PMC

E/sox2:EGFP⁺ NSCs give rise to neurons and glia upon differentiation in vitro. A, E/sox2:EGFP⁺ cells were differentiated in DMEM/F12/1% FBS at passage 1. The resultant progeny included both neurons (βIII-tubulin, red) and astrocytes (GFAP, green). B–D, At passage 3, E/sox2:EGFP⁺ cells still readily differentiated into neurons and astrocytes (B, C), as well as O4-defined oligodendrocytes (D). Scale bars: A, C, D, 50 μm; B, 100 μm.

AdE/Sox2:EGFP⁺ cells coexpress other neural progenitor and stem cell antigens. A, B, When immunostained immediately (2 h) after AdE/sox2:EGFP-based FACS, EGFP⁺ cells expressed sox2 protein (A), as well as nestin (B). C, By 3 d after FACS, E/sox2:EGFP⁺ cells almost uniformly expressed sox2, musashi1, and nestin, while approximately one-third of the population expressed GFAP. D–K, Corresponding immunofluorescent (top row) and phase (bottom row) images of E/sox2:EGFP⁺ cells immunostained for sox2 (D, E), nestin (F, G), musashi-1 (H, I), and GFAP (J, K), at 3 d after FACS. Scale bar: D–K, 50 μm.

JAK-STAT and Notch pathway expression profiles in E/sox2:EGFP⁺ cells. A, Like the MAPK and wnt pathways, the JAK-STAT pathway was identified as differentially expressed in E/sox2:EGFP⁺ cells. This heat map shows only those members of the JAK-STAT pathway that were significantly differentially expressed in E/sox2:EGFP⁺ vs E/sox2:EGFP⁻ cells (>3-fold change, 5% FDR). B, Manual analysis of differentially expressed genes identified a large number of Notch-related transcripts. See for further details. As in , the color key indicates relative expression on a log₂ scale, with red and green indicating high- and low-level gene expression, respectively.

Sox2⁺ NPCs can be isolated and enriched using AdE/sox2:EGFP-based FACS. A–D, Human fetal NPCs were sorted after their infection by AdE/sox2:EGFP. A, B, The FACS gating threshold was set using nontransduced fetal VZ/SVZ cells as a nonfluorescent control. C, D, E/sox2:EGFP⁺ cells were sorted based on their expression of EGFP (P5 region), while E/sox2:EGFP⁻ cells were collected as the nonfluorescent population (P4). Infection of cells with a control virus, AdP/CMV:lacZ, revealed no significant autofluorescence (not shown). E–H, Live E/sox2:EGFP⁺ and E/sox2:EGFP⁻ cells, imaged 2 h after sort. Virtually every cell in the EGFP⁺ P5 gate expressed detectable fluorescence, while none of the cells in the EGFP⁻ P4 gate did so. Scale bar: E–H, 30 μm.

Sox2 is expressed by neural progenitor cells within the VZ/SVZ of human fetal forebrain. A, A cross section of human forebrain ventricular wall at 16 weeks g.a., exhibiting dense concentrations of sox2⁺ cells (red) in the VZ and IZ (DAPI nuclear counterstain, blue). Within the VZ, sox2⁺ cells coexpressed the neural progenitor markers nestin (B, C) and musashi (D, E). A relatively small fraction of VZ cells also coexpressed sox2 with GFAP (F, G), although most GFAP⁺ cells at this stage were noted in the SVZ; these included both sox2⁺ and sox2⁻ examples (G). Scale bars: A, 50 μm; B–G, 25 μm.

E/sox2:EGFP⁺ NSCs exhibit telomerase activity and maintain long telomere length. A, Telomerase enzymatic activity was examined in the purified E/sox2:EGFP⁺ and E/sox2:EGFP⁻ populations by TRAP assay. CHAPS lysis buffer, a negative control, was substituted for a cell extract to check for primer-dimer PCR artifacts and/or PCR product carry-over contamination. TSR8 is a quantification standard that estimates the total product generated, expressed in relative terms as TPG units. Telomerase enzymatic activity was significantly higher in E/sox2:EGFP⁺ cells (23.9 units in this run) than in matched E/sox2:EGFP⁻ cells (9.6 units). Telomerase-positive control cells (PCC; included in the TRAPeze kit) were included as standardization controls (116.9 units). B, Telomeric lengths were examined in the purified E/sox2:EGFP⁺ and E/sox2:EGFP⁻ populations, by TLA. Freshly isolated E/sox2:EGFP⁺ cells exhibited telomeric lengths of 15–17 kb (mean, 16.3 kb), which did not significantly differ from those of E/sox2:EGFP⁻ cells (mean, 15.9 kb) at this relatively early gestational stage.

