cystine, and reduced forms. Under the condition of an intracellular reduced state determined by BME + BHA, cystine uptake is most likely a compensatory mechanism to maintain the adequate redox potential in these cells. Cystine is the most oxidized intracellular thiol/disulfide couple, and such a situation may have homeostatic purposes as it can serve as an oxidant signal to control normal cell function [48]. It is possible that redox modulation can drive neuronal differentiation, but non-physiological changes are more likely related to cell damage [49,50,51]. A recent work demonstrated that MSC treated with reduced glutathione apparently differen- tiated into neurons [52]. The induction protocol in this paper used serum-free culture medium, which we demonstrated to be sufficient to induce the morphological changes in MSC formerly believed to be neurons. The electrophysiological data indicate that chemically-induced cells with neuronal phenotype do not exhibit intrinsic properties of neurons. The absence of action potential suggests that either the cells are immature neurons or they are not neurons. Recently, using a protocol described by Woodbury et al. [9], Wenisch et al. [30] studied the intrinsic properties of the induced MSC and showed that they were unable to fire spontaneously or evoke action potential. However, Wenisch et al. [30] concluded that these cells could be immature neurons, based on some ultra- structural characteristics similar to that of neurons. Although immature neurons may not fire an action potential, they exhibit ion voltage-gated channels producing K+ and Na+ currents in response to depolarization. The membrane perme- ability to K+ and Na+ increases during depolarization of excitable cells [53] and currents could be identified using the voltage-clamp technique. According to our results, chemically-induced MSC from adult rat bone marrow did not present K+ or Na+ currents (in contrast to what was recorded in immature neurons). Moreover, chemically-induced MSC are electrophysiologically different from fibroblasts, since they present a class of maxi K+ channels [54]. An interesting observation is that non-induced bone marrow MSC have voltage-gated K+ and Na+ channels [55]. But when these cells are chemically induced they lost this characteristic. It is possible that the redox imbalance caused by the chemical compounds interfere in MSC ion channel function/activation. Some ion channels are modulated or modified by oxidative stress, resulting in modified ion conductance (K+, Ca2+ or Na+) [49,50]. The abnormal function of these ion channels can alter the membrane potential. Moreover, increased concentration of intracellular Ca+ can trigger apoptosis and K+ efflux causes cell shrinkage [56]. Thus, cell shrinkage, altered activity of ion channels and cell death are all closely related phenomena [57]. Following MSC chemical induction, we could clearly notice the interplay between these three events. Holistically, our data allow us to suggest that bone marrow MSC are damaged in the presence of toxic substances that constitute one specific chemical induction protocol and, conse- quently, present abnormal responses. The expression of neural proteins could be one of these abnormalities that may not be necessary (e.g. adult Purkinje cells do not express NSE protein in their somas) [58], and is definitely not sufficient (current results) to classify any single cell as a neuron. Indeed, even the most distinctive neuronal feature, i.e., the ability to fire action potentials, References Induced MSC Are Not Neurons is not present in every single neuron (e.g., bipolar retinal cells lack this capacity). The development of the nervous system depends on the expression of specific genes in particular places and periods. Thus, considering the current conditions (adult rat bone marrow MSC and one specific chemical induction protocol), it is very unlikely that, in a few hours of induction, MSC transdifferentiate into mature (or even immature) neurons. Conclusion Our results indicate that after a specific protocol of chemical induction, adult rat derived MSC display some neuronal characteristics (morphology, expression of neural proteins), a number of which can also be seen in other stressed or damaged cells. However, this protocol of chemical MSC induction does not cause these cells to display neuronal electrophysiological proper- ties. Therefore, we suggest that this specific treatment leads to cytoskeletal alterations that are more likely the culprit in cellular stress and cell death. Hence, we conclude that, even though it may be possible to produce neurons from the multipotent cells present in the MSC population, chemically-induced acute transdifferen- tiation, as used here, is not likely a means to achieve this goal. Nevertheless, it is still plausible that other methods of induction and/or some other sources of MSC are able to achieve a successful neuronal differentiation in vitro. Lastly, we would like to stress the importance of electrophys- iological approaches to determine stem cell differentiation into neurons. Morphological changes and protein expression are not conclusive in the stem cell research field and, therefore, the plasticity of stem cells requires careful validation. Supporting Information File S1 culture. Found at: doi:10.1371/journal.pone.0005222.s001 (0.03 DOC) Supplemental information about the neuronal primary MB Figure S1 Negative controls for the immunocytochemistry method. The primary antibody was lacking to verify the specificity of secondary antibodies. A) Secondary biotinylated antibody anti- mouse IgG; B) Secondary biotinylated antibody anti- rabbit IgG. Found at: doi:10.1371/journal.pone.0005222.s002 (2.64 MB TIF) Acknowledgments We thank Ivone de Paulo for helping in the histological procedures, Dr. Catia A. Pereira for giving the primary cultures of cortical neurons, Prof. Beatriz Amaral Castilho for the donation of the NIH 3T3 cell line, Prof. Jeannine Aboulafia and Prof. Viviane L.A. Nouailhetas for helping in the electrophysiological studies, Prof. Helena B. Nader for the laminin. We also thank Michael Williams and Solange F. Williams from University of Maryland for reviewing and editing the text. Author Contributions Conceived and designed the experiments: GFB TTS MEC ACdO LMNK VD RMO LEM. Performed the experiments: GFB TTS MEC FLM GJM ACdO LMNK. Analyzed the data: GFB TTS MEC FLM GJM ACdO LMNK VD RMO LEM. Contributed reagents/materials/analysis tools: GJM ACdO VD RMO. Wrote the paper: GFB TTS LEM. 1. Abouelfetouh A, Kondoh T, Ehara K, Kohmura E (2004) Morphological differentiation of bone marrow stromal cells into neuron-like cells after co- culture with hippocampal slice. Brain Res 1029: 114–119. 2. Bonilla S, Silva A, Valdes L, Geijo E, Garcia-Verdugo JM, et al. (2005) Functional neural stem cells derived from adult bone marrow. Neuroscience 133: 85–95. PLoS ONE | www.plosone.org 10 April 2009 | Volume 4 | Issue 4 | e5222