Effective adenovirus-mediated gene transfer into neural stem cells derived from human embryonic stem cells.

Human embryonic stem cell-derived neural stem cells (hESC-NSCs) are an attractive cell type for studying aspects of brain development and pathology. To develop the full potential of this model system, it is important to establish a reliable methodology for the manipulation of gene expression in hNSCs. To address this issue, we used an adenoviral vector with a CMV promoter-driven green fluorescent protein (GFP) reporter gene (Ad5-GFP). We optimized conditions for Ad5-GFP infection and assessed the efficiency of infection of whole and dissociated embryonic stem cell (ESC)-derived neurospheres as well as the effect of adenoviral vectors on cell surface marker expression, proliferation, and differentiation potential. Our results demonstrate that most neurosphere cells ( approximately 70%) express the coxsackie and adenovirus receptor and can be infected with Ad5. More specifically, the CD133+ hESC-NSC population could be infected more efficiently than the CD133 population and both populations expressed GFP at high levels. At low multiplicity of infection (MOI < 25), the virus had no significant effect on stem cell marker expression (CD133 and Nestin), cell survival, cell proliferation rate, or differentiation potential. This model system provides a practical new approach to study human NSC function in the context of neurodegenerative and neoplastic disorders.

Classic and desmoplastic medulloblastoma: complete case reports and characterizations of two new cell lines.

Medulloblastoma (MB) is the most common type of brain tumor affecting children. These tumors are a significant cause of childhood mortality and morbidity, and more effective and less invasive treatment options are urgently required. To achieve these aims, it will be critical to develop a more comprehensive understanding of the molecular pathogenesis of MB. At present, there are relatively few well-characterized MB cell lines available to the research community for the study of MB molecular and cellular biology. Here we present the case reports of two children diagnosed with classic and desmoplastic MB, and describe the characteristics of two new MB cell lines derived from these individuals. A number of genes encoding components of the sonic hedgehog (SHH) and WNT pathways were up-regulated in the desmoplastic relative to the classic MB cell line consistent with aberrant activation of these pathways in desmoplastic MB. These cell lines represent an additional resource for the analysis of diverse aspects of MB biology.

Aberrant over-expression of a forkhead family member, FOXO1A, in a brain tumor cell line.

BACKGROUND: The mammalian FOXO (forkhead box, O subclass) proteins are a family of pleiotropic transcription factors involved in the regulation of a broad range of cellular processes critical for survival. Despite the essential and diverse roles of the FOXO family members in human cells and their involvement in tumor pathogenesis, the regulation of FOXO expression remains poorly understood. We have addressed the mechanisms underlying the high level of expression of the FOXO1A gene in a cell line, PER-453, derived from a primitive neuroectodermal tumor of the central nervous system (CNS-PNET). METHODS: The status of the FOXO1A locus in the PER-453 CNS-PNET cell line was investigated by Southern blotting and DNA sequence analysis of the proximal promoter, 5'-UTR, open reading frame and 3'-UTR. FOXO1A expression was assessed by conventional and quantitative RT-PCR, Northern and Western blotting. RESULTS: Quantitative real-time RT-PCR (qRT-PCR) data indicated that after normalization to ACTB mRNA levels, canonical FOXO1A mRNA expression in the PER-453 cell line was 124-fold higher than the average level of five other CNS-PNET cell lines tested, 24-fold higher than the level in whole fetal brain, and 3.5-fold higher than the level in fetal brain germinal matrix cells. No mutations within the FOXO1A open reading frame or gross rearrangements of the FOXO1A locus were detected. However, a single nucleotide change within the proximal promoter and several nucleotide changes within the 3'-UTR were identified. In addition, two novel FOXO1A transcripts were isolated that differ from the canonical transcript by alternative splicing within the 3'-UTR. CONCLUSION: The CNS-PNET cell line, PER-453, expresses FOXO1A at very high levels relative to most normal and cancer cells from a broad range of tissues. The FOXO1A open reading frame is wild type in the PER-453 cell line and the abnormally high FOXO1A mRNA expression is not due to mutations affecting the 5'-UTR or proximal promoter. Over expression of FOXO1A may be the result of PER-453 specific epimutations or imbalances in regulatory factors acting at the promoter and/or 3'-UTR.