Abstract
Neural stem cells (NSCs) have the ability to self-renew, and are capable of differentiating into neurons, astrocytes and oligodendrocytes. Here, we examined the effects of mitogens, fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF), on NSCs prepared from mouse ES cells by the neural stem sphere (NSS) method. Cultured with mitogens, the NSCs were promoted to proliferate by the mitogens in dose-dependent manners. Affinity of the NSCs for FGF-2 was lower than that for EGF, but promotion effects by FGF-2 was higher than that by EGF. The promotion effects by FGF-2 and EGF were not additive. Doubling time of the NSCs cultured with FGF-2 was 15.1h. Subculture of the cells could be stably passaged at least 5 times. Irrespective of the mitogen added to the culture, the NSCs expressed high levels of mRNAs of the marker genes for NSCs, Nestin, Musashi-1, and mRNAs of two types of FGF receptors and EGF receptor, FGFR2, FGFR3, and EGFR, respectively. In contrast, the NSCs expressed low levels of mRNAs of the marker genes for neurons, astrocytes and oligodendrocytes, NF-M, GFAP and MBP, respectively, and mRNAs of FGF receptor-4, FGFR4. The gene expression levels of the NSCs were stably maintained during culture at least 5 passages. These results confirm that the NSCs prepared from ES cells maintain potential of active proliferation under culture conditions containing FGF-2 or EGF.
