Human Umbilical Cord Mesenchymal Stem Cells Differentiation Into Neuron-like Cells With IGF-1in Vitro

Objectives: To identify the biological characteristics of human umbilicalcord mesenchymal stem cells (hUMSCs), and explore the condition and effectof insulin-like growth factor1(IGF-1) induce hUMSCs differentiation intoneuron-like cells in vitro. Laying the foundationg of theoretical andexperimental for induce hUMSCs differentiation into neuron-like cells andclinical applications.Methord: Take the umbilical cords from full-term caesarean sectionpatients were flushed by D-Hank's and to remove the umbilical artery andumbilical vein. The umbilical cord was then cut into pieces of1mm3, anddigesting the pieces by collagenase to obtain mononuclear cells. Then the cellswere cultured in DMEM/F12medium that contain20%FBS,2ng/ml EGF,25mM L-Glu,100U/ml Penicillin,100μg/ml Streptomycin. We ovserved thecells' morphological changes, once adherent cells reached approximately80％～90％confluence, they were digested by collagenase and replated underthe same culture conditions to amplify. Collecting the cells and the surfaceantigens of hUMSCs were detected by FACS, that include D73CD90CD105CD19CD34CD45CD11and HLA-DR (HLA-II). The primary hUMSCs werepassaged as a rat of1:1marked P1. We ovserved the cells'morphologicalchanges, once adherent cells reached approximately90％confluence, andpassaged as a rat of1:2or1:3. HUMSCs of P4were induced to differentiateinto neural like cells using the following method. HUMSCs were put intothree different concentration(25ng/ml,50ng/ml,100ng/ml) of IGF-1inductionmedium. We ovserved the cells'morphological changes and took photos everytwo hours, and choose ten vision of high magnification. We took count of thetypical neuron-like cells to account the rat of differentiation. We use theImmunocytochemistry to detect the cell phenotype and took photo to analyse the rat of positive cells.Results: After24h, most of the P1cells got adherent, and becamelozenge, triangle, oval and other forms. After7-8d the cells had syncretizeabout80％～90％, the hUCMSCs were shown swirling arrangement by lowmagnification. Primary hUCMSCs could be amplified at least15passages andstill maintain a strong vitality. Surface antigens of hUCMSCs of P3, P5, P10were detected by FACS. The results showe that hUCMSCs express CD73,CD90, CD105and not express CD34, CD45, CD19, CD11b, HLA-DR.The hUCMSCs began to contract and stretched out the finger-likesynapses at30min after induction with50ng/ml and100ng/ml IGF-1. Then thecell morphology changed more obviously in100ng/ml group, at the sametime, a few cells got death.4h later, cells in the group of50ng/ml and100ng/ml become to display many branches.8h later the processescontinued elaborate and to display many branches, some branch of differentcells connected with each other, forming a net. While the a lot of cells died in100ng/ml group. And the group of25ng/ml only a few cells becomemorphology change.24h later, cells in100ng/ml group only a few still alive,in50ng/ml group cells shown more typical neuron-like cells.Immunocytochemistry showed that induced hUCMSCs express GFAP, NSEand Nestin.Conclusion:HUCMSCs have high level of proliferative capacity in vitro.HUCMSCs express CD73, CD90, CD105and not express CD34, CD45,CD19, CD11b, HLA-DR. HUCMSCs could be induced differentiation intoneuron-like cells with IGF-1in vitro.