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.
Bertram
Cornelia M
CM
Brain Tumour Research Program, Telethon Institute for Child Health Research and Centre for Child Health Research, University of Western Australia, Perth, Australia.
Hawes
Susan M
SM
Egli
Simone
S
Peh
Swee Lim
SL
Dottori
Mirella
M
Kees
Ursula R
UR
Dallas
Peter B
PB
D000071916:AC133 Antigen; D000256:Adenoviridae / Q000235:genetics*; D015703:Antigens, CD / Q000378:metabolism; D015415:Biomarkers; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*
D000071916:AC133 Antigen; D015703:Antigens, CD; D015415:Biomarkers; D006023:Glycoproteins; D007381:Intermediate Filament Proteins; C577484:NES protein, human; D009419:Nerve Tissue Proteins; D064231:Nestin; C000606023:PROM1 protein, human; D010455:Peptides