Human Cortical Glial Tumors Contain Neural Stem-Like Cells Expressing Astroglial and Neuronal Markers In Vitro TATYANA N. IGNATOVA, 1 VALERY G. KUKEKOV, 1 ERIC D. LAYWELL, 1 OLEG N. SUSLOV, 1 FRANK D. VRIONIS, 2 AND DENNIS A. STEINDLER 1 * 1 Departments of Neuroscience and Neurosurgery, McKnight Brain Institute and Shands Cancer Center, University of Florida, Gainesville, Florida 2 NeuroOncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida KEY WORDS neural stem cell; glial tumors; neural cell diversity; serum and anchorage withdrawal; pleiotropic growth factors; nestin; Survivin; Delta; Jagged ABSTRACT Neural stem cells from neurogenic regions of mammalian CNS are clono- genic in an in vitro culture system exploiting serum and anchorage withdrawal in medium supplemented with methyl cellulose and the pleiotropic growth factors EGF, FGF2, and insulin. The aim of this study was to test whether cortical glial tumors contain stem-like cells capable, under this culture system, of forming clones showing intraclonal heterogeneity in the expression of neural lineage-specific proteins. The high frequencies of clone-forming cells (about 0.1–10  10 -3 ) in clinical tumor specimens with mutated p53, and in neurogenic regions of normal human CNS, suggest that the ability to form clones in this culture system is induced epigenetically. RT-PCR analyses of populations of normal brain- and tumor- derived sister clones revealed transcripts for nestin, neuron-specific enolase, and glial fibrillary acidic protein (GFAP). However, the tumor-derived clones were different from clones derived from neurogenic regions of normal brain in the expression of transcripts specific for genes associated with neural cell fate determination via the Notch-signaling pathway (Delta and Jagged), and cell survival at G2 or mitotic phases (Survivin). Moreover, the individual glioma-derived clones contain cells immunopositive separately for GFAP or neuronal -III tubulin, as well as single cells coexpressing both glial and neuronal markers. The data suggest that the latent critical stem cell characteristics can be epigenetically induced by growth conditions not only in cells from neurogenic regions of normal CNS but also in cells from cortical glial tumors. Moreover, tumor stem-like cells with genetically defective responses to epigenetic stimuli may contribute to gliomagenesis and the develop- mental pathological heterogeneity of glial tumors. GLIA 39:193–206, 2002. © 2002 Wiley-Liss, Inc. INTRODUCTION Within the neurogenic subependymal zone (SEZ) and hippocampus throughout life, multipotent glial cells have been found to play a role in persistent neurogen- esis (Doetsch et al., 1999; Seri et al., 2001). We have previously demonstrated that neural stem cells (NSCs) from these neurogenic regions are clonogenic in an in vitro culture system exploiting serum and anchorage withdrawal in medium supplemented with methyl cel- lylose (MC) and the pleiotropic growth factors epider- mal growth factor (EGF), fibroblast growth factor 2 (FGF2), and insulin (Kukekov et al., 1997, 1999). We have shown also that astrocytes from different brain areas during a critical period in development express specific attributes of NSCs (Laywell et al., 2000). Pro- liferation and differentiation potential of progenitor T.N. Ignatova and V.G. Kukekov contributed equally to this study. Grant sponsor: NIH/NINDS; Grant number: NS37556; Grant sponsor: the McKnight Brain Institute; Grant sponsor: Department of Neurosurgery and Shands Cancer Center. *Correspondence to: Dr. Dennis A. Steindler, McKnight Brain Institute, Shands Cancer, and Program in Stem Cell Biology, University of Florida, 100 S. Newell Drive, P.O. Box 100244, Gainesville, Fl 32610. E-mail: steindler@mbi.ufl.edu Received 18 December 2001; Accepted 8 April 2002 DOI 10.1002/glia.10094 Published online 11 June 2002 in Wiley InterScience (www.interscience.wiley. com). GLIA 39:193–206 (2002) © 2002 Wiley-Liss, Inc.