until the end of the experiment. The duration and amplitude of each current level were determined using idealized records from the original data, constructed through the recognition of the transitions between distinct levels. NPo values, where N is the number of channels in the patch available to open and Po is the open probability of the channel, were calculated by the ratio of the mean current to the unitary single-channel current. The mean current was obtained from the amplitude current distribution histogram, using the following expression: Imean = A1f1+A2f2+..... Anfn where A1, A2, and An represent the area under the Gaussian curve for each current level (f1, f2, fn) present in the patch. Voltage and current were registered with the Axopatch 200 amplifier (Axon Instruments/Molecular Devices Corp., Union City, CA). Whole-cell voltage-clamp data were filtered at 1 kHz and cell-attached data were filtered at 5 kHz (3 dB, 8-pole Bessel filter), and monitored with pClamp 6.0 software (Axon Instru- ments/Molecular Devices Corp.). The resulting currents and voltage were analyzed using Clampfit 9.0 (Axon Instruments/ Molecular Devices Corp.). Ethical issues This work was developed under the approval of the Ethics Committee of Federal University of Sa˜o Paulo (UNIFESP), process 01093-06. Results Acquisition of neuronal-like morphology by MSC subjected to chemical induction is due to retraction of cytoplasmic processes Adult rat bone marrow-derived MSC were isolated and successfully expanded, displaying characteristic morphology and colony formation [34]. Morphological alterations were observed a few hours after MSC induction following the protocol proposed by Woodbury et al. [9] (figure 1), thus reproducing the effects described previously. Those cells showed morphological similar- ities to neurons. Changes in cell shape were gradual and due to retraction of cytoplasmic processes. A long period of induction (24 h) caused complete processes retraction and cells acquired a round shape, leading to detachment from the culture plate surface. To evaluate cell death we performed Annexin V and PI staining, as demonstrated below. To properly evaluate cell death and detachment, experiments were done at 8 h post-induction, when 80% of the initial amount of cells was likely to be alive and 82% displayed a neuronal-like shape (figure 1). Acquisition of neuron-like morphology also occurred for MSC cultured in the absence of fetal bovine serum for 24 h (likely to represent a stressful condition) (figure 2). After chemical induction and re-exposure to fetal bovine serum, cells reacquired their typical normal morphology, with a spread-out cytoplasm, and there were no longer cells with neuronal-like morphology (data not shown). As demonstrated in figure 1D, NIH 3T3 fibroblasts also adopted neuronal phenotype after exposure to the chemical induction protocol. Our data suggest that MSC display morpho- logical alterations as a response to the chemical compounds used for the induction of neuronal fate or to the lack of normal supporting substances (such as fetal bovine serum). Expression of neural proteins is not restricted to chemically-induced MSC Immunocytochemistry results showed that induced cells with neuronal-like morphology were positively stained for NF-200, S100, b-tubulin III, NSE and MAP-2 proteins (figure 2). MSC in Induced MSC Are Not Neurons Figure 1. Morphological comparisons between non-induced MSC, chemically-induced MSC, chemically-induced NIH 3T3 and primary neurons. A) Schematic illustration of the chemical neuronal induction protocol. B–D) Phase-contrast microscopy images showing morphological aspects. B) MSC have a spread-out cytoplasm in standard medium; C) after induction for 8 h cells exhibited neuronal-like morphology; D) fibroblast NIH 3T3 after 8 h induction adopted neuronal-like morphology; E) primary neuronal cultures. Scale bar = 100 mm. doi:10.1371/journal.pone.0005222.g001 the serum-free condition also stained for all the markers that appeared in the chemical induction group, except for NeuN. NeuN immunostaining of chemically-induced MSC had low intensity compared to neurons from rat brain section (figure 2). Cell staining was found at a similar intensity level in the entire cytoplasmic extension of induced MSC as well as in their extension processes, while the localization of these proteins is normally restricted to specific neuronal regions (dendrites: b-tubulin III; axons: MAP-2). Culture of neural cells expressed all of the expected markers including GFAP that did not appear in any of the other MSC culture conditions. The counting of immunostained cells in bone marrow mononuclear fraction, non-induced, serum-free and chemically- induced MSC shows the gene expression profile of neural markers (figure 3). All other neural markers used in this work were found in mononuclear cell fraction, except MAP-2. In standard culture conditions, MSC (non-induced) did not express MAP-2 and NeuN, but a small fraction of cells were positive for b-tubulin III, NF-200 and S100. The proportion of positive staining increased in serum-free condition for b-tubulin III, MAP-2, NF-200 and NSE, when compared to non-induced MSC. Finally, chemical induction always had a higher proportion of positive cells than non-induced, with a remarkable increase in the percentage of NeuN, NF-200 and S100 staining compared to the serum-free condition. Considering the Western Blot analysis (figure 4), samples from non-induced NIH 3T3 (lane 1) and non-induced MSC (lane 3) PLoS ONE | www.plosone.org 4 April 2009 | Volume 4 | Issue 4 | e5222