Induced MSC Are Not Neurons Figure 5. Cell death analysis of MSC. Graphic representations of the percentage of MSC that are: A) live cells (Annexin2 PI2); B) necrotic (Annexin2 PI+); C) early apoptotic (Annexin+ PI2); or D) late apoptotic (Annexin+ PI+) in the non-induced (control, CTR), serum-free (SF), pre-induced (BME) or chemically-induced (for CI4 h, CI8 h and CI24 h). Mean6standard error. p,0.05, # different from every other group; * different from non- induced and serum-free. doi:10.1371/journal.pone.0005222.g005 High demand over redox circuitry during chemical induction In order to evaluate whether the chemical compounds used in the neuronal induction could promote alterations in the redox balance, we analyzed the Cys and Hcy cellular contents. No cellular change in Hcy content was observed in chemically- induced MSC. However, total Cys content increased almost four times in induced MSC as compared to non-induced cells (figure 6). Neuronal-like MSC do not exhibit neuronal electrical properties To determine whether neuronal-like cells derived from adult rat MSC were electrically active, we performed patch-clamp record- ings to study the presence of K+ and Na+ currents (whole-cell voltage-clamp), voltage-gated channels (cell attached) and the ability of cells to fire action potentials (whole-cell current-clamp). We observed that both neurons from E17 cortex primary culture (n = 6) and untreated MSC (n = 5) exhibited K+ currents (Figue 6 A1, A2, B1, B2). However no K+ current was detected from chemically-induced MSC with neuronal-like morphology (n = 7) (figure 7 A3, A4, B1, B2). These cells also did not present Na+ current when compared with controls (neurons from primary culture n = 6; chemically-induced MSC n = 10) (figure 8 A1, A2). We observed that both, the immature neurons from primary culture and the chemically-induced MSC were unable to fire action potentials when depolarizing currents were injected in current-clamp recordings (n = 3; n = 5) (figure 8 B). Lastly, cell-attached voltage-clamp recordings were performed to evaluate the presence of voltage-gated channels in the cell membrane (figure 7 C). Here again, MSC subjected to the chemical treatment differed from embryonic rat neurons. The activation of K+ channels in response to membrane depolarization (+60 mV) occurred only in primary cultured neurons (NPo = 0.04) (n = 3) (figure 7 C1), but no activation was observed for chemically-induced MSC (NPo = 0) (n = 5) (figure 7 C2). Discussion The potential of adult bone marrow MSC to become neurons is controversial. When cells are removed from their niche and propagated in vitro, they lose many of their original properties (proliferation rates, gene expression, morphology, cell-cell inter- Figure 6. Ratio between Hcy e Cys contents obtained for non- induced (control, CTR) and chemically-induced MSC. No cellular change in Hcy content was observed in the induction protocol (p = 0.23). Total Cys content increased almost four times (p = 0.00006) in chemically-induced MSC compared to non-induced ones. Data represented as mean6standard deviation. doi:10.1371/journal.pone.0005222.g006 PLoS ONE | www.plosone.org 7 April 2009 | Volume 4 | Issue 4 | e5222