Isolation, Culture and Differentiation of the Human Fetal Retinal Progenitor Cells

Abstract

Abstract: : Purpose: To isolate, culture and identify human fetal retinal progenitor cells (HRPCs) in vitro from human embryonic retina, and, study their differentiation in different culture conditions.Methods: HRPCs were isolated from neural retinas at the 16^th–20^th week of human gestation embryo and cultured in serum–free media. The cellular differentiation was initiated in the media containing serum and growth factors (EGF and bFGF) in vitro and in the mouse eyecup with monolayer of retinal pigment epithelium (RPE) that mimic the neuronal retinal environment in vivo. The expression of specific proteins, including nestin, GFAP, rhodopsin, syntaxin PKCα and Thy1 (the markers for progenitor cells, glial cell, rod photoreceptors, amacrine, bipolar, and ganglion cells respectively), were investigated by immunohistochemistry for identification of the cultured cells. The mRNA levels of nestin gene in HRPCs and differentiated retinal cells were detected by real time RT–PCR analysis. Results:HRPCs could be isolated and cultured from human embryonic retina. They were capable to divide into multiple generations. With serum and factors (EGF and bFGF) in culture medium, these stem cells or progenitors could differentiate into several types of cells that express GFAP, rhodopsin, syntaxin, PKCα, and Thy1, respectively. While cultured in mouse eyecup with monolayer of RPE, HRPCs could express preceding markers simultaneously. Real time RT–PCR showed differentiated HRPCs downregulating the expression of nestin significantly. Conclusions: Our results indicated that human fetal neural retina at the 16^th–20^th weeks of gestation harbored neural retinal progenitor cells. These cells may have the potential of self–renewal and multi–differentiation. The RPE may play an important role in differentiation of HRPCs in vitro.

Keywords: regeneration • retinal culture • retinal development