Human Retinal Progenitor Cells Integrate as Photoreceptors After Transplantation Into the Subretinal Space of Mice

Abstract

Purpose: : Photoreceptor loss due to retinal degenerative diseases or injury is the leading cause of untreatable blindness. Work in rodents has demonstrated that transplanted retinal progenitor cells integrate as photoreceptors into the degenerating or injured retinas, thus suggesting a potential therapeutic approach for treatment of similar degenerative conditions in human. Here we investigate the integration, differentiation of cultured human retinal progenitor cells (hRPCs) transplanted into the subretinal space of mice with degenerating or laser injured retinas.

Methods: : hRPCs harvested from the neural retinas of human fetal eyes (12 to 18 weeks gestational age) were grow in Rencell media supplemented with epidermal growth factor (10ng/ml) and basic fibroblastic growth factor (20ng/ml). hRPCs were transfected with the GFP gene through retroviral infections. Two µl of hRPCs suspension, containing ~2 x 10⁵ cells, were injected into the subretinal space of Rhodopsin -/- mice, SCID mice and B6 mice with retinal photocoagulation (spot size: 350 µm; power: 100 mW; duration: 100 ms; 10 spots per eye). Mice were sacrificed by CO₂ inhalation 1 to 3 weeks after transplantation. Immunohistochemistry study was performed with the following primary antibodies against human mitochondria, GFP, nestin, Ki67, GFAP, rhodopsin, recoverin, peripherin-2, rom-1. Retinal sections were viewed on a confocal microscope. Cell morphometry was performed to assess the integration of donor cells.

Results: : Prior to transplantation, cultured hRPCs labeled positively with nestin, Ki67, GFAP, recoverin, and rhodopsin in vitro. One to 3 weeks after transplantation, a subset of hRPCs survived as allografts in the subretinal space without exogenous immune suppression. Grafted hRPCs migrated and incorporated into the recipient neural retina. Some integrated hRPCs co-labeled with human mitochondria and photoreceptor marker rhodopsin and recoverin. In a few cases, integrated hRPCs developed morphological features of mature photoreceptors.

Conclusions: : Transplanted hRPCs migrate and integrate into the mice retina, differentiate into photoreceptors. These results demonstrate that many of the findings seen with rodent RPCs can be duplicated with hRPCS, suggestive of the potentiality of hRPC transplantation for treating retinal degenerative diseases.

Keywords: retina • retinal degenerations: cell biology • transplantation