ArticleInternational journal of ophthalmology2016
Promotion on the differentiation of retinal Müller cells into retinal ganglion cells by Brn-3b.
Article in International journal of ophthalmology, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
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Who cites it
2 citing papers in PubMed, 3 citations in OpenAlex.
- TRIM9 promotes Müller cell-derived retinal stem cells to differentiate into retinal ganglion cells by regulating Atoh7.In vitro cellular & developmental biology. Animal · 2023Article
- Evidence of Müller Glia Conversion Into Retina Ganglion Cells Using Neurogenin2.Frontiers in cellular neuroscience · 2018Article
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
aimTo investigate the role of Brn-3b in differentiation process of stem cells derived from retinal Müller cells into the ganglion cell.
methodsThe passage culture method of Müller 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 cell 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 Müller cells were cultured for 3-7d, then, the percentage of ganglion cells was counted by immunofluorescence staining.
resultsFACS demonstrated the purity of retinal Müller 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 Müller cells. Western blot analysis further confirmed the expression of nestin and Ki-67 in the cell spheres but not in the Müller 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 effective proliferation. The statistics result showed the difference between Brn-3bsiRNA group and Brn-3b control siRNA group or the control group was significant (F=15, P<0.05), while the difference between Brn-3b control siRNA group or the control group was not statistically significant (P>0.05).
conclusionThe repeated incomplete pancreatic enzyme digestion method is an efficient and practical method to purify retinal Müller cells. Retinal stem cells were successfully cloned in the dedifferentiational medium. Retinal Müller cells are accessible sources of retinal stem cells. Brn-3b is an important regulatory gene in stem cells differentiated into retinal ganglion cell.
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