Skeletal Muscle Neural Precursor Figure 5. Proliferation of neural stem cells derived from FDB skeletal muscle. A. Unsorted nestin-GFP+ and Tuj1+ cells from FDB muscle were exposed to EdU on culture day 7. Nuclear DNA was stained with Hoechst 33342. Scale bar = 100 mm. B. Percent of nestin-GFP+/Tuj1+ cells exhibiting EdU incorporation at days 7, 10, and 14 in culture. The fourth column corresponds to nestin-GFP+ cells sorted on culture day 7, cultured for 48 hours, and fixed (n = 3 preparations). EdU (10 mM) was administered 2 hours before cells were fixed. Data are mean 6 SEM. doi:10.1371/journal.pone.0016816.g005 publication, nestin-GFP+ cells were sorted and cultured at an earlier time. Their lack of neurogenic capacity indicates that neural progenitor cells need the influence of nestin-GFP- cells to develop a neural phenotype. Nestin-GFP+/Tuj1+ neural progenitors originate from interstitial but not myofiber satellite cells Nestin-GFP+ cells are located beneath the basal lamina in a myofiber satellite position and the interstitium of the skeletal muscle. In our culture conditions, nestin-GFP+ cells attached to myofibers are quiescent satellite cells, based on Pax7+ immuno- reaction, and exhibit exclusive myogenic potential. Analysis of skeletal muscle cross-sections from the nestin-GFP transgenic mouse shows population of GFP+/Pax7- cells, which suggests that cells other than quiescent satellite cells express GFP under nestin intron II control in the native muscle. These cells may correspond to activated satellite, neural progenitor, or some other cells. We ruled out the possibility that satellite cells produce neural cells because myogenic cells lose nestin-GFP expression by days 4–5 in culture, as reported previously [25], and satellite cells released enzymatically from myofibers differentiate into a myogenic but not neurogenic lineage, based on prolonged culture (4 month), morphology, staining/immunocytochemical analysis, and time- lapse experiments. This prolonged culture stains positive for desmin and MyoD but not for Tuj1, which indicates that satellite cells do not give rise to neural precursor cells. Recently, using the same transgenic mouse used here [40], nestin was shown to identify sympathetic nervous system-innervated perivascular stromal cells, which supports the concept that muscle nestin- GFP+/Tuj1+ neural cells derive from interstitial mesenchymal stem cells. Advantages of our experimental approach, biological significance, and potential therapeutic contribution Neural progenitor cells derived from adult skeletal muscle have been reported previously [7,11,41,42]. These studies provided the groundwork for analysis of neurogenic cells in peripheral tissue; however, some limitations must be overcome to move ahead with PLoS ONE | www.plosone.org 9 February 2011 | Volume 6 | Issue 2 | e16816