Optimization of induced differentiation of human neural stem cells into dopaminergic neurons by factorial design

Abstract

Objective To induce the differentiation of human neural stem cells (hNSCs) into dopaminergic neurons. Methods From a total of 5 kinds of inducing factors including aFGF, dbcAMP, pramipexole (PRX) , TPA (phorbol-12-myristae-13-acetate) , forskolin, single factor experiments were used to screen significant factors which play an important role in inducing hNSCs into dopaminergic neurons, and significance tests were tested by student's t. Factorial design was used to evaluate the inducing effect of the selected significant factors and to find out how these factors affect the induced dopaminergic neuron positive ratio. Statistical significance was tested by analysis of variance. Finally, the optimal concentration of each significant factor was determined by using the steepest-ascent method. Results According to single factor experiments, aFGF, dbcAMP and TPA were considered to be important factors in inducing differentiation of dopaminergic neurons form hNSCs. Inducing effect of aFGF, dbcAMP and TPA was modeled by factorial design. These results showed that aFGF and dbcAMP were positive factors and TPA was negative factor at the initial concentrations. The ratio of concentration changes in aFGF, dbcAMP and TPA should be 0.29 : 0.90 : -0.39. The stepwise of aFGF, dbcAMP and TPA used in the steepest-ascent method was selected as 11 ng/ml, 35 μmol/L and -15 nmol/L, respectively. Then, the optimal concentration of aFGF, dbcAMP and TPA was achieved at 166 ng/ml, 310 μmol/L and 10 nmol/L. The optimal dopaminergic neuron positive ratio was (6.56±0.07) ﹪, which increased by 23.8﹪ comparing to control group. Conclusion Factorial design can effectively optimize the differentiation of hNSCs into dopaminergic neurons. Key words: Neural stem cells; Dopamine; Neurons