Efficient non-viral transfection of adult neural stem/progenitor cells, without affecting viability, proliferation or differentiation.
BACKGROUND
Neurogenesis occurs in defined areas of the adult mammalian brain, including the dentate gyrus of the hippocampus. Rat neural stem/progenitor cells isolated from this region retain their multipotency in vitro and in vivo after grafting into the adult brain. Molecular signalling and lineage selection in these cells may be examined using genetic manipulation. However, valid analysis requires that this manipulation should not affect cellular viability, proliferation or differentiation.


METHODS
We screened several transfection protocols to develop a method which met these criteria. We then tested the effects of transfection on viability, proliferation and differentiation into the three neural lineages: neurons, astrocytes and oligodendrocytes.


RESULTS
In initial testing, ExGen500 and FuGene6 efficiently transfected adult neural stem/progenitor cells, in vitro. After optimisation, these agents transfected 16% and 11% of cells, respectively. FuGene6-treated cells did not differ from untransfected cells in their viability or rate of proliferation, whereas these characteristics were significantly reduced following ExGen500 transfection. Importantly, neither agent affected the pattern of differentiation following transfection. Both agents could be used to genetically label cells, and track their differentiation into the three neural lineages, after grafting onto ex vivo organotypic hippocampal slice cultures.


CONCLUSIONS
These data demonstrate that non-viral transfection may be used to genetically manipulate neural stem/progenitor cells, without adversely affecting their growth or perturbing lineage selection. Such a method is valuable for examining the molecular mechanisms of cell fate determination in vitro. Furthermore, this protocol may be exploited in the development of cell-based gene therapy strategies.
Tinsley
R B
RB
Arvid Carlsson Institute for Neuroscience, Department of Clinical Neuroscience, Medicinaregatan 11, Box 432, Göteborg University, Göteborg 40530, Sweden. rogan.tinsley@neuro.gu.se
Faijerson
J
J
Eriksson
P S
PS
D000818:Animals; D001253:Astrocytes / Q000502:physiology; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D005240:Feasibility Studies; D008055:Lipids / Q000235:genetics / Q000494:pharmacology; D009474:Neurons / Q000502:physiology*; D009836:Oligodendroglia / Q000502:physiology; D051381:Rats; D013234:Stem Cells / Q000502:physiology*; D014162:Transfection / Q000379:methods*
C411955:FuGene; D008055:Lipids