Abstract
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.
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Acknowledgements
Supported by Project ALS, the Human Frontiers Scientific Program, the National Multiple Sclerosis Society, and the Mathers Charitable Foundation. We thank Drs. Theo Palmer and Fred Gage for pNIT-EGFP plasmid and retrovirus, Dr. James Goldman for advice on its use, Drs. Rebecca Baergen and Brad Poulis for assistance in identifying appropriate samples, and Drs. Melissa Carpenter, Kazunobu Sawamoto and Katsuhiko Mikoshiba for valuable discussions, Testu Yoshida for assistance in the preparation of E/nestin:EGFP adenovirus, and Drs. Neil Hackett and Erik Falck-Pedersen for pJM17 and pAdCMV-HSgD, respectively.
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Supplementary Figure 1.
AdE/nestin:EGFP and AdP/musashi1:hGFP vectors. (A) In the plasmid separation cassette E/nestin:EGFP, EGFP was placed 3' to the heat shock protein-68 basal promoter, and this was placed under the regulatory control of the nestin second intronic enhancer. (B) Adenoviral E/nestin:EGFP was constructed to include E/nestin:hsp68:EGFP in a DE1 DE3 adenovirus. The detailed construction of this virus will be reported elsewhere (A. Suzuki and H. Okano, unpublished data). (C) In the plasmid separation cassette P/musashi1:hGFP, hGFP was placed 3' under the regulatory control of the mouse musashi1 promoter. (D) Adenoviral AdP/musashi1:hGFP was constructed to include P/musashi1:hGFP in a DE1 adenovirus. (GIF 37 kb)
Supplementary Figure 2.
Retroviral tagging of FACS-purified human neural stem cells. This schematic outlines the strategy by which we retrovirally tagged and clonally propagated FACS-purified neural progenitor cells. (GIF 44 kb)
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Keyoung, H., Roy, N., Benraiss, A. et al. High-yield selection and extraction of two promoter-defined phenotypes of neural stem cells from the fetal human brain. Nat Biotechnol 19, 843–850 (2001). https://doi.org/10.1038/nbt0901-843
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DOI: https://doi.org/10.1038/nbt0901-843
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