At day 5, the population of nestin+/Tuj1+ cells increased to 88.860.8%, while the nestin-GFP+/MyoD+ population disap- peared completely, as confirmed by time-lapse experiments (see below). At day 6, 9660.4% of cells were nestin+/Tuj1+, while from day 7 to day 14, they represented 100% of nestin-GFP+ cells. This co- expression indicates that nestin-GFP+ fluorescence in cells cultured for 7–14 days defines a population of neural progenitor cells in the skeletal muscle of the adult mouse. These experiments were repeated three times using 4 nestin-GFP transgenic mice, 8 FDB muscles, and 40 coverslips per experiment. Four microscope fields were analyzed per coverslip (160 fields total) at 20X magnification for cell quantification. Note that the culture medium used for these experiments contains chicken embryo extract, which is a cocktail of multiple trophic factors and mitogenic cues, and no specific neurotrophic factors were added. This medium is normally used for myogenic cultures and to support muscle cell proliferation and differentiation [29]. To assess its importance, we used cultures without embryo extract and obtained similar results (data not shown), which implies that it is not essential for commitment to neural cells. Laminin coating on dishes made an obvious difference. For prolonged periods, nestin-GFP+ cells in culture did not adhere to uncoated dishes, indicating that they need laminin as an attachment matrix. Fibroblasts, myogenic cells, and pericytes may contribute to the significant number of MyoD-/Tuj1- cell nuclei in our cultures. Pericytes, a population of a smooth-muscle actin+ and CD31+ cells may contribute to the pool of nestin-GFP+ fluorescent cells during days 1–6, but they do not react to Tuj1 (see below). Approximately all nestin-GFP+ cells co-expressed Tuj1 from day 7 on, so they could not be pericytes. Nestin-GFP+ cells, expressed as a fraction of all cells, decreased during the first 4 days in culture. At days 1–4, this fraction was 19.362.5%, 16.560.5%, 9.461.4%, and 5.260.8%, respectively, which may be explained by either a relative increase in nestin- GFP- cells or decreased nestin-GFP+/MyoD+ cells, corresponding to satellite cells and/or nestin-GFP+/MyoD- cells. After day 4, the fraction of nestin-GFP+ outpaces negative cells, being 5.862.2%, 9.761.3%, 14.765.0%, and 54.9619.0% at days 5–7 and 14, respectively. Neural nestin-GFP+ cells do not derive from muscle satellite cells Investigating nestin-GFP+ cells in EDL muscle shows that most of them overlap with Pax7 positive cells, although a small fraction does not (arrows in Fig. 2A). We speculate that this fraction of nestin-GFP+/Pax7- cells are interstitial and gives rise to our neural cells in culture (see below,). In this preparation, we have not detected Tuj1+ cells (data not shown). Satellite cells are considered the main source of stem cells in postnatal skeletal muscle, located beneath the myofiber basal lamina [30], but there are other progenitor cells in interstitial skeletal muscle [31]. Nestin-GFP+ cells remain attached as satellite cells to the myofiber after fine dissection and enzymatic dissociation with collagenase type-1. Quiescent satellite cells are typically defined by nestin-GFP [25] and Pax-7 [32]; their immunostaining coincides with Hoechst- based nuclear localization (Fig. 2B). We followed satellite cells attached to single FDB fibers, using a time-lapse technique that takes advantage of their nestin-GFP fluorescence. Figure 2C illustrates one of 6 individual FDB fibers in which a satellite cell in phase-contrast (a, c, e, g, h) and fluorescence (b, d, f) was analyzed 4, 50, 80, 154, and 162 h after the experiment started. These snapshots of the complete record Skeletal Muscle Neural Precursor show that at 4 h, the satellite cell is attached to the FDB fiber (a, arrow), and GFP fluorescence can be clearly discerned (b). At 50 h, the satellite cell moves to a border of the myofiber (c), and GFP fluorescence starts to fade (d). At 80 h, the myofiber shrinks, and the rounded satellite cell becomes an elongated myogenic cell (e), while nestin-GFP fluorescence is difficult to detect (f), as reported previously [25]. At 154 (g) and 162 h (h), the parent myofiber has died and moved out of the microscope’s field, while the new muscle cell remains with both ends retracted (g) or, finally, elongated (h). We also used a pure satellite cell culture to determine whether satellite cells differentiate into neural cells (Fig. 2Da). These experiments used EDL fibers because they are longer and therefore have more satellite cells than FDB. Fibers showing GFP fluorescent satellite cells and no evidence of contamination were cultured in the medium reported above for 4 months. Satellite cells formed desmin+ myoblasts and myotubes exclusive- ly; neural-like cells were completely absent from all cultures analyzed. To further examine the presence of neural precursor cells in our purified culture, we immunostained it with Tuj1 antibody and counterstained with Hoechst. All cells in the culture were Tuj1-, and most were MyoD+ (Fig. 2Db). These data, together with time-lapse experiments, support the conclusion that myofiber satellite cells do not give rise to neural precursor cells. Nestin-GFP+ cells isolated from skeletal muscle can form neurospheres Neural progenitor cells—multipotent cells from which central nervous system neurons and glia arise—often aggregate into neurospheres, which exhibit the capacity for self-renewal [33]. Neurospheres derived from FDB muscle were characterized by morphological and immunohistochemical analysis. Nestin-GFP+/ Tuj1+ cells typically formed them around day 7 in culture (n = 5 cell cultures). Most of the differentiated neurosphere-derived cells (neuron-like cells) displayed bipolar or tripolar morphology, some with 4 or 5 processes (Fig. 3A). Neurospheres spontaneously attached to the bottom of culture dishes and gave rise to cells with neural morphology. Nestin–GFP+ cells cultured from the skeletal muscle grew as monolayers in big clusters and exhibited a distinct phenotype characterized by a small cytoplasm with aligned multipolar extensions (Fig. 3B). From day 7 on, multipolar cells expressed Tuj1 protein and nestin-GFP in fairly even amounts. Brightfield images also show the predominance of a neuronal phenotype, characterized by small bright somata and two or more pronounced dendrite or axon-like processes. Incipient cell-cell contact was detected at the ends of the dendrite-like processes (Fig. 3C); however, desmosoms or fused plasmalemmas were never found [33]. Some nestin-GFP+ cells exhibited 5 or more processes (Fig. 3D). These characteristics were not evident in other cell types in the culture. A population of skeletal muscle nestin-GFP+ cells exhibit glutamate-evoked membrane currents To investigate whether nestin-GFP+ cells obtained from unsorted FDB muscle culture express the ion channels typically found in neurons, we challenged them with glutamate in the whole-cell configuration of the patch-clamp. Figure 4 shows typical responses to flushing 500 mM L-glutamate over nestin- GFP+ cells with multipolar neural processes like those depicted in Figure 3D. The membrane potential was held at 260 mV, and the intracellular recording solution was devoid of Mg2+ (see Methods). Figure 4A shows inward currents in response to three glutamate pulses. Pulse durations are indicated above the traces. PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e16816