High-yield selection and extraction of two promoter-defined phenotypes of neural stem cells from the fetal human brain.
Neural stem and precursor cells reside in the ventricular lining of the fetal forebrain, and may provide a cellular substrate for brain repair. To selectively identify and extract these cells, we infected dissociated fetal human brain cells with adenoviruses bearing the gene for green fluorescence protein (GFP), placed under the control of enhancer/promoters for two genes (nestin and musashi1) that are expressed in uncommitted neuroepithelial cells. The cells were then sorted by fluorescence-activated cell sorting (FACS) on the basis of E/nestin- or P/musashi1-driven GFP expression. Both P/musashi1:hGFP- and E/nestin:EGFP-sorted cells were multipotent: limiting dilution with clonal expansion as neurospheres, in tandem with retroviral lineage analysis and xenograft to E17 and P0-2 rat forebrain, revealed that each phenotype was able to both self-renew and co-generate neurons and glia. Thus, fluorescent genes placed under the control of early neural promoters allow neural stem cells to be specifically targeted, isolated, and substantially enriched from the fetal human brain.
Keyoung
H M
HM
Department of Neurology and Neuroscience, Cornell University Medical College and New York Presbyterian Hospital, New York, NY 10021, USA.
Roy
N S
NS
Benraiss
A
A
Louissaint
A
A
Suzuki
A
A
Hashimoto
M
M
Rashbaum
W K
WK
Okano
H
H
Goldman
S A
SA
D000256:Adenoviridae / Q000235:genetics; D001921:Brain / Q000196:embryology*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000235:genetics; D064231:Nestin; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D013234:Stem Cells / Q000502:physiology*; D016378:Tissue Transplantation
D007381:Intermediate Filament Proteins; D008164:Luminescent Proteins; C491297:MSI1 protein, human; C491296:Msi1 protein, rat; C577484:NES protein, human; D009419:Nerve Tissue Proteins; C577486:Nes protein, rat; D064231:Nestin; D016601:RNA-Binding Proteins; D049452:Green Fluorescent Proteins