Induced MSC Are Not Neurons Figure 3. Proportion of positive stained cells for neural proteins in rat bone marrow mononuclear cells and in non-induced, serum- free and chemically-induced MSC. For each neural marker, statistically significant differences (ANOVA, p,0.05) between cell types/conditions appears under brackets. doi:10.1371/journal.pone.0005222.g003 expressed only the 66 KDa NeuN isoform, while bone marrow mononuclear cells (lanes 7, 8, 9) and brain (lane 10) extracts had all three NeuN isoforms (66, 46, 48 KDa). The antibody against b- tubulin III cross reacts with the rat tubulin, and explained the 55 KDa band. Only bone marrow mononuclear cells (lanes 7, 8, 9) and brain (lane 10) samples had the 48 KDa band corresponding to the b-tubulin III expression. NF-200 expression increased after MSC chemical induction (lanes 5, 6) and is also present in bone marrow mononuclear cells (lanes 7, 8, 9). A small increase in NF- 200 expression was observed after serum deprivation of NIH 3T3 cells (lane 2) and of MSC (lane 4). Actin (43 KDa) was used as an internal control. Chemical induction leads to apoptosis Flow cytometry analysis for Annexin V and PI indicated that during the chemical induction process there was an increase in cell death (figure 5). Although serum deprivation for 24 h is characterized as a stressful condition for MSC, it was not sufficient to trigger cell death mechanisms. Furthermore, serum-free MSC show the same proportions of live and dead cells as non-induced MSC. At the end of the chemical induction protocol (24 h) we found a significant percentage of dead cells (44.8%; figure 4A; ANOVA p,0.05, followed by Newman-Keuls as compared to every other group, p,0.01). Our results also demonstrated that most of cells died due to apoptotic events during chemical induction. Indeed, Annexin V2/PI+ cells, likely to indicate necrotic elements, were less frequent than Annexin V+ (both PI+ and PI2) likely to indicate apoptotic events. Putative early apoptotic cells (Annexin V+/PI2) increased after exposure to BME and BHA/DMSO at 4 h, and were augmented after exposure to BHA/DMSO at later times with the highest value occurring at 24 h (ANOVA p,0.05, followed by Newman-Keuls all groups as compared to non-induced group, and chemically- induced for 24h as compared to every other group, p,0.01; figure 4C). The number of late apoptotic cells (figure 4D) significantly increased after BME pre-induction treatment (11%) as compared to non-induced MSC and at the 24 h induction time point (24.2%) as compared to every other group. We have thus found a number of significant differences between non-induced and chemically-induced MSC groups for live and apoptotic cells (early or late apoptotic; figure 5). Figure 4. Analysis of the expression of NeuN, NF-200 and b-tubulin III by Western Blot. Lanes: 1) non-induced NIH 3T3; 2) chemically-induced 3T3; 3) non-induced MSC; 4) serum-free MSC; 5 and 6) chemically-induced MSC for 8 h; 7, 8 and 9) freshly extracted bone marrow mononuclear cells; 10) brain tissue (positive control). Actin (43 KDa) was used as an internal control. doi:10.1371/journal.pone.0005222.g004 PLoS ONE | www.plosone.org 6 April 2009 | Volume 4 | Issue 4 | e5222