Effective Adenovirus-Mediated Gene Transfer Into Neural Stem Cells Derived From Human Embryonic Stem Cells

Abstract

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.

Gene Expression Analyses of the Spatio-Temporal Relationships of Human Medulloblastoma Subgroups during Early Human Neurogenesis

“…Neurospheres consisting of ∼30% CD133+ NSCs and ∼70% CD133- neural progenitor cells (NPCs) [23] , [26] , were dissociated using a trypsin-based neural tissue dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were centrifuged (400×g) and resuspended in FACS buffer (PBS supplemented with 0.5% bovine serum albumin (BSA) (Sigma) and 2 mM ethylene diamine triacetic acid (EDTA) (Sigma).…”

Identification of suitable endogenous control genes for microRNA expression profiling of childhood medulloblastoma and human neural stem cells

“…Dissociation of neurospheres and isolation of CD133+ NSCs by flow cytometry was performed as described previously [64]. Enrichment of CD133- NPCs was 100% for both hES3 and MEL1 ESC lines, with the enrichment of CD133+ NSCs being 81.1% and 97% for the hES3 and MEL1 ESC lines, respectively.…”

Integrated Analysis of miRNA and mRNA Expression in Childhood Medulloblastoma Compared with Neural Stem Cells

“…Neurospheres were dissociated using the trypsin-neural dissociation kit (Miltenyi Biotec, North Ryde, Australia) as described previously [41]. Dissociated NSC suspensions were washed and re-suspended in FACS buffer (1xPBS supplemented with 0.5% BSA (Sigma, Castle Hill, Australia) and 2 mM EDTA (Sigma, Castle Hill, Australia).…”

“…Dissociated NSC suspensions were washed and re-suspended in FACS buffer (1xPBS supplemented with 0.5% BSA (Sigma, Castle Hill, Australia) and 2 mM EDTA (Sigma, Castle Hill, Australia). Cells were blocked in Fc block (Miltenyi Biotec, North Ryde, Australia) for three min at room temperature and CD133+ NSCs were isolated as described [41]. FACs enrichment of CD133− NPCs was 100% for both hES3 and MEL1 ESC lines, with the enrichment of CD133+ NSCs 81.1% and 97% for the hES3 and MEL1 ESC lines, respectively.…”