Preliminary Study Of New Method For Establishing Mouse Embryonic Stem Cell Line And TFAG Promoting The Differentiation Of Embryonic Stem Cells To Neural Cells

Objective To investigate the probability of deriving mouse inner cell mass and obtaining ES cells by the technique of cultivation of uterus tissue pieces of 1-3 days implantation embryo and the effect of TFAG on differentiation of embryonic stem cells to neural cells.Methods The mouse uterus of 1-3 days implantation embryos were separated,sheared and cultivated. Inner cell masses(ICM) were dissociated and passaged in two days according to routine method of ES cells digesting and passaging. Embryonic bodies were cultured from the mouse embryonic stem cells in the absence of LIF and MEF layer, digested into single cell, and then replanted into adhesive tissue cultivation dishes with different groups of RA and TFAG. After inducing differentiation of these cells for four days, we observed cell morphology through microscope, counted the viable cells in trypan blue solution; then identified these cells by immunohistochemistry with rabbit anti-mouse NF200 antibody and anti-mouse GFAP antibody after culturing these cells for two days in the absence of RA and TFAG.Results 1. Using this method, a stain of mouse embryonic stem cells line passaged ten generations has been established. AKP staining of the cells was strongly positive, the expression of Oct-4 mRNA was also strongly positive manifested by RT-PCR.2. RA was added in each group (5 10-7 mol/L) with the same concentration, the amount of viable cells in the control group without TFAG was 5.5 104ml, 43% of the transformed neural cells was neuron and 12% was glia cells. Compared with the control group, the amount of viable cells were as 11.9, 6.2, 2.8, 1.2 times as the amount of the control when the concentration of TFAG was 5 10-3g/L , 5 10-4g/L , 5 10-5g/L , 5 10-6g/L separately. The proportion of transformed neural cell was 1.9, 1.6, 1.4, 1.1 times of the proportion of the control. There was no difference in transformed glial cells between the control group and the groups adding different concentration of TFAG.Conclusions 1. The undifferentiating inner cell masses can be derived and mouse embryonic stem cells can be obtained by the cultivation of uterus tissue pieces of 1-3 days implantation embryos. The experiment protocols of new method are simplified and the difficulty of culture technique is lowered.2. TFAG can speed up the growth of transformed neural cells; TFAG in high concentration can induce EB cells transforming intoneuron.