Induce and differentiation of pig Müller cells into photoreceptors in vitro / 中华实验眼科杂志

ABSTRACT

Background Recent studies indicated that rat and mouse Müller cells can be induced and differentiated into photoreceptor-like cells in vitro, but it is not known whether this also happens to adult pig Müller cells nowadays.Objective This study was to test whether adult pig Müller cells can be differentiated to the retinal photoreceptors (the primary transmission neurons of the retina) in vitro.Methods Müller cells were isolated from the neural retina of adult pig eyes and cultured and passaged.The 3rd and 4th generation of cells were themonolayerly cultured,and the cells forced to form spheres in suspension in altra-low adherent dishes for 2-3 days first and then reseeded in normal adherent plates,and both of them were cultured in a specifically formulated medium to induce the differentiation of retinal photoreceptor.The cells was verified by immunocytochemistry.Cell morphology and immunofluorescence staining were utilized to measure the efficacy of the differentiation.Results The 2nd,3rd and 4th generation of Müller cells expressed glutamate synthetase (GS) , a specific maker of Müller cells.Inaddition, the 3rd generation of cells also expressed glial fibrillary acidic protein (GFAP) and another specific maker of Müller cells.Three visual fields under fluorescence-microscope were randomly chosen to calculate the average positive ratio of rhodopsin,a specific marker of mature photoreceptors.The photoreceptor differentiation ratios of the 2nd generation of cells for monolayer culture only and with additional sphere suspension culture were (27.99±6.53 (％ and (16.54±3.40) ％ , respectively.With passages, the number of rhodopsin positive cells gradually decreased, and the intensity of rhodopsin expression gradually weakened.The directed rhodopsin positive ratios of the 2nd,3rd and 4th generation of cells from sphere formation were (56.23±7.32)％ , (36.26 ±8.55)％ and (12.68 ±3.18)％ , respectively.Although the rhodopsin expression was weakened over passages,the differentiated cells were more slender and elongated.There was no statistic ally significant difference between different groups (F =2.618, P =0.099).Conclusions Adult pig Müller cells can be differentiated into retinal photoreceptors in vitro.The morphology of the differentiated cells appears moreslender and elongates if the sphere-induced differentiation method is used and/or the directed differentiation time is further extended.