the tissue environment and capacity to integrate with the host tissue [53]. Several studies have indicated a higher rate of neurogenesis when a neuronal phenotype was already induced before cells were implanted [54]. Neural progenitor cells transplanted into the injured rat spinal cord favored differentiation into astrocytes but not neurons [55]. Late-stage precursors and immature neurons have been transplanted into the adult neocortex and induced pyramidal neurons to degenerate; the engrafted cells were found to follow pyramidal cell differentiation, as they were already committed to neuronal lineage [56]. Taken together, these results indicate the need for differentiation protocols before grafting. Our results imply that, to certain extent, skeletal muscle-derived neural stem cells expressing nestin are highly proliferating and can be used for remodeling and repair processes [57,58]. Because they can be isolated based on their nestin-GFP fluorescence, prolifer- ation capacity, and differentiation into neurons, they can be used as a source of neural progenitor cells for therapeutic purposes in animal models of neural injury or degeneration. Nestin-GFP- cells promote appearance of the neural phenotype Neural phenotype differentiation depends on some factors secreted by co-cultured cells, such as myoblasts and fibroblasts. We observed that not only nestin-GFP- cells, but also their medium, induce a neural phenotype, indicating that factors important for neuronal progenitor differentiation may be soluble, diffusible, and secreted by neighboring cells. These results are comparable to those published for co-cultures of neuronal progenitors and conditioned medium from bone marrow stromal cells (BMSCs) [59]. Fibroblasts, myoblasts, or any other cell type derived from muscle interstitium may be the source of the molecules that influence muscle interstitial cells to evolve into a neural lineage. The main group of molecules regulating cell differentiation and proliferation are growth factors. Members of the transforming growth factor-b (TGFb) family may influence premature neuronal differentiation [60]. Fibroblast growth factors (FGFs) are critical References 1. Addis RC, Bulte JW, Gearhart JD (2008) Special cells, special considerations: the challenges of bringing embryonic stem cells from the laboratory to the clinic. Clin Pharmacol Ther 83: 386–389. 2. Li JY, Christophersen NS, Hall V, Soulet D, Brundin P (2008) Critical issues of clinical human embryonic stem cell therapy for brain repair. Trends Neurosci 31: 146–153. 3. Salgado AJ, Oliveira JT, Pedro AJ, Reis RL (2006) Adult stem cells in bone and cartilage tissue engineering. Curr Stem Cell Res Ther 1: 345–364. 4. Zipori D (2006) The stem state: mesenchymal plasticity as a paradigm. Curr Stem Cell Res Ther 1: 95–102. 5. Aguiari P, Leo S, Zavan B, Vindigni V, Rimessi A, et al. (2008) High glucose induces adipogenic differentiation of muscle-derived stem cells. Proc Natl Acad Sci U S A 105: 1226–1231. 6. Zavan B, Giorgi C, Bagnara GP, Vindigni V, Abatangelo G, et al. (2007) Osteogenic and chondrogenic differentiation: comparison of human and rat bone marrow mesenchymal stem cells cultured into polymeric scaffolds. Eur J Histochem 51(Suppl 1): 1–8. 7. Alessandri G, Pagano S, Bez A, Benetti A, Pozzi S, et al. (2004) Isolation and culture of human muscle-derived stem cells able to differentiate into myogenic and neurogenic cell lineages. Lancet 364: 1872–1883. 8. Fernandes KJ, McKenzie IA, Mill P, Smith KM, Akhavan M, et al. (2004) A dermal niche for multipotent adult skin-derived precursor cells. Nat Cell Biol 6: 1082–1093. 9. Fernandes KJ, Kobayashi NR, Gallagher CJ, Barnabe-Heider F, Aumont A, et al. (2006) Analysis of the neurogenic potential of multipotent skin-derived precursors. Exp Neurol 201: 32–48. 10. Gingras M, Champigny MF, Berthod F (2007) Differentiation of human adult skin-derived neuronal precursors into mature neurons. J Cell Physiol 210: 498–506. 11. Arsic N, Mamaeva D, Lamb NJ, Fernandez A (2008) Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages. Exp Cell Res 314: 1266–1280. Skeletal Muscle Neural Precursor for midbrain/hindbrain [61] and forebrain [62] cell survival. A number of vasculature-related growth factors have been shown to regulate neural stem cell and progenitor proliferation, most notably PEDF [63] and vascular endothelial growth factor [64]. Factors secreted by blood vessels, such as the brain-derived neurogenic factor (BDNF), are known to influence neuronal stem cell behavior [65–66]. BDNF can also be secreted by myofibers, which play an active role as an endocrine organ [67]. Many of these factors may originate from any of the nestin-GFP- cells in our skeletal muscle-derived cultures. Additionally, strong differen- tiation was only observed after cells adhered to laminin-coated dishes, indicating that they require a matrix anchorage. Although necessary, laminin was not sufficient to induce a neural phenotype, as nestin-GFP+ did not give rise to neural progenitor cells when cultured in isolation. In summary, prolonged nestin-GFP+ expression in skeletal muscle-derived cultures provides a unique tool to obtain predifferentiated neural progenitor cells. Whether these cells differentiate into multiple phenotypes or an exclusive neuron subclass is unknown. Whether they are suitable for transplanta- tion, retain the capacity to engraft, develop specific neuronal functions, and allow tissue repair are subjects of ongoing research in our lab. The fact that an individual can be the donor and host of these cells circumvents the immunological and ethical concerns impeding cell therapy. Acknowledgments The authors would like to thank Jackson Taylor and Dr. Tan Zhang for their useful comments, the Comprehensive Cancer Center Cell Sorting Facility, and Micromed of WFUSM. Author Contributions Conceived and designed the experiments: AB OD. Performed the experiments: AB Z-MW MLM. Analyzed the data: AB GNE MLM OD. Contributed reagents/materials/analysis tools: GNE OD. Wrote the paper: AB GNE OD. 12. Silva AT, Wardhaugh T, Dolatshad NF, Jones S, Saffrey MJ (2008) Neural progenitors from isolated postnatal rat myenteric ganglia: expansion as neurospheres and differentiation in vitro. Brain Res 1218: 47–53. 13. Le Douarin NM, Calloni GW, Dupin E (2008) The stem cells of the neural crest. Cell Cycle 7: 1013–1019. 14. Lendahl U, Zimmerman LB, McKay RDG (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60: 585–595. 15. Mignone JL, Kukekov V, Chiang AS, Steindler D, Enikolopov G (2004) Neural stem and progenitor cells in nestin-GFP transgenic mice. J Comp Neurol 469: 311–324. 16. Yamaguchi M, Saito H, Suzuki M, Mori K (2000) Visualization of neurogenesis in the central nervous system using nestin promoter-GFP transgenic mice. Neuroreport 11: 1991–1996. 17. Schultz SS, Lucas PA (2006) Human stem cells isolated from adult skeletal muscle differentiate into neural phenotypes. J Neurosci Methods 152: 144– 155. 18. Zammit PS, Golding JP, Nagata Y, Hudon V, Partridge TA, et al. (2004) Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Biol 166: 347–357. 19. Carlson BM, Dedkov EI, Borisov AB, Faulkner JA (2001) Skeletal muscle regeneration in very old rats. J Gerontol A Biol Sci Med Sci 56: B224–233. 20. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch- clamp techniques for high-resolution current recording from cells and cell-free patches. Pflugers Arch 391: 85–100. 21. Nowak L, Bregestovski P, Ascher P, Herbet A, Prochiantz A (1984) Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307: 462–465. 22. Joshi HC, Cleveland DW (1989) Differential utilization of beta-tubulin isotypes in differentiating neurites. J Cell Biol 109: 663–673. 23. Lee MK, Rebhun LI, Frankfurter A (1990) Posttranslational modification of class III beta-tubulin. Proc Natl Acad Sci U S A 87: 7195–7199. PLoS ONE | www.plosone.org 14 February 2011 | Volume 6 | Issue 2 | e16816