Skeletal Muscle Neural Precursor Figure 3. Neurospheres derived from muscle FDB cultures. A. Neurospheres formed on day 8 in culture are nestin-GFP+ and Tuj1+. Nuclei were stained with Hoechst 33342. B. Nestin-GFP+ cells grow as monolayers after fusing into neurospheres. C. Nestin-GFP+ cells form networks. D. Nestin-GFP+/Tuj1+ cells exhibit neural morphology, with many processes. Right panels for B, C, and D are brightfield images. Images in B-D were taken on culture day 8. Scale bar = 100 mm for all pictures, including insets in panel A. doi:10.1371/journal.pone.0016816.g003 was recorded in 2 cells (data not shown). Differences in response kinetics may result from variable perfusion efficiency and/or channel expression or modulation. Figure 4C shows the 21 cells tested between days 4 and 14 in culture. Approximately 50% did not respond to glutamate pulses; 4 cells responded with a current greater than 300 pA, while the remaining 5 cells showed a current smaller than 200pA. The graph shows a small or no response to glutamate at days 4 and 5, increasing at days 12–14, which indicates the progressive differentiation and expression of ion channels in culture. Nestin-GFP+/Tuj1+ cells proliferate To determine whether they proliferate, nestin-GFP+ cells were cultured using the FDB preparation described above. On day 7, they were incubated with 10 mM EdU (5-ethynyl-29 deoxyuridine) for 2 h prior to fixation and immunostaining. Results were similar to those achieved with an EdU concentration of 20 mM (data not shown), which indicates that the 10 mM EdU used in most experiments was not a limiting factor in assessing cell proliferation. Approximately 28% of nestin-GFP+/Tuj1+ were also EdU positive (Fig. 5A, B). These data support the concept that these cells proliferate. To confirm that they retain their proliferative capacity, EdU was examined at days 10 and 14: 2660.6% and 2961.3% cells incorporated EdU, respectively (n = 3 FDB muscles from 3 mice) (Fig. 5B). These differences were not statistically significant. Skeletal muscle neural progenitor cells can be purified from 7-day FDB skeletal muscle cultures based on nestin- GFP expression To investigate whether nestin-GFP+ cells can survive and proliferate in isolation from other cells in FDB cultures, they were sorted based on nestin-GFP fluorescence at day 7 and cultured. Flow cytometry showed that nestin-GFP+ cells represent approx- imately 5% of the presorted cell population (n = 3 preparations from 3 different cultures; Fig. 6A, C). We also sorted GFP- cells as a control. To verify the purity of our sorted cells, they were re- analyzed by cell sorting, which confirmed that all were GFP+ (Fig. 6B). Sorted cells were plated on dishes precoated with laminin and cultured using the medium described above (Methods). Sorted nestin-GFP+ cells exhibit neural markers After 48 hours in culture, nestin-GFP+ sorted cells from the FDB muscle were examined for Tuj1, neurofilament-L (NF-L), and neurofilament-H (NF-H) expression in 4 FDB from 2 mouse cultures, which gave 4 dishes, each containing 5 coverslips; 100 cells were counted per stain. All nestin-GFP+ cells were positive for these markers (Fig. 7A). These cells survive at least 2 weeks in culture and retain proliferative capacity, as demonstrated by the number that incorporated EdU (2560.7%) (Fig. 5B). All nestin- GFP- cells were negative for these markers (Fig. 7B). Nestin-GFP+ cell immunostaining that omitted the primary antibody was negative for the three neural markers (data not shown). Endothelial cell, pericyte, and Schwann cell markers in unsorted FDB muscle-derived nestin-GFP+ neural cells We examined whether unsorted cells exhibit endothelial cell, pericyte, and Schwann cell markers in FDB muscle-derived nestin- GFP+ neural cells using CD31, a smooth-muscle actin, and S100b antibodies, respectively, at day 10 in culture. Figure 8A shows that nestin-GFP+ cells were negative to these three markers. The lack of immunoreactivity to these antibodies was confirmed in 4 FDB from 2 mouse cultures; 100 cells were counted per dish. We used a PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e16816