E/sox2:EGFP⁺ VZ/SVZ stem and progenitor cells express a transcript signature distinct from E/sox2:EGFP⁻ cells. A, Following one-way ANOVA, sorted cell fractions and VZ/SVZ tissue RNA profiles were compared by post hoc t test and differentially expressed genes identified at 5% FDR and more than fourfold change between groups. The numbers of significantly different genes between comparisons were then plotted on a Venn diagram. Many more differentially expressed genes were found between E/sox2:EGFP⁺ cells to either E/sox2:EGFP⁻ cells or tissue, than between E/sox2:EGFP⁻ cells and tissue. The degree of overlap between sox2⁺ and either sox2-depleted or tissue was large, with >60% of genes shared. B, Bar graph of cell type-selective gene expression by E/sox2:EGFP⁺ cells. Expression ratios were calculated following normalization against the E/sox2:EGFP-depleted cells. E/sox2:EGFP⁺ cells expressed high levels of neuroepithelial progenitor and radial glial or astroglial associated transcripts. In contrast, expression of proneural and neuronal transcripts was significantly depleted from E/sox2:EGFP⁺ cells. Markers of glial progenitor and oligodendrocyte lineage cells were not enriched in E/sox2:EGFP⁺ cells. * indicates more than fourfold change and significance at 5% FDR, in transcript levels of E/sox2:EGFP⁺ and E/sox2:EGFP⁻ cells.

E/sox2:EGFP⁺ cells exhibit differential expression of pathway specific transcripts. A, Parametric gene set enrichment analysis of KEGG signaling pathways was performed on the profiles of E/sox2:EGFP-sorted (E/sox2:EGFP⁺), -depleted (E/sox2:EGFP⁻), and -unsorted VZ/SVZ dissociates (n = 4 each). Using a linear modeling approach, significant KEGG pathways were identified between E/sox2:EGFP⁺ and E/sox2:EGFP⁻ cells at 5% FDR. The heat map visualization shows relative enrichment within each pathway; red indicates overexpression and blue lower expression relative to the mean of E/sox2:EGFP⁻ profiles. B, The MAP kinase signaling pathway was significantly depleted according to KEGG pathway analysis. The heat map shows expression of those MAPK pathway genes whose expression differed significantly between E/sox2:EGFP⁺ and E/sox2:EGFP⁻ cells (>3-fold change, 5% FDR). Furthermore, activators of the MAPK pathways were identified as significantly enriched in E/sox2:EGFP⁺ following GO-based GSEA. C, The wnt signaling pathway was significantly overexpressed in E/sox2:EGFP⁺ cells. As in B, the heat map shows only significant differentially expressed genes. GO-GSEA further confirmed the overrepresentation of wnt genes in E/sox2:EGFP⁺ cells (q < 0.05). The color key indicates relative expression on a log₂ scale, with red indicating high expression and green low expression values.

E/sox2:EGFP⁺ cells give rise to self-renewing neurospheres in sustained culture. A–H, E/Sox2:EGFP⁺ cells isolated from human forebrain VZ/SVZ (16–17 weeks g.a.) initiated neurospheres rapidly after FACS. A, B, Single E/sox2:EGFP⁺ cells 4 h after FACS, then as seen at 2 d (C, D), 4 d (E, F), and 15 d (G, H) after FACS. No such rapid initiation of neurospheres was observed in the E/sox2:EGFP⁻ fractions, though low numbers were observed to arise in some cultures by 2 weeks in vitro (not shown); these likely reflected the occasional false-negative FACS assignment, of sox2⁺ cells sorted into the E/sox2:GFP⁻ fraction, as well as the incomplete infection of the initial dissociates by the adenoviral E/sox2:EGFP vector. Of note, most E/sox2:EGFP⁺ cells remained fluorescent for at least 4 weeks after FACS, though their intensity of EGFP expression weakened with time thereafter. I, The mean number of neurospheres per 100,000 plated cells generated in E/sox2:EGFP⁺ and E/sox2:EGFP⁻ populations was determined 2 weeks after FACS. J, E/sox2:EGFP⁺-derived neurospheres were maintained in vitro for 6 months, during which the incidence of neurospheres appeared to peak at 3 months. Scale bar: A–H, 100 μm.

A 0.4 kb sox2 neuroepithelial core enhancer identifies human neural progenitor cells. A, Schematic of sox2 gene coding region and its upstream and downstream regulatory elements. The N2 region is a conserved POU binding domain located upstream of the Sox2 gene, which acts as a core enhancer that specifies sox2 expression to both mouse and chick forebrain neural stem and progenitor cells. B, The 0.4 kb neural-specific enhancer, lying