Abstract
Abstract: :
Purpose: The ability to regenerate and repair damage to the retina is at the center of hopes for the therapeutic use of stem cells in the eye. Neural stem cells have shown some ability to repair injured CNS, and might serve to provide precursors to retinal neurons, including photoreceptor cells. Methods: Neural stem cells were isolated from 14d embryos collected from mice which express beta–galactosidase (beta–gal) using the arrestin promoter, which directs expression to rod photoreceptor cells in the retina. The stem cells were expanded in vitro, cultured as neurospheres, and transferred to adult, syngeneic mice via inoculation into the anterior chamber (AC) of the eye, deposited in brain, or inoculated by the intraperitoneal (IP) route. The retinas of some mice were injured with autoimmune disease prior to stem cell transfer. At 2 months post–grafting, eyes and other tissues were collected and examined to determine if stem cell progeny were present in retina, and if they expressed beta–gal. Results: Four of 8 AC–inoculated recipients showed evidence of Bluo–gal staining in the retina, and two of two IP–inoculated recipients also exhibited staining in the retina. All but one recipient of both the IP and AC stem cell inoculation routes was also PCR positive for beta–gal DNA in the spleen. Retinal injury was not required for stem cell engraftment. Beta–gal–positive cells were found in all layers of the retina, and were concentrated in peripheral retina. The cells did not express beta–gal if inoculated into the brain, but survived in the brain for at least two months. Conclusions: The results show that a reporter gene known to be expressed in differentiated retinal rod photoreceptor cells due to the activity of the arrestin promoter, and unique to the visual cascade, is expressed in normal mice by neural stem cells isolated from transgenic mice bearing the arrestin/beta–gal promoter/reporter construct. The presence of beta–gal DNA in the spleen two months after inoculation suggests that the cells migrated out of and took up residence in other organs.
Keywords: retina • regeneration • transgenics/knock-outs