Induced MSC Are Not Neurons Figure 8. Electrophysiological recordings of Na+ current and AP firing ability. A) Representative voltage-dependent Na+ current recorded during a 100-ms hyperpolarizing pulse, followed by 200-ms depolarizing steps (10-mV increments) (see inset) for primary culture neuron (A1), and chemically-induced MSC (A2). B) Membrane potential of chemically-induced MSC recorded under current clamp. Note the inability to fire AP with depolarizing current steps (50 or 1000 ms). doi:10.1371/journal.pone.0005222.g008 to stress; ii) adult rat bone marrow mononuclear cells also express some neural markers irrespective of being subject to chemical induction; iii) apoptosis rates are increased over induction time; iv) redox circuitry is under high demand during chemical induction; v) adult rat bone marrow MSC using this specific neuronal induction protocol do not present electrical properties of fetal neurons. It was previously described that some bone marrow cell populations, including MSC, express neural markers even before any neural induction protocol [3,38–42]. These data are in agreement with our findings for adult bone marrow mononuclear cells, where we found S100, b-tubulin III, NeuN, NSE and NF- 200 expression by immunocytochemistry and/or Western Blot analysis. It has been suggested that an overlap between the genetic programs of bone marrow adult stem cells and neural stem cells [38,42] exists, but this does not mean that the same proteins play the same roles in different cell types. NRSF/REST mediates the transcription repression of neuron- specific genes in non-neuronal cells [43,44]. The transcriptional repressor neuron-restrictive silencer factor has a critical role in the specific control of connexin36 in insulin-producing cell lines, indicating that connexin36 participates to the neuronal phenotype of the pancreatic beta-cells [45]. In order to explain the presence of neural markers in bone marrow mononuclear cells, we can hypothesize that neuronal gene expression in a specific subpopu- lation could be due to release of neuron-restrictive silencer element. When bone marrow mononuclear cells were plated for in vitro expansion, a subpopulation was selected by the property of adhesion to plastic surface. The resulting adherent MSC present low levels or do not express any of the tested neural proteins. Whether this is a feature of this subpopulation or cells down- regulate neural protein expression after plating is still unknown [46]. But when fetal bovine serum was removed or cells were chemically induced with specific compounds, the expression of these proteins became high. In fact, immunocytochemistry experiments showed that cells with neuronal-like morphology express neuronal proteins. Other groups also have demonstrated that chemical induction triggers the increase in the transcription and expression of some genes that code for neuronal proteins [9,27], as well as serum-free media, when fetal bovine serum is removed [35]. As we observed, this increased gene transcription/ expression may be related to stressful factors [26]. For example, NSE is increased after brain injury and in cancers [47]. Measures of cell death during this specific induction process are indicative of toxicity of the chemical compounds used. Here we show that most cells die within 24 h of chemical induction, in agreement with other studies [29]. We can consider a gradient of cellular stress in which the mild fraction triggers the expression of genes that might help cells to survive; and a severe, long-lasting cellular stress culminates with cell death. Here we can compare the mild stress to the serum-free media, and the severe to the chemical induction. In this way, it is possible we had found less positive cells for some neural markers after chemical induction than in serum- free MSC because the stained cells already died after 8h induction. Accordingly, the toxic environment should trigger abnormal responses in the cells, far from what would constitute a physiological response. Altered total cysteine content in these cells can also be interpreted in this context. In the presence of a S-adenosylmethi- onine synthetase inhibitor, i.e., DMSO, the increase in cysteine content must be related to increased cysteine uptake. Cells are able to take cysteine from the culture media both in its oxidized, PLoS ONE | www.plosone.org 9 April 2009 | Volume 4 | Issue 4 | e5222