Skeletal Muscle Neural Precursor Figure 6. Nestin-GFP+ cell sorting. Representative scatter plots for 7-day FDB cultures, before (A) and after (B) FACS. GFP fluorescence was plotted against the number of cells. We selected cells with very high GFP fluorescence (P2). A. Sorting yielded approximately 5% of total GFP events and a large population of GFP- cells. B. Re-analysis of GFP+ cells after sorting. C. Percent of nestin-GFP+ cells before and after FACS. Data are mean 6 SEM. doi:10.1371/journal.pone.0016816.g006 their clinical application. A heterogeneous population of endothelial, vascular smooth, skeletal, and cardiac muscle cell progenitors was isolated, but after in vitro differentiation, neurally committed cells did not exceed 10% of the total [7]. Probably due to its multipotentiality, the skeletal muscle-derived culture was therapeutically ineffective, as demonstrated by lack of neural regeneration after cell engraftment in spinal cord lesions [7]. In contrast, our approach isolates a pure neural progenitor cell population, expressing nestin-GFP and Tuj1 from skeletal muscle cultures and shows a promising capacity to induce neural regeneration. Cells other than neural progenitors may express nestin-GFP in our skeletal muscle-derived culture, but we demonstrated that all nestin-GFP+ cells are neural progenitors from day 7 on. Pericytes are unlikely to contaminate our cultures, based on the absence of their typical phenotype, which is flat and fibroblast-like [43], and lack of overlap with nestin-GFP+ cells after one week in culture. Neurosphere formation, immunoreactivity to neural markers, and survival in culture This work shows that nestin-GFP+ neural progenitor cells can expand into neurospheres. Our primary muscle-derived cell cultures showed the floating nestin-GFP+ spherical aggregates characteristic of neurospheres [44,45]. Neurospheres attached and detached from the culture substrate, but in contrast to previous publications [33], their composition and the cells’ morphology were more homogenous, supporting the similarity of their stem- like properties. These cells co-express nestin, which is a marker of neural progenitor cells [46], and Tuj1, commonly used as an axonal marker in young neurons [24,46]. After 8 days, the neurospheres retain nestin and Tuj1 expression and continuously produce outgrowing cells. One of the defining features of neural progenitors and stem cells in general is their ability to sustain multiple rounds of amplification. Nestin-GFP+/Tuj1+ cells derived from skeletal muscle cultures PLoS ONE | www.plosone.org 10 February 2011 | Volume 6 | Issue 2 | e16816