Neural Stem Sphere Method: Induction of Neural Stem Cells and Neurons by Astrocyte-Derived Factors in Embryonic Stem Cells In Vitro

We have developed a novel method to produce large numbers of neural stem cells and neurons directly from embryonic stem (ES) cells. The method is composed of three culture stages. In the first stage, floating ES cell colonies are cultured in astrocyte-conditioned medium to directly differentiate neural stem cells from ES cells, resulting in neural stem spheres. In the second stage, neural stem spheres are cultured as adhesive cells to produce neural stem cells by cell migration. In the third stage, neural stem cells are cultured as monolayers, which proliferate in the presence of fibroblast growth factor 2 and epidermal growth factor and differentiate into neurons in astrocyte-conditioned medium.