Induce and differenctiation of marrow mesenchymal stem cell into photoreceptor-like cell in vitro and microenvironment / 中华实验眼科杂志

ABSTRACT

Background Marrow mesenchymal stem cells (MSCs) has been successful induced to differentiate into corneal cells,retinal ganglion cells (RGCs) and retinal neuron-like cells in recent years,but there are few studies about MSCs induced into photoreceptor cells and their microenvironment.Objective The aim of this study was to explore the induce and differentiation of bone marrow mesenchymal stem cells (BMSCs) into photoreceptor-like cells in vitro and microenvironment.Methods The second generation of human BMSCs strain and RPE cells strain were cultured and passaged,respectively,and the fourth generation of BMSCs and the third generation of RPE cells were used in the experiment.BMSCs were cocultured using the mesenchymal stem cells medium (MSCM) contained 20 μg/L basic fibroblast growth factor (bFGF),20 μg/L epithelial growth factor (EGF)and 20 μg/L brain derived neurotrophic factor (BDNF) with RPE cells to induce the differentiation of BMSCs in the induced group,and BMSCs were cultured in MSCM only in the control group.The morphology of induced and differentiated cells were observed under the inverted microscope.Inmmunocytochemistry was used in induced for 3-,5-,7-day cells to detect the expression rate of rhodopsin protein for the identification of phenotype of the differentiated cells.RT-PCR was used in induced for 5-,7-day cells to detect the expressions of rhodopsin mRNA and recoverin mRNA.Results Cultured BMSCs grew well with the spindle shape,and passaged RPE cells showed the uniform size and polygon shape with the abundant pigment in the cells.Some induced cells appeared to be the neuron-like cells with round shape and long prominence and the secondary reticular branches.The expression rates of rhodopsinin the cells were (5.83±0.29)％,(20.36±0.32)％ and (29.80±2.30)％ in the third,fifth and seventh day after induce,which were significantly higher than (0.71 ±0.35) ％,(2.56±0.24) ％ and (2.32±0.42) ％ of control cells (t3 d =41.510,t5d =107.290,t7 d =30.036,P＜0.01).The grey scales of rhodopsin mRNA and recoverin mRNA were significantly elevated in the induced and differentiated cells compared with control cells in the fifth and seventh day (rhodopsin mRNA:t5 d =103.506,t7 d =122.584,P＜0.01 ; recoverin mRNA:t5 d =106.674,t7 d =189.992,P＜0.01).Conclusions BMSCs can be successfully induced to differentiate into photoreceptor cells after cocultured by conditioned medium with RPE cells.