BACKGROUND: A combination of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), human heregulin-beta-1, beta-mercaptoethanol retinoic acid and forskolin has been reported to induce the differentiation of rat bone marrow stromal cells into myelinating Schwann-like cells. OBJECTIVE: To investigate the inducing effects of a combination of bFGF, PDGF, human heregulin-beta-1, beta-mercaptoethanol retinoic acid and forskolin on neural stem cell differentiation by one- and two-step methods.DESIGN, TIME AND SETTING: A cytobiology experiment was performed at the Department of Histology and Embryology, Medical School of Nantong University, and Jiangsu Province Key Laboratory of Neuroregeneration, China, between August 2005 and January 2007.MATERIALS: A total of 30 healthy Sprague Dawley rat embryos at gestational days 14-16 were selected. bFGF, PDGF, human heregulin-beta-1, beta-mercaptoethanol, retinoic acid, and forskolin were purchased from Sigma, USA. METHODS: Passage 3 rat neural stem cells were cultured by a one-step method in serum-free medium plus 10 ng/mL bFGF, 5 ng/mL PDGF, 200 ng/mL heregulin-beta-1, 35 ng/mL all-trans retinoic acid, and 5 μmol/L forskolin or by a two-step method in serum-free medium plus 35 ng/mL all-trans retinoic acid for 72 hours, followed by serum-free medium plus 10 ng/mL bFGF, 5 ng/mL PDGF, 200 ng/mL heregulin-beta-1 and 5 μmol/L forskolin. The control condition consisted of 10% fetal bovine serum alone or 20 ng/mL bFGF alone.MAIN OUTCOME MEASURES: Differentiated cells were identified by immunocytochemical staining for microtubule associate protein-2 (MAP2) and S100 protein. Geometric parameters and sodium ion currents of the differentiated cells were measured by image analysis and whole-cell patch-clamp techniques, respectively.RESULTS: Compared with the two-step culture method, neuronal-like cells exhibited longer processes and a similar appearance to mature neurons using the one-step method. The percentage of MAP2 positive cells induced by the one-step method was significantly greater than the serum-alone group (P < 0.05). Furthermore, the MAP2 positive cells induced by the one-step method had greater surface areas, cell body perimeters, and longer process than cells induced by serum-alone and bFGF-alone (P < 0.05). There were no significant differences in these parameters between the one-step and two-step methods (P > 0.05). In addition, 80% of the induced neuronal-like cells from the one-step method and 20% from the two-step method displayed inwardly-evoked currents. CONCLUSION: The combination of bFGF, PDGF, human heregulin-beta-1, beta-mercaptoethanol retinoic acid and forskolin successfully induced neuronal differentiation from neural stem cells, with the one-step induction being more effective than the two-step method.