Human Umbilical Cord Mesenchymal Stem Cells Differentiation Into Neuron-like Cells With Berberine In Vitro

Objectives: To identify the biological characteristics of human umbilicalcord mesenchymal stem cells (hUMSCs), and explore the condition and effectof berberine induce hUMSCs differentiation into neuron-like cells in vitro.Laying the foundation of theoretical and experimental for inducing hUMSCsdifferentiation into neuron-like cells and clinical applications.Methord: The umbilical cords taken from full-term caesarean sectionpatients were flushed by D-Hank’s then remove the umbilical artery andumbilical vein. The remainder umbilical cords were cut into pieces of1mm3,and digest the pieces by0.2%collagenase to obtain mononuclear cells. Thenthe cells were cultured in DMEM/F12medium that contains20%FBS,2ng/mlEGF,25mM L-Glu,100U/ml Penicillin,100μg/ml Streptomycin. We observedthe cells’morphological changes. Once adherent cells reached approximately80％～90％confluence, they were digested by collagenase and replantedunder the same culture conditions to amplify. We collected the digested cellsand FACS was used to detect the surface antigens of hUMSCs, that includeCD11b, CD19, CD34, CD45, CD73, CD90, CD105and HLA-DR (HLA-II).The primary hUMSCs were passaged at a ratio of1:1to P1. The cells’morphological changes and growth rate were observed and recorded, onceadherent cells reached approximately90％confluence again, they werepassaged at a ratio of1:2or1:3. The cell growth curve was plotted accordingto data collected.HUMSCs of P4were induced to differentiate into neural-like cells usingthe following method. HUMSCs were cultured in medium containing fourdifferent concentration (25μg/ml,50μg/ml,100μg/ml,200μg/ml) of berberine.HUMSCs were inoculated in five poly-L-lysine-coated six-well plates (eachfor a group of six-well plates) at a density of3×104/well. Once adherent cells reached approximately70-80%confluence, medium in experimental groupswere replaced with serum-free low glucose DMEM/F12medium containingberberine of different concentrations(200μg/ml,100μg/ml,50μg/ml,25μg/ml),while the midium in control group had the same ingredients except berberine.The cells’ morphological changes and growth rate were observed and recordedthrough inverted phase contrast microscope once every12hours for sevendays. We used the Immunocytochemistry to detect the cell phenotype and tookphotoes to analyse the rat of positive cells expressing NSE, GFAP and Nestin.Results are expressed as mean±standard deviation.Results: After12h, most of the P1cells were adherent with morphologyof triangle, diamond, oval, dots, etc. With the incubation time, long spindlehomogeneous cells gradually increased in the number. After7-8d the cells hadsyncretize about80％～90％, the hUMSCs shown radial pattern and swirlingarrangement at low magnification. Primary hUMSCs could be amplified atleast15passages that still maintain a strong vitality. Surface antigens ofhUMSCs of P3, P5and P10were detected by FACS. The results show thathUMSCs expressed CD73, CD90, CD105and not expressed CD11b, CD19,CD34, CD45, HLA-DR.The hUMSCs began to contract and stretched out the finger-like synapsesat24h after induction with200μg/ml and100μg/ml berberine. And the cells’morphology changed more obviously soon afterwards. The hUMSCs in50μg/ml and25μg/ml groups did not represent initial diversification.36h later,cells in200μg/ml and100μg/ml groups become to display many bipolarprotrusions.48h later, cellular protrusions in200μg/ml and100μg/ml groupsbecame more slender than before, even up to2-5. And some cellularprotrusions gradually developed into a multi-polar, coming into contact witheach other to form typical neuron-like network structure. At this point, cells inthe50μg/ml group also became to change but not in the group of25μg/mlberberine.60h later, cells in200μg/ml and100μg/ml groups shown moretypical neuron-like cells, at the same time, a few cells began shedding, floatingin the group of200μg/ml berberine. In50μg/ml group, the number of cells forming protrusion also increased slightly than before. However, cells in25μg/ml group had not yet shown significant changes.72h later, there was notsignificantly increasing of cells with morphological changes in each group.With the continuing role of induced fluid, differentiated cells showed moreand more typical neuron-like morphology, while there were more floating anddeath cells especialy in the group of200μg/ml berberine. In100μg/ml and50μg/ml groups, surviving cells showed more typical neuron-like morphology:Contraction of the cell cytoplasm is diricted to the nucleus.The cell bodiesdiopter is enhanced with the formation of a bipolar, multi-polar protrusion.Protrusion elongation is more pronounced than before.2,3level protrusionswere visible, some of which were stretching radially. Until in the fifth day,these typical neural-like cells began to significantly shed, float in100μg/mland50μg/ml groups and there were only few cells alive in the200μg/ml group.Immunocytochemistry showed that induced hUMSCs express GFAP, NSE andNestin. At different induction concentration, expression of each marker wasvarying according to time,and the positive expression rates are as follows: in200μg/ml group, NSE (15.0±3.9,81.4±5.8,57.7±7.7,39.1±4.3,25.4±6.8,8.3±4.0,0.0±0.0)，GFAP (15.0±4.0,81.3±8.1,56.3±5.6,39.3±5.4,27.1±6.9,8.6±3.2,0.0±0.0)，Nestin (14.8±4.0,80.3±7.3,55.9±5.6,38.6±4.9,25.4±6.8,7.9±3.5,0.0±0.0); in100μg/ml group, NSE (6.4±3.2,50.4±4.7,87.7±4.6,86.6±5.7,85.2±5.0,64.5±6.6,37.2±5.4)，GFAP (6.9±3.7,50.8±5.7,87.5±4.0,87.0±5.9,85.3±4.7,66.1±5.0,38.3±4.9)，Nestin (6.3±3.2,50.1±5.0,87.2±4.1,86.6±5.6,84.8±5.3,64.4±6.5,37.3±5.3); in50μg/ml group, NSE (0.0±0.0,3.6±2.3,14.3±5.3,13.8±4.4,15.6±6.3,15.8±6.5,15.6±5.7)，GFAP (0.0±0.0,3.5±3.0,12.4±6.3,13.8±4.7,15.0±4.2,15.2±5.3,15.1±4.1)，Nestin（0.0±0.0,2.8±3.3,12.9±6.5,14.5±5.9,14.9±4.8,15.4±5.6,15.2±5.1). Immunofluoresce-nce staining showed the same positive expression of GFAP, NSE, Nestin;Immunohistochemistry and immunofluorescence staining of the control groupwere negative.Conclusion:HUMSCs have high level of proliferative capacity in vitro.HUMSCs express CD73, CD90, CD105and not express CD11b, CD19, CD34, CD45, HLA-DR. HUMSCs could be induced differentiation into neuron-likecells with berberine. Concentration of berberine100μg/ml group is the mostobvious. And the longest survival time was observed in this group.