Laboratory Investigation Inhibition of glioma cell proliferation by neural stem cell factor Tsuyoshi Suzuki 1 , Shuichi Izumoto 1 , Kouichi Wada 1 , Yasunori Fujimoto 1 , Motohiko Maruno 1 , Mami Yamasaki 2 , Yonehiro Kanemura 2 , Takuya Shimazaki 3 , Hideyuki Okano 3 and Toshiki Yoshimine 1 1 Department of Neurosurgery, Osaka University Medical School, Suita, Osaka, Japan; 2 Departments of Neurosur- gery, Osaka National Hospital, Osaka, Japan; 3 Department of Physiology, School of Medicine, Keio University, Tokyo, Japan Key words: cell proliferation, glioma, glioma cell, neural progenitor cell, neural stem cell Summary Neural stem cells (NSC) have unique differentiation-, proliferation-, and motility properties. To investigate whether they secrete factors that interfere with the proliferation of glioma cells, we grew glioma cells in conditioned medium (CM) obtained from cultures of neurospheres including neural stem / progenitor cells (NSPC) isolated from embryonic (E14)- or adult mouse brain or fetal human brain. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and BrdU-labeling assays showed that CM from NSPC (NSPC/CM) contained factor(s) that inhibited the proliferation of glioma cells by 28–87%. Filter-fractionation of NSPC/CM revealed that the 50,000–100,000 nominal molecular weight limit (NMWL) fraction contained the inhibitory activity. On the basis of these observations we transplanted 203G glioma cells and/or NSPC into the intrathecal space of the cisterna magna of mice to investigate whether NSPC interfere with the proliferation of glioma cells in vivo. Mice transplanted with both 203G and NSPC survived significantly longer than did mice transplanted only with 203G. We concluded that NSPC secrete factor(s) that may control glioma cell proliferation. Introduction Stem cells are undifferentiated cells capable of prolifer- ation, self-maintenance, the production of a large number of differentiated functional progeny, and the regeneration of injured tissue [1]. In the brain, neural stem cells (NSC) exhibit unique differentiation-, prolif- eration-, and motility characteristics. Neural progenitor cells (NPC) engineered as therapeutic vehicles expressing cytokine genes work effectively in the glioma-bearing murine brain [2,3]. Aboody et al. used migratory NSC to deliver therapeutic genes and vectors to refractory, migratory, and invasive murine brain tumors [2]. Because the treatment-resistance of gliomas is not related to the proliferation of glioma cells, but rather to their ability to migrate and insinuate into normal neural tissue, new strategies for the treatment of malignant glioma are needed. In the course of neuronal development, various cytokines are expressed at each stage of neuronal differentiation. Soluble peptide factors influence the survival, growth, and phenotypic differentiation of cells. The list of factors known to affect the developing nervous system has expanded well beyond the neuro- trophins and now includes the interleukin-6 family of cytokines, insulin-like growth factors (IGF), platelet- derived growth factor (PDGF), transforming growth factor beta (TGF-b), glial-derived growth factor, and heparin-binding factors [3]. In the glioma-bearing brain, NSC may secrete factors that interfere not only with other stem cells but also with glioma cells [4]. NSC may also stimulate surrounding normal brain tissue to produce various cytokines, and they may produce factors that could be used to control the proliferation of glioma cells. Many factors regulate the activities of NSC. For example, the protein kinase C (PKC) substrate GAP-43 is expressed in neural precur- sor cells [5], and sonic hedgehog regulates the differen- tiation of central nervous system (CNS) precursor cells [6]. Bone morphogenic protein (BMP) negatively regulates the signals that promote development of epidermal growth factor (EGF)-responsive stem cells [7,8]. Stem cell factor is trophic for certain neurons derived from the neural crest and cerebral cortex and regulates the activities of astroglia, oligodendroglia, and microglia [9]. These factors are secreted by NSC and are necessary for their maintenance. We investigated whether NSC interfere with glioma cell proliferation, whether humoral factors secreted by NSC interfere with glioma cells, and whether physical interactions between NSC and glioma cells affect the characteristics of glioma. We also studied whether NSC affect glioma cells in vivo by transplanting NSC and glioma cells intrathecally via the cisterna magna of mice. Materials and methods NCS culture from brain NSC were cultured as described previously [10]. Briefly, the brain striatum was dissected from adult mice, day 14 Journal of Neuro-Oncology (2005) 74: 233–239 Ó Springer 2005 DOI 10.1007/s11060-004-7118-5