision when the function of the macula is decreased. The retinal pigment epithelium (RPE) plays a major role in the etiology. In this project we aim to transfer stem cell derived-RPE in an animal model for retinal degeneration, to regain vision.

Methods : Human Embryonic Stem cells (hESCs) and induced Pluripotent Stem Cells (iPSCs) were differentiated into RPE in vitro on matrigel and artificial Bruch’s membranes (scaffolds). Different types were selected as potential carrier for the differentiation and transplantation procedures. Characterization of differentiating hESC/iPSC-RPE included immunohistochemistry, flow cytometry, microarray, and semi-quantitative PCR. C57BL/6 mice were anesthetized (0.08mL/10 mg) using Ketamine (10 mg/mL) and Xylazine (1 mg/mL). Subretinal injection of a cell suspension (50.000 cells) and transplantation of cells on scaffolds were done using, respectively, a 33G needle and 23-27G instruments. As a proof of principle, we transferred hESC/iPSC-RPE, ARPE-19 cells and controls (no cells) into genetically blind (RPE65 knockout) mice. For baseline measurements, 3M-old RPE65 mice were followed using SLO/OCT (Scanning laser ophthalmoscopy/Optical coherence tomography) and ERG (Electroretinography) to check structure and function of the retina. ERG responses were analyzed using the a- and b-waves (amplitudes; latency time).

Results : hESCs and iPSCs successfully differentiated into RPE on matrigel and scaffolds, usually within 30-40 days. They showed the expression of different specific RPE markers (RPE65, BEST1, MITF, MERTK, ZO-1). A phagocytosis assay showed that the RPE lines were able to digest rod outer segments. Microarray analysis showed that hESC-RPE, native RPE and human Iris epithelium are similar, but also showed distinct (functional) features. After hESC/iPSC-RPE cell transfer, mice were followed using SLO/OCT and ERG. We verified that the RPE65 mice have no retinal degeneration (yet) but that photopic and scotopic amplitudes are absent, a stage of disease potentially allowing documentation of improvement on natural history.

Conclusions : We developed a pre-clinical pipe-line, in which we differentiate stem cells to RPE, transfer these cells in a suitable animal model and evaluate function by non-invasive inspection (SLO/OCT and ERG).

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.