Effect of Basic Fibroblast Growth Factor on Adult Human Retinal Stem Cell Proliferation in vivo

Abstract

Abstract: : Purpose: Many vital tissues of the eye are unable to regenerate after injury or degeneration. Adult human retinal stem cells have recently been isolated from the ciliary body pigmented epithelium of cadaver eyes. When grown in suspension the cells form neuronal spheres and express nestin. When plated on laminin the cells assume neuroretinal morphologic phenotypes and express neuroretinal markers. Unfortunately the prevalence of sphere forming stem cells in the ciliary body pigmented epithelium is relatively small. Cell populations need to be expanded in culture in order to permit further investigation and facilitate possible therapeutic applications. Basic fibroblast growth factor (bFGF) has been shown to stimulate the growth of other mammalian retinal progenitor cells in culture. Our aim is to ascertain the effect bFGF on adult retinal stem cell proliferation. Methods: Adult retinal stem cells were harvested from cadaver eyes obtained from the Illinois Eye Bank. Eyes were bisected along the equator, and the vitreous and lens were removed exposing the ciliary processes. After treating with dispase the pigmented epithelium was peeled off of the ciliary processes. The cells were disassociated in trypsin, washed, and then plated in 24 well plates at a density of 1X10⁴/ml in serum free defined growth medium (D–MEM F12 with N2). bFGF 100ng/ml was added to half of the cultures. All cells were incubated at 37°C. After 10 days the number of neuronal spheres were counted using an inverted microscope. Results: Retinal stem cells were isolated from a human donor eyes with an average yield of 3x10⁵. Isolated cells in cultures with and without bFGF formed undifferentiated neuronal spheres of pigmented cells in suspension. Cells grown in the presence of bFGF did appear to form larger spheres. After 10 days the mean number of spheres per well (n=10) in cultures was 40.8 ±10.2 without bFGF, and 95.2±17.2 for bFGF containing cultures. A t–test demonstrated this difference to be statistically significant with a p<0.001. Conclusions: bFGF can be used to stimulate the growth of adult human retinal stem cells isolated from ciliary body pigmented epithelium in vivo. The long–term effects of bFGF remain to be investigated. These adult human stem cells remain an exciting possible therapeutic modality for previously untreatable ocular degenerative disease.

Keywords: retinal pigment epithelium • retinal culture • regeneration