Skeletal Muscle Neural Precursor Figure 8. Unsorted nestin-GFP+ cells from FDB cultures do not exhibit endothelial cell, pericyte, or Schwann cell markers. Unsorted FDB-derived cells were seeded on precoated dishes with laminin and grown for 10 days. A. A CD31 antibody identified a population of endothelial cells that did not overlap with nestin-GFP+ cells. Similarly, an a smooth-muscle actin antibody recognized a population of pericytes. Nestin-GFP+ cells do not react with the S100b antibody for Schwann cells. B. A primary Schwann cell culture shows positive immunostaining when exposed to S100b but no immunostaining when incubated with Tuj1. Scale bar = 100 mm. doi:10.1371/journal.pone.0016816.g008 Nestin-GFP+/Tuj1+ cells exhibit additional neural markers and proliferation Nestin is expressed in both embryonic and adult CNS stem cells. In embryonic CNS, its expression is closely associated in space and time with the proliferation of neuronal progenitor cells [14]. It is also expressed in adult stem cells both in vivo and in vitro under nondifferentiating conditions [49]. To confirm that our cells are neural precursors, other markers were tested. After 7 days in culture, these cells expressed Neu-H and Neu-L, evidencing predifferentiation at later stages, but they still expressed nestin- GFP, a stem cell marker. As neurons differentiate from their precursors, they downregulate nestin expression, which is replaced by other tissue-specific intermediate filaments, such as neurofila- ments [14]. The fact that nestin-GFP fluorescence does not disappear after two weeks in culture indicates that neural cells have not completely matured. That nestin expression can be re- induced after it disappears in various degenerative and regener- ative conditions in the fully differentiated organism has been reported [50]. A population of cells in the brain cortex co- expresses bromodeoxyuridine in Tuj1+ and Neu-L cells, suggest- ing generation of neurons with proliferative capacity [51–52]. Tuj1 is initially expressed in immature neurons and persists as they mature [23]. Likelihood of success in transplanting predifferentiated neural progenitor cells Our method can efficiently isolate immature neural progenitor cells from skeletal muscle culture. Predifferentiated neural precursor cells, cultured after sorting, may have a better chance for success in proliferating and replacing damaged tissue than more immature cluster cells or neurospheres. Restricting progen- itor cell lineage may limit tumor formation, with better response to PLoS ONE | www.plosone.org 12 February 2011 | Volume 6 | Issue 2 | e16816