Figure 4. Glutamate-evoked membrane currents in neural multipolar cells under voltage-clamp. A. Typical inward currents in response to three 500 mM L-glutamate pulses in neural cells from FDB muscle culture. The membrane potential was held at 260 mV. Glutamate pulses are depicted above the current traces. The dashed lines represent baseline. B. Response to glutamate application for 1 s and desensitization during agonist application. C. Glutamate-evoked current as a function of culture time (days 4–14). Data points represent individual cells. doi:10.1371/journal.pone.0016816.g004 Schwann cell primary culture as a positive control for the S100b antibody to confirm that nestin-GFP+/Tuj1+ cells lack of peripheral glial marker expression. Additionally, Schwann cells did not express Tuj1 (Fig. 8B). To look for myelin production in culture, we stained cells with toluidine blue (1%) and sodium borate (1%), following published procedures [34], and observed no specific staining (n = 2 preparations, data not shown), supporting the concept that nestin-GFP+ cells are not Schwann cells or their precursors. Nestin-GFP+ neural stem cells derive from interstitial progenitor cells in skeletal muscle To address the source of nestin-GFP+/Tuj1+ neural stem cells, immediately after isolating hindlimb muscles, we sorted two populations, GFP+ and GFP-, and cultured them separately under Skeletal Muscle Neural Precursor different conditions (Fig. 9A–C). Nestin-GFP+ cells represent a small fraction of all sorted cells (Fig. 9D), as reported above. A comparison between nestin-GFP+ cells sorted from pooled hindlimb muscles and FDB muscles is not straightforward. Pooled hindlimb cells were sorted immediately after dissociation, normalized to the number of events, and characterized by a significant number of floating cells, while FDB muscle cells were sorted after 7 days in culture, normalized to the number of nuclei, and floating cells were absent. These methodological differences affect whether these two preparations yield variable nestin-GFP+ cells and require further examination. Neither positive nor negative cells gave rise to cells with neural phenotype after 8 days in culture (Fig. 9E), which agrees with previous observations [25]. However, when co-cultured, approximately 12 nestin-GFP+/ Tuj1+ cells/dish showed 2 or more processes. To determine the source of these neural cells, either nestin-GFP+ or nestin-GFP- cells were co-cultured with dissociated FDB muscle fibers from wild-type mice. While the negative cell co-culture produced no cells with neural morphology, that with positive cells produced approximately 17 cells/dish with neural morphology, supporting the concept that nestin+/Tuj1+ cells arise from nestin-GFP+ progenitors residing in skeletal muscle. To exclude the influence of myofibers, before culture, FDB preparations were filtered through a 40 mM strainer that retained the myofibers. After 7 days in culture, cells with neuronal phenotype co-expressing nestin-GFP and Tuj1 appeared normal (data not shown). To test the action of putative factors secreted by nestin-GFP- cells on nestin-GFP+ cell morphology, the medium from the FDB cultures was added to nestin-GFP+ cultured cells. Approximately 17 cells/dish showed neural phenotype, indicating that the medium contains soluble ‘‘factor(s)’’ that enable nestin-GFP+ cells to adopt a neural phenotype. Discussion Nestin-GFP and Tuj1 expression markers can identify neural progenitor cells from adult skeletal muscle In this study, starting at day 2 in culture, we isolated neural progenitor cells from adult skeletal muscle-derived cultures based on nestin-GFP and Tuj1 expression. These cells also expressed light and heavy neurofilament as additional neural properties but not CD31, a smooth-muscle actin, or S100b, which indicates that they express typical neural but not endothelial cell, pericyte, or Schwann cell markers. Here, we propose that the continuous expression of transgenic nestin-GFP in muscle cultures provides an invaluable tool to isolate a population of predifferentiated neural cells with therapeutic potential. Neural stem cells isolated from the embryonic central nervous system express nestin, a neuro-ectodermal cell marker [14]. After a few days in vitro, when plated on a coated surface, they spontaneously express classical markers of more differentiated neural cells, such as Tuj1 [35], and recapitulate major features of central nervous system neural cell generation [36,37]. Based on these observations, we conclude that cells that express nestin and Tuj1 are neural progenitors. Under our culture conditions, nestin- GFP+/Tuj1+ cells seem to produce only predifferentiated neural cells, possibly from a reduced capacity to produce other cell types [38]. Whether they might produce a more diverse cell population in animals of different species or age is not known. Previous studies showed GFP+ neural progenitors in the brain [15] but not the skeletal muscle of the nestin-GFP transgenic mice used in this work [39], which may be explained by differences in cell isolation and culture [25]. In our work, cells sorted after 7 days in culture were already predifferentiated, while in Day’s PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e16816