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[Study on the differentiation of retinal ganglion cells from rat Müller cells in vitro].

OBJECTIVE
To certify the ability of retinal Müller cells for producing neural stem cells in vitro and to find a method that can aquire more retinal ganglion cells from these stem cells.


METHODS
Müller cells were isolated from rat retina, and proliferating cells were expanded in serum-containing medium. The third or fourth passage of cells were identified by RT-PCR and Immunocytochemistry analysis.For dedifferentiation, the cultured cells were transferred to the sphere-culture medium composed of DMEM/F-12 supplemented with N2, bFGF and EGF. After 3 - 5 days, the culture media were substituted with BDNF, RA and 5% FBS and culture was continued for 7 - 10 days. At last, cells in this two stages were identified by immunocytochemical analysis.


RESULTS
Approximately (95.17 ± 2.68)% of cells in the culture were Müller cells as revealed by expressing glutamate-aspartate transporters (GLAST) and glutamine synthetase (GS) immunoreactivities. RT-PCR analysis also revealed that the culture was enriched for Müller cells and not contaminated with other retinal cells. After 3 - 5 days cultured in the the sphere-culture medium, the Müller cells became round and differentiate to neurospheres. (95.26 ± 1.35)% of cells in the neurosphere were positively reacted for Nestin, and (90.33 ± 4.12)% for BrdU. Neurospheres cultured for 7 - 10 days with 5% FBS, BDNF and RA can redifferentiate to various new cells. And the expression of Thy1.1 which is a marker of retinal ganglion cells was observed in (21.14 ± 1.49)% of these cells.


CONCLUSIONS
Adult rodent Müller cells can generate clonal neurospheres, which consist of proliferating and multipotent cells, and redifferentiate to ganglion cells. This study may provide a novel tool in the study on stem cells and contribute to therapies for neural regeneration in retina.