electrophysiological responses [23–25]. Thus, chemically-induced adult bone marrow MSC transdifferentiation is very unlikely to occur. Other reports suggest that neural differentiation induced by chemical compounds results from cytoplasmic retraction triggered by stress, which confers a neuronal-like morphology to the cells after treatment [26,27]. However, these authors did not define the stressful conditions triggered by chemical induction. Despite the limited mechanistic data supporting this chemical induction method, it is still widely used by a considerable number of researchers [3,5,8,28–30]. The objectives of present study are to evaluate the potential of a protocol based on chemical reagents in generating neurons from rat bone marrow MSC, and elucidate the electrical properties and molecular characteristics of these cells. Given that DMSO is an inhibitor of S-adenosylmethionine synthetase, an enzyme involved in homocysteine metabolism, and that BME is a potent disulfide bond reducer, changes in this metabolic route and redox state may be important determinants of cellular fate. Materials and Methods MSC Culture Primary adult rat bone marrow MSC were isolated according to the method of Azizi et al. [17]. Briefly, Wistar adult male rats, 2 months old, were sacrificed in a CO2 chamber, according to the Animal Care and Use Committee of the Federal University of Sa˜o Paulo. Tibias and femurs were dissected, the ends of the bones were cut, and 10 mL of DMEM (Gibco, Grand Island, NY, USA) were injected into the central canal of the bone to extrude the marrow. After Histo-Paque (density of 1.077 g mL21) (Sigma, St. Louis, MO, USA) purification of whole bone marrow, mononuclear cells were isolated and cultured at a density of 26105 cells cm22 in DMEM supplemented with 20% fetal bovine serum (Gibco) and 1% penicillin/streptomycin/amphotericin (Gibco). After 24 h, non- adherent cells were removed, and the medium was changed every 3–4 days until the culture became 80% confluent. The cells were tripsinized, split 1:4 and passaged up to three times. Neuronal Cultures Rat neural precursor cells (NPC) [31] and primary culture of rat fetal neurons from the brain cortex [32] were obtained as described previously [31,32] (for details see supplementary data on file S1) and served as positive controls. NPC were used to simulate the differentiation of neurons from stem cells. Primary neuronal cultures were used as a common source of neurons. Fibroblast Culture NIH 3T3 fibroblasts were maintained in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin/amphotericin. These cells were used as negative controls in many experiments. Chemical Induction Primary MSC were chemically induced to become neuronal- like cells in vitro according to the methods of Woodbury and colleagues [9]. Briefly, approximately 80% confluent MSC cultures were pre-induced for 24 h with DMEM, 20% fetal bovine serum, and 1 mM BME (Sigma). The pre-induction medium was then removed, and changed to serum-free induction medium consisting of DMEM containing 2% DMSO (Sigma) and 200 mM BHA (Sigma) for 4, 8 or 24 h. Control Groups Matching MSC cultures were kept in standard medium (non- induced group, DMEM + 20% fetal bovine serum), or in serum-free Induced MSC Are Not Neurons condition (DMEM only) for 24 h, or were induced for 8 h and then returned to the standard medium overnight. NIH 3T3 fibroblasts, used as controls, were treated for 8 h with the chemical induction components (induced NIH 3T3), or kept in standard medium (non- induced NIH 3T3). The mononuclear fraction of bone marrow, after separation in Histo-Paque, was used in immunofluorescence and Western Blot assays. As positive controls for the neuronal differentiation protocol, NPC and primary neuronal culture were obtained as described above. Images of all groups were taken periodically with an inverted phase contrast microscope coupled to a digital camera (microscope model IX51-RC; Olympus, Japan) to record possible morphological changes in culture. Immunocytochemistry NPC and all MSC groups were seeded into 24-well plates containing round coverslips. Chemically induced MSC were incubated in induction medium for 8 h. After treatment, cells were fixed with 4% paraformaldehyde in 0.1 M PBS (pH 7.2) for 30 minutes and processed for immunocytochemistry to assess for cell differentiation, as described below. Nonspecific antibody reactions were blocked with 5% normal goat serum (Gibco) for 1 h at room temperature and cells were permeabilized with 0.1% Triton X-100 (Sigma). Next, cells were incubated overnight at 4uC with primary antibodies diluted in blocking solution directed against GFAP (Dako, Denmark, 1:500), NeuN (Chemicon, Temecula, CA, USA, 1:500), b-tubulin III (Sigma, 1:500), MAP- 2 (Chemicon, 1:250), NSE (Dako, 1:200), NF-200 (Sigma, 1:100) and S100 (Sigma, 1:100). After three washes with PBS, cells were incubated with the respective anti-mouse or anti-rabbit biotiny- lated secondary antibodies (1:200; Vector Laboratories, Burlin- game, CA, USA) for 1 h at room temperature, followed by 1 h of incubation in avidin-biotin peroxidase complex (1:100; Vector Elite Kit) at room temperature. Diaminobenzidine (0.05%; Sigma) with nickel chloride (0.04%) was used as chromogen, with reactions sustained for 10 minutes at room temperature. Cells were then coverslipped with Entellan (Merck, Germany) on glass slides. NPC were used as positive controls for immunolabeling; for negative control primary antibodies were omitted (figure S1). Cells were analyzed under a bright field microscope (Nikon Eclipse E600 FN, Nikon, Japan) and images were captured by a coupled camera system using ACT-1 software (v.2.2, Nikon). This experiment was performed independently for three times using two coverslips of plated cells for each marker (n = 6). Cells were counted and the proportion of positive stained cells was estimated by dividing the number of positive cells by the total cells number in the same field. Statistical analysis consisted of ANOVA (95% of confidence interval) followed by Newman-Keuls post-hoc test. Immunofluorescence The bone marrow mononuclear cell fraction of 5 adult rats (3 to 6 months old) was obtained after separation of total bone marrow in Histo-Paque, as described above. Cells were immediately fixed in 1% paraformaldehyde and distributed on gelatinized glass slides by cytocentrifugation at a density of 56105 cells mL21 in 200 mL per slide. Cells were washed and incubated in blocking solution with primary antibodies against GFAP, b-tubulin III, NeuN, NSE, NF- 200 and S100, followed by fluorescent secondary antibodies (Cy3 conjugated; Jackson Immuno Research, West Grove, PA, USA, 1:1000), in the same conditions as described for immunocyto- chemistry. Nuclei were counterstained with DAPI and slides were coverslipped with paraphenylenediamine. Cell images were taken by Axiocam MRm camera system (Carl Zeiss Inc., Thornwood, NY, USA) coupled to a fluorescent microscope (Axiovert 200M, PLoS ONE | www.plosone.org 2 April 2009 | Volume 4 | Issue 4 | e5222