Nestin-GFP Transgene Reveals Neural Precursor Cells in Adult Skeletal Muscle Alexander Birbrair1,2, Zhong-Min Wang 1 , Maria Laura Messi1, Grigori N. Enikolopov3, Osvaldo Delbono1,2* 1 Department of Internal Medicine-Gerontology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, United States of America, 2 Neuroscience Program, Wake Forest University School of Medicine, Winston-Salem, North Carolina, United States of America, 3 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America Abstract Background: Therapy for neural lesions or degenerative diseases relies mainly on finding transplantable active precursor cells. Identifying them in peripheral tissues accessible for biopsy, outside the central nervous system, would circumvent the serious immunological and ethical concerns impeding cell therapy. Methodology/Principal Findings: In this study, we isolated neural progenitor cells in cultured adult skeletal muscle from transgenic mice in which nestin regulatory elements control GFP expression. These cells also expressed the early neural marker Tuj1 and light and heavy neurofilament but not S100b, indicating that they express typical neural but not Schwann cell markers. GFP+/Tuj1+ cells were also negative for the endothelial and pericyte markers CD31 and a-smooth muscle actin, respectively. We established their a) functional response to glutamate in patch-clamp recordings; b) interstitial mesenchymal origin; c) replicative capacity; and d) the environment necessary for their survival after fluorescence- activated cell sorting. Conclusions/Significance: We propose that the decline in nestin-GFP expression in muscle progenitor cells and its persistence in neural precursor cells in muscle cultures provide an invaluable tool for isolating a population of predifferentiated neural cells with therapeutic potential. Citation: Birbrair A, Wang Z-M, Messi ML, Enikolopov GN, Delbono O (2011) Nestin-GFP Transgene Reveals Neural Precursor Cells in Adult Skeletal Muscle. PLoS ONE 6(2): e16816. doi:10.1371/journal.pone.0016816 Editor: Marcello Rota, Brigham & Women’s Hospital, United States of America Received September 26, 2010; Accepted January 11, 2011; Published February 3, 2011 Copyright: 2011 Birbrair et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The present study was supported by grants to Osvaldo Delbono from the National Institutes of Health/National Institute on Aging (AG13934, AG033385, and AG15820), the Muscular Dystrophy Association (MDA #33149), and the Wake Forest University Claude D. Pepper Older Americans Independence Center (P30-AG21332) and by the National Institutes of Mental Health and Ira Hazan Fund to Grigori N. Enikolopov. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: odelbono@wfubmc.edu Introduction Hopes for finding a therapy to replace lost or defective neuronal and/or glial cells, thus healing neural lesions or degenerative diseases, rest mainly on stem cells as transplantable active precursors. Primarily derived from embryonic and fetal tissues, their clinical use is limited by ethical and technical concerns related to heterologous transplants [1,2]. Autologous tissues would be a valid alternative, but neural precursors from the central or peripheral nervous system cannot be obtained without potentially harmful consequences for the donor. Interest in adult human stem cells arises from recent work demonstrating their ability to differentiate into various lineages [3,4,5] [6]. Multipotent cells, reminiscent of embryonic neural stem cells, have been isolated from several postnatal tissues, including skeletal muscle, skin, gut, heart, fat, and dental pulp, with promise for applications in research and therapy [7,8,9,10,11] [12,13]. Several markers are used to identify neuronal precursor cells in the nervous system; one is nestin [14], an intermediate protein classified as a type IV neurofilament, which, together with microfilaments and microtubules, constitutes most of the cytoskeleton. Previous work has shown that the nestin- GFP transgene is a useful marker of neurogenesis throughout life [15], and GFP fluorescence can be used to trace the migration and differentiation of newly generated neurons in the mammalian central nervous system [16]. The persistence of neural stem cells in skeletal muscle raises the possibility of their use for a variety of research purposes and therapies. To identify and isolate a transplantable population highly enriched in neural precursor cells, we used a transgenic mouse in which nestin regulatory elements control GFP expression [15]. Here, we show that nestin is co-expressed with other neuronal stem cell markers in the skeletal muscle and that its presence can facilitate their identification and isolation from adult skeletal muscle or neurosphere cultures by fluorescence-activated cell sorting (FACS). Previous studies using immunostaining, RNA extraction, western blot, and antibody-based FACS revealed that stem cells residing in adult muscle differentiate into neurons [7,11,17]. However, these procedures prevent their use for culture, cloning, or transplant. More important, functional identity, unequivocal source, replicative capacity, and survival after FACS are unknown. PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e16816