AIM: To investigate the role of Brn-3b in differentiation process of stem cells derived from retinal Muller cells into the ganglion cell. METHODS: The passage culture method of Muller cells from retina of newborn Sprague Dawley rats was carried out by repeated incomplete pancreatic enzyme digestion method. The cells were detected by fluorescence- activated ceil sorter (FACS), immunohistochemistry technology and reverse transcription-polymerase chain reaction (RT-PCR) to determine the purity. The third passage of cells was induced in the serum-free dedifferentiation medium. The expression of the specific markers Ki-67 and nestin of retinal stem cells was measured by RT-PCR and Western blot. The cell proliferation of retinal stem cells was detected by 5- Ethynyl-2'-deoxyuridine (Edu) staining. The cells were randomly divided into 5 groups as follows: group A: Brn-3bsiRNA group; group B: Brn-3b control siRNA group; group C: pGC-Brn-3b-green fluorescent protein (GFP) group; group D: pGC-GFP group; group E: control group (without any handling). The purified Muller cells were cultured for 3-7d, then, the percentage of ganglion cells was counted by immunofluorescence staining. RESULTS: FACS demonstrated the purity of retinal Muller cells was more 97.44%. A few spherical cell spheres appeared. Immunofluorescence staining showed that stem cells within the spheres were positive for retinal stem cell-specific markers nestin (red fluorescence, 92.94%±6.48%) and Ki-67 (green fluorescence, 85.96%± 6.04% ). Meanwhile, RT-PCR analysis showed cell spheres in the culture to have expressed a battery of transcripts characteristic of stem cells such as nestin and Ki-67, which were absent in the Muller cells. Western blot analysis further confirmed the expression of nestin and Ki-67 in the cell spheres but not in the Muller cells. Edu staining showed most of the nuclei within the cell spheres were stained red (82.80%±6.65%), suggesting the new cell spheres had the capacity for e
Zhen-Kai WuLan CaoXue-Yong ZhangWei-Tao SongXiao-Bo Xia