Abstract
Purpose :
The implantation of retinal progenitor cells (RPCs) is under investigation for the therapy of retinal degeneration diseases. However, methods to increase cell growth, promote survival, and inhibit differentiation during in vitro culture remain a major challenge in the production of quality RPCs for implantation. This study aimed at optimizing the culture conditions to obtain high yield of quality RPCs.
Methods :
1). Human retinas were isolated from fetal eyes at 18-20 gestational weeks, sheared into small cell aggregates by forcing them through a 25-gauge needle several times, and cultured in suspension in RPC medium for 24 hours. 2). RPC were then cultured in different conditions: co-culture with polarized human embryonic stem cell-derived (HES)-RPE cells, surface-attached culture, and suspension culture. 3). RPCs cultured in different conditions and at certain time points were collected and analyzed for gene profiles by qPCR.
Results :
Using newly isolated retinas as a standard, RPCs in suspension culture expressed the highest level of photoreceptor marker genes, such as arrestin and rhodopsin, while RPC co-cultured with polarized HES-RPE cells expressed the highest level of Ki67 (proliferation marker gene) among the three groups after 1 month of culture. RPC in attached culture only expressed higher levels of neuronal and photoreceptor marker genes at 15 days of culture, but decreased the expressions of those genes at 1 month of culture.
Conclusions :
The co-culture of RPCs with polarized RPE promotes RPC growth and inhibits RPC differentiation; while suspension culture increases RPC photoreceptor differentiation. Future studies will focus on how culture conditions (co-culture and suspension culture) may be used sequentially, to achieve the most efficacious RPG for successful retinal integration, differentiation and function.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.