Rat Bone Marrow Mesenchymal Stem Cells Were Induced To Differentiate Into Neuron-like Cells Exposed To Retina Extract In Vitro

ObjectiveThe development of advanced diagnostic and therapeutic equipment make it possible to cure the reversible eye diseases such as cataract and corneal diseases eventually.However,some retinal and optic nerve diseases just as retinitis pigmentosa,age-related macular degeneration and glaucoma optic neuropathy still remain to be irreversible.Based on the effective lowering of IOP,we can use different medicines to treat the damage of optic nerve and protect the visual function of patients with glaucoma.In addition to neuroprotective treatment,the replenishing retinal cells by transplanted cells have been paid increasing attention as another way to treat neurodegenerative disease.Extensive transplantation studies have been conducted by the use of a variety of cell sources,including brain-derived neural progenitor cells,retinal progenitor cells,and bone marrow mesenchymal stem cell (BMSCs).In this Study,we investigated if BMSCs can differentiate into neuron-like cells and the proper condition.It can establish a method to treat glaucoma optic neuropathy and other neurodegenerative disease by providing seed-cells.MethodsBecause of its autologous characteristic,relative ease of isolation,and its less controversial nature,we chose BMSC as the cells of neuron differentiation in vitro.It included 2 parts:1.Isolation,culture and purification of BMSCs;cell characterization;multipotency of BMSCs:adipogenic differentiation and chondrogenic differentiation were performed.2.In vitro the BMSC were preinduced by bFGF and then induced byβ-BM and retinal extract.The morphological changes were observed under phase contrast microscope,the specific markers of induced cells were identified immunocytochemically with Nestin,NF,NSE,GFAP antibodies.Results1.Isolation,culture and characterization of BMSCs (1)Pure BMSCs is harvested by adherence sieving from rat bone.After culturing for primary BMSCs for 3 days,BMSCs were characterized by spindle-shaped appearance and proliferated rapidly for 7 days,part of BMSCs of spindle-shaped appearance changed to polygon-shaped gradually.(2)Flow Cytometry BMSCs cells expressed CD29,CD44,CD90.In contrast,no expression of the hematopoietic lineage markers CD31,CD34,and CD45.(3)Multilineage potential BMSCs can differentiate toward the osteogenic,adipogenic lineages induced by certain inducers in vitro.2.Neuronal Introduction(1)Induction byβ-BM1)After exposed to 5 mmol/Lβ-BM for 1h,the cells began to contract their cytoplasm towards nucleus and acquired condensed refractile soma,extending multiple processes.After 5h,cells exhibited typical neuronal morphologies and there were intercrosses of cell processes.The Nestin positive rate of the induced cells was statistically higher than those of NSE and NF(P＜0.01).The NSE positive rate had no statistical difference between the two groups exposed toβ-BM for 1h and 5h(P＞0.01).The Nestin positive rate of the 1h group was statistically higher than that of the 5h group,the NF positive rate of two groups were in contrast.2)After exposed to 10 mmol/Lβ-BM for 30min,BMSCs exhibited neuronal morphologies.The positive rates of the 1h group were NSE＞NF＞Nestin (P＜0.001).The Nestin positive rate of the 5h group was statistically significant lower than those of NSE and NF(P＜0.001).The positive rates of NSE and NF in the 1h were lower than the 5 h group(P＜0.001),but the positive rate of Nestin was in contrast.In all groups,the GFAP positive rates in any time were statistically significant lower than other antibodies(P＜0.001).(2)Induction by retina extract BMSC were induced to neuron-like cells by two different densities of retina extract.In contrast toβ-BM,the differentiation occurred slowly.The neuronal-like cells survived for more than 7 days.The Nestin positive rate was higher than those of NSE and NF in different concentration groups exposed to retina extract for 24 h(P＜0. 01).Exposed to retina extract for 3 days,the Nestin positive rate was statistically lower than those of NSE and NF(P＜0.01)and it continued for 7 days.NSE and NF:The positive rate of the 24 h group was lower than those of the 3d and the 7d group(P＜0.01).There was no difference between the 3d and the 7d groups. The positive rate in higher concentration group was higher than the lower one.Nestin: The longer the induced time,the lower positive rate.The positive rate was higher in higher concentration group.In all groups,the GFAP positive rates in any time were statistically significant lower than other antibodies(P＜0.001).Conclusion1.β-BM could induce BMSCs to differentiate into neuronal-like cells in vitro. 5mmol/L concentration was better than 10 mmol/L.The induced cells expressed Nestin in early time,later expressed NF and NSE.2.Retina contract could also induce BMSCs to differentiate into neuronal-like cells in vitro.The induced cells expressed similar markers to the cells exposed toβ-BM and survived longer.But the differentiation rate is lower than those ofβ-BM.