Notch signaling pathway inhibitor promotes differentiation of Müller cell-derived retinal stem cells into retinal ganglion cells

Abstract

Objective To observe the role of Notch signaling pathway inhibitor in differentiationprocess of stem cells derived from retinal Muller cells into the ganglion cell.Methods Retinas of Sprague Dawley rat at postnatal 10-20 days were dissociated from eye balls.The third passage of Muller cells was used in this experiment,which cultured by repeated incomplete pancreatic enzyme digestion method.The retinal Muller cells were induced in the serum-free dedifferentiation medium.The cell proliferation state was observed under an inverted microscope.The expression of the specific markers Nestin and Ki-67 of retinal stem cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.The positive rate of nucleus was detected by Edu.The retinal stem cells was divided into Gamma secretase inhibtor-Ⅰ (GSI) group and control group,the rate of ganglion cells was counted by using immunofluorescence staining.Results The cell proliferation had gathered to form a sphere.Immunofluorescence staining showed that the expressions of Nestin and Ki-67 were (92.94 ± 6.48％) and (85.96±6.04％) respectively.Edu positive rate of nucleus was (82.80±6.65)％.RT-PCR and Western blot further confirmed the high expression of Nestin and Ki-67 in the cell spheres but not in the Muller cells.The positive rate of ganglion cells were (16.98±2.87)％ and (11.17±0.71)％ in GSI group and control group respectively,with the significant difference (t =3.210,P =0.002).Conclusion Notch signaling pathway is an important regulatory gene in stem cells differentiated into retinal ganglion cell. Key words: Microglia; Retinal ganglion cells; Stem cells; Receptors, notch; Animal experimentation