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 5μmol/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 5μmol/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. 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 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 c ells 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, 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- ) 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 with longer processes and a similar appearance to mature neurons using the one-step method. percentage of MAP2 positive cells induced by the one -sThe tep 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 There was no significant differences in these parameters between the one-step and two-step methods (P> 0.05). Addition, 80% of the induced neuronal-like cells from the one-step method and 20% from the two-step method 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.