Monosialotetera-hexosyl Ganglioside Induces Human Umbilical Cord Mesenchymal Stem Cells To Differentiate Into Neuron-like Cells In Vitro

Objective:After isolation,characterization and immortalization, human umbilical cord mesenchymal stem cells(h UMSCs) were investigated for their potential to be induced to differentiate in vitro into neuron-like cells by GM1.Methods:First,get the newborn baby’s umbilical cord,remove the umbilical artery and vein among it and cut the left interstitial tissue into small blocks.Then,place the small blocks,which has been collagenase digested,in the mixture containing fetal bovine serum,nutritional factors and penicillin- streptomycin in high glucose DMEM culture base. After that, the full amount mixture which soaked the cultured primary cells changed after 12 hours, half the amount changed after 24 hours,and from then on every 3 days changed once,to observe the the cells’ growth state, adherent and the count cells’ growth density using an inverted microscope every time and note down and photograph. When the observed cells in culture flasks grow radially or arrang swirling,etal bovine serum was added to terminate digestion,then after digestion with trypsin,1:2or1:3 ratio passaged, denote as P1.And again denote as P2, P3 and so on. And draw a cells growth curve.Obtain the cells in the logarithmic growth phase denoted as P3,P5,P10, which were trypsinized first, resuspended to a single cell suspension which was rinsed with PBS buffer, the cell count density is achieved by cell counting plate,put them in 10 average EP tubes by the amout of greater than 105, dropping monoclonal antibody CD73,CD90 and CD105, CD19, CD34, CD45, CD11,and histocompatibility antigens HLA-DR(MHC-II),with anti-mouse Ig G1-PE and Ig G1-FITC monoclonal antibody as isotype control.After amplification, cells of the 4th passage were divided into four experimental groups(A,B,C and D) which were treated with L-DMEM culture medium containing GM1 at 50, 100, 150 and 200 μg/m L, respectively. The control group(E)contained L-DMEM only. The inductive effect of GM1 on h UMSCs to differentiate into neuron-like cells was observed by monitoring the cell morphological changes every hour with an inverted phase contrast microscope for 6 h. Expression levels of microtubule-associated protein 2(MAP-2),neurofilament protein(NF-H) and glial fibrillary acidic protein(GFAP)were measured after 6 h of induction by immrnohistochemistry, and the proportion of positive cells was determined.Results: Most of the primary cells were adherent 12 h after exposure to GM1.The cells first appeared as diamond or polygon shapes.They gradually grew into long spindle shapes and finally exhibited a radiating or swirling pattern. The cells maintained a strong proliferative capacity after continuous passage.By flow cytometric analysis, cultured h UMSCs in the 3rd, 5th and 10 th passages expressed CD73,CD90 and CD105 but not CD11 b,CD1,CD34, CD45 or HLA-DR.After 6 h of induction,neuron-like cells were present in all experimental groups with oval-shaped cell bodies and protruding neurites.These neuron-like cells in the experimental groups were positive for MAP-2, NF-H but negative for GFAP expression.Group C(150 μg/m L GM1) had the highest positive expression rate of MAP-2 and NF-H, while no changes in these markers were found before and after induction in the control group.Conclusion:GM1 induced h UMSCs to differentiate into neuron-like cells effectively,with the optimal concentration at 150 μg/m L.