Differentiation Of Mononuclear Cells Derived From Human Umbilical Cord Blood Into Neuron-like Cells In Vitro

Traditional viewpoint believed that neurons do not regenerate in adult mammalian brain and spinal cord. So central neural tissues can not regenerate, when they were injured or degenerated. Find of neural stem cells (NSCs) changes this traditional viewpoint. NSCs are a multipotent primordial cells and have the capacity of self-renewal, moreover can generate neurons, astroglias and oligodendrocytes. So far, NSCs are mainly acquired from embryo, but there are ethical and legal limitations acquiring NSCs from embryo. It is very important to search other original NSCs. Investigation showed that mesenchymal stem cells (MSCs) of marrow could differentiate into neurons and expressed neural-specific markers in vivo or vitro, but amount of stem cells are little in adult marrow, moreover decrease following aging, and separation and purification of stem cells are very difficult, and transplantation canresult in serious immunoreaction. These disadvantages limit to a great extent application of MSCs, so it is necessary to look for other abundant source, separating and purifying easy, and weak immunoreaction origins of NSCs.Umbilical cord blood is ideal experimental material, because of abundant source, separating and purifying easy, moreover ABO and HLA antigens uncreated on umbilical cord stem cells surface, so not to induce immunoreaction. Investigation showed that stem cells umbilical cord blood-derived could also differentiate into neurons, astroglias and oligodendrocytes. There are low-level expression of Nestin, human MNCs after cultivated in vitro, but there is not study about expressions of nestin in fresh isolated and cultivated with cytokinesso as stimulator MNCs. My experiment is to explore this aspect and farther probe into differetiation into neurons, to examine change of expression of T mRNA, which has important function for structural integrity and functional keep of central neural system, during MNCs differentiating into neurons and astroglias in vitro, to explore actions of bFGF and EGF promoting proliferation and differentiation of neural stem cells from umbilical cord blood MNCs in vitro, to offer abundant cells source for MNCs as seed cells to treat neural damnification and degenerationMethods:MNCs were isolated by centrifugation over Lymphoprep (density 1.077/ml) and divided into tow parts. One part were cryopreserved in liquid nitrogen, the other were cultivated with 1 106/ml density in T-75 tissue culture flasks in humidified atmosphere with 5% CO2 at 37 癈. Cells cultivated were divided 4 groups.I group: un-cultivated MNCsII group: H-DMEM and 20 % fetal bovine serum ( control)III group: H-DMEM and 20 % fetal bovine serum and bFGF (concentration 20ng/ml.)IV group: H-DMEM and 20 % fetal bovine serum and EOF (concentration 20ng/ml.)V group: H-DMEM and 20 % fetal bovine serum and bFGF and EGF (eachconcentration 20ng/ml.)Half exchanging medium per 3d, in 14d, collecting cultivated cells and resuscitating cryopreserved, both MNCs respectively were used to detected the expressions of neural stem cells specific and neural specific markers: Nestin and i, NF-M and MAP2 by RT-PCR; Preparation of immunocytochemic slices : Cells were cultured with 1 l06/ml density in 6-well plates, slice previously dealt with polylysine then placed in wells. EOF and bFGF were added in culture medium as mitogen stimulating growth. After cultivated 14d, Nestin, NF-M, NSE and MAP2 are examined by immunocytochemistry. Proliferative ability of MNCs are determined with MTT method.Results:1. Fresh isolated MNCs are small cyton roundish. After cultivated 14d, cells without cytokines were main the acerose shape, but cells with cytokines, which were similar to neurons, were big cyton having some thick and long cytodenrites, which closer cytodenrite inosculated reticulation.2. In nu-cultivated MNCs, positive cells of anti-Nestin and anti-MAP2were respective 0.5%, 1%( Under 40譵ultiple, random counting some views positive cells and total cells, positive cells percentage= positive cells amount/total cells amoun...