Modeling Wet AMD Using Patient-Specific iPSC-Derived RPE Cells

Abstract

Purpose: : Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the Western world, yet its pathogenesis is poorly understood and there are few suitable in vitro models for this disease. We plan to create a patient-specific in vitro model of wet AMD using induced pluripotent stem cells (iPSCs).

Methods: : Eligible patients received a complete eye exam, SD-OCT and FA studies, as well as genetic analysis to determine genotypes in HTRA1 and CFH that are implicated in AMD. Fibroblasts were obtained using a skin punch biopsy and cultured. Human iPSCs were generated by lentiviral transfection of four transcription reprogramming factors (OCT3/4, SOX2, LIN28, and NANOG). Pluripotency was assessed by expression of stem cell markers and teratoma formation. The iPSC colonies were picked and cultured in feeder-free conditions and then induced to differentiate into retinal pigment epithelium (RPE).

Results: : The patient selected for this study was a 73 year-old Caucasian female with bilateral wet AMD due to choroidal neovascularization. She has a 100-pack year smoking history and was found to harbor a homozygous risk rs11200638 AA genotype in HTRA1 and a heterozygous C/T risk allele at rs2274700 in CFH. iPSC colonies obtained from this patient were positive for typical pluripotency cell markers: SSEA4, TRA-1-60, TRA-1-81, OCT3/4, SOX2, and NANOG. The iPSC colonies also formed teratomas that had characteristics of all three germ layers when injected subcutaneously into SCID mice.

Conclusions: : We have successfully obtained iPSCs from an AMD patient and are now in the process of differentiating them into RPE cells, which will serve as an excellent model for studying the disease mechanisms of AMD, and particularly the effects of single mutations at the cellular level.

Keywords: retinal pigment epithelium • retinal degenerations: cell biology • age-related macular degeneration