Skeletal Muscle Neural Precursor Figure 7. Sorted nestin-GFP+ cells from FDB cultures show neural markers. Sorted GFP+ (A) and GFP- (B) cells were seeded in parallel on dishes precoated with laminin and grown for 48 hours until attached. The first column shows immunostaining for Tuj1, NF-L, or NF-H, while the second shows their corresponding nestin-GFP fluorescence. Nuclei were stained with Hoechst. Merged images are shown on the far right. Scale bar = 100 mm. doi:10.1371/journal.pone.0016816.g007 proliferated at a rate approximately one-fourth that of cell propagation. Moreover, these cells survive for at least two weeks and maintain their proliferation rate even when cultured after sorting. With EdU, we detected a skeletal muscle neural progenitor cell proliferation rate similar to that reported for neural progenitor cells in the hippocampus [47] and spinal cord [48]. Functional response to glutamate Functional analyses of our predifferentiated neural progenitor cells revealed that a population of nestin-GFP+ cells isolated from skeletal muscle had the electrophysiological characteristics of neurons. Glutamate-evoked currents shared some properties with those reported in native mouse central neurons [21]: they were evoked in a 1 mM Ca2+ solution; the current was inward at negative potentials; it responded to repeated glutamate challenges in a typical way; and it desensitized during prolonged glutamate application. Additionally, the number of cells responding to the agonist increased with time in culture. These recordings agree with a previous report in which intracellular Ca2+ concentration increased in response to glutamate [42]. The increased respon- siveness of nestin-GFP+/Tuj1+ cells in culture supports their progressive differentiation and expression of ion channels. PLoS ONE | www.plosone.org 11 February 2011 | Volume 6 | Issue 2 | e16816