Membrane properties of rat embryonic multipotent neural stem cells Jingli Cai, Aiwu Cheng, Yongquan Luo, Chengbiao Lu, Mark P. Mattson, Mahendra S. Rao and Katsutoshi Furukawa Laboratory of Neurosciences, Gerontology Research Center, National Institute on Aging, Baltimore, Maryland, USA Abstract We have characterized several potential stem cell markers and defined the membrane properties of rat fetal (E10.5) neural stem cells (NSC) by immunocytochemistry, electro- physiology and microarray analysis. Immunocytochemical analysis demonstrates specificity of expression of Sox1, ABCG2/Bcrp1, and shows that nucleostemin labels both progenitor and stem cell populations. NSCs, like hematopoi- etic stem cells, express high levels of aldehyde dehydroge- nase (ALDH) as assessed by Aldefluor labeling. Microarray analysis of 96 transporters and channels showed that Glucose transporter 1 (Glut1/Slc2a1) expression is unique to fetal NSCs or other differentiated cells. Electrophysiological examination showed that fetal NSCs respond to acetylcholine and its agonists, such as nicotine and muscarine. NSCs express low levels of tetrodotoxin (TTX) sensitive and insen- sitive sodium channels and calcium channels while expres- sing at least three kinds of potassium channels. We find that gap junction communication is mediated by connexin (Cx)43 and Cx45, and is essential for NSC survival and proliferation. Overall, our results show that fetal NSCs exhibit a unique signature that can be used to determine their location and assess their ability to respond to their environment. Keywords: calcium, gap junctions, glucose transporter, ion channel, precursor, progenitor. J. Neurochem. (2004) 88, 212–226. Stem cells are multipotent cells capable of self-renewal by asymmetric division to generate daughter cells, progenitors and differentiated cell populations. Stem cells have been isolated from almost all tissues and are classified based on the tissue from which they have been purified, and on the types of cells into which they can differentiate. An emerging idea is the concept of a stem cell state, as defined by a characteristic stem cell signature profile, which is modulated by the stem cell niche, the environment in which the stem cell resides (Lin 2002). The competence of a cell to differentiate is regulated by extracellular signals. A stem cell interacts with its environment through cell surface receptors that activate signaling pathways. Multiple signaling pathways active in stem cell populations have been recog- nized and include those activated by cytokines (reviewed in Ip 1998), neurotransmitters (Ma et al. 2000), gap junction communication (Rozental et al. 1998), chemokines (Reiss et al. 2002) and extracellular matrix (ECM) molecules (Lin and Bertics 1995; Sommer and Rao 2002; Lobo et al. 2003). What has become clear from multiple studies is that environmental context regulates the stem cell response to a particular factor. Thus, the effect of a particular factor does not depend only on the expression of the appropriate signaling pathway, but also on the overall state of the cell that integrates information from a variety of competing signaling pathways (reviewed in Sommer and Rao 2002). Neurosphere cultures, while valuable for selecting and enriching for stem cells, are not a pure population of stem cells and several different strategies for cell selection have been described. Using a variety of stem cell markers, we have been able to show that at early developmental stages the neural tube consists of a relatively homogenous population of stem cells that express nestin, Sox2, ABCG2, telomerase, FGFR4, exhibit high telomerase activity, do not express Received June 23, 2003; revised manuscript received September 24, 2003; accepted September 25, 2003. Address correspondence and reprint requests to Katsutoshi Furukawa, LNS, GRC, NIA/NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224, USA. E-mail: furukawaka@grc.nia.nih.gov Abbreviations used: ACh, acetylcholine; ALDH, aldehyde dehy- drogenase; bFGF, basic fibroblast growth factor; CEE, chicken embryo extract; Cx, connexin; ECM, extracellular matrix; GA, 18a-glycyrrhe- tinic acid; GRP, glial-restricted precursor; HSC, hematopoetic stem cells; MMLV, moloney murine leukemia virus; NRP, neuron-specific precur- sor; NSC, neural stem cells; PCR, polymerase chain reaction; TTX, tetrodotoxin. Journal of Neurochemistry , 2004, 88, 212–226 doi:10.1046/j.1471-4159.2003.02184.x 212 Ó 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 212–226