Resveratrol Induces Human Umbilical Cord Mesenchymal Stem Cell To Differentiate Into Neuron-Like Cell In Vitro

Objective: To culture the human umbilical cord mesenchymal stem cells which have been isolated from neonatal umbilical cord in vitro to observe the growth and form of hUC-MSCs,and analysis the cellular phenotype;To study the condition and effect of Resveratrol inducing hUC-MSCs to differentiate into neuron-like cell with different concentration;To observe the change of morphology and detect the expression of mRNA and protein.To offer support of theory and technology for basal and clinical therapy in future.Methods: We get the human umbilical cord from full-term cesarean fetus and used D-Hank's to rinse them fully after removing the umbilical artery and umbilical vein,then cut the umbilical cord mesenchymal tissue into tissue block that size is 1mm3.Then we digest the tissue block by 0.2% collagenase?,and put them into the DMEM/F12 which contains 20% FBS,2ng/ml EGF,25 mM L-Glu,100U/ml penicillin,100?g/ml streptomycin.Observed the morphology and growth tendency of primary cells.When the fusion rate of cells reached 80%-90%,we added 0.25% trypsin-1mM EDTA into culture medium to digest cells and passaged.We used flow cytometry to detect the expression of surface antigen CD105,CD73,CD90,CD11 b,CD45,CD34,CD19 and histocomp-atibility antigens HLA-DR(MHC-II),IgG1-PE and IgG1-FITC were used as isotype control.The primary hUC-MSCs were passaged at a ratio of 1:1 marked as P1.The change rate of cell morphology and growth was recorded dynamically,the adherent cells were passaged as a rate of 1:2 or 1:3 when they reached approximately 90% confluence.According to these,we plotted the growth curve.The hUC-MSCs of P5 were passaged into six-well plates and 25cm2 culture bottle,and cultured by complete medium.We assigned one six-well plate and three 25cm2 culture bottles into a group randomly.The first three groups were experimental groups containing Resveratrol inductive liquid that was compounded with L-DMEM culture medium and the fourth group was control group that contained L-DMEM culture medium only.The concentration of Resveratrol inductive liquid in experimental groups is7.5mg/L,15mg/L and 30mg/L.When the cell fusion rate reached 70%-80%,we changed the complete medium to Resveratrol inductive liquid.The volume added into six-well plates was 2ml and the 25cm2 culture bottle had 4ml,then we put them in incubator for 24 hours.We observed the number of neuron-like cells and total cell count with the inverted phase contrast microscope at 10 non-overlapping view.We calculated the positive rate of neuron-like cells with X ±s and detected the expression of GFAP,Nestin and NSE with hUC-MSCs induced in six-well plates by immunohistochemical method at the fourth hour.Then choosing the appropriate concentration,we used hUC-MSCs induced in 25cm2 culture bottles to detect the mRNA and protein expression of GFAP,Nestin and NSE with Western blot and RT-PCR at 2th,4th,6th,12 th,24th hours after inducing.Results: The most primary hUC-MSCs were adherent after culturing 12 hours,looked like trigon or fusiform.The longer the time passed,the more the number of cells were,so that adherent cells grew to bigger and long spindle.The cells merged completely with growing to spiral fashion or radial till the5 th day.After several passages or more,the cells still kept exuberant proliferation capacity relatively.We analyzed the surface antigen of P2,P5 and P10 hUC-MSCs with flow cytometry,the result showed that CD105,CD73 and CD90 were expressed positively,CD11 b,CD45,CD34,CD19 and HLA-DR were not.After inducing,the cells in group 15mg/L had no obvious change on morphology at the first hour.There were only little cells shrinking and some cells floated after 4 hours.At the sixth hour,only 5% cells shrinked into roundness or oval with refraction strengthening and stretched out two or moreprotuberances which had some branches from cell bodies.After 12 hours,there were no more neuron-like cells appearing and the number even decreased with a few cells breaking away.The positive rate of neuron-like cells in group 30mg/L reached 50% after inducing 1 hour.The number of multilevel differentiated cells were more than group 15mg/L observably.The length of protuberances were more longer.Each cell relied on the protuberances and branches interweaving into a network.There were about70% neuron-like cells and very few floating cells in the view at the fourth hour.After 6 hours we could see 80%~90% neuron-like cells and the ratio was not increasing significantly after inducing 12 hours.The rate of neuron-like cells decreased to 70%~80% at the 24 th hour and there were more floating cells at the same time.There was no significant change in group 7.5mg/L and control group before and after induction.After inducing 4 hours,the result of immunohistochemical method showed that the expression of Nestin and NSE in group 15mg/L and group 30mg/L were positive,and GFAP was negative.We could see brown cell bodies and purple nucleus.The result of Western blot and RT-PCR indicated that the mRNA and protein expression of GFAP was negative.There were little mRNA and protein expression of Nestin and NSE in the blank group.And they all were increasing gradually as the time were more longer,then decreased gradually.Moreover,the highest expression of Nestin was at the fourth hour,and NSE was the sixth.Conclusions: After isolating and purifying from the umbilical cord,the hUC-MSCs can be cultured and passaged steadily in vitro and maintain higher activity;The hUC-MSCs co-express CD105,CD73 and CD90,but not express CD11 b,CD45,CD34,CD19 and HLA-DR;Resveratrol can induce hUC-MSCs to differentiate into neuron-like cells in vitro and the relatively suitable concentration is 30mg/L.By inducing,the Nestin and NSE of neuron-like cells were positive and the GFAP was negative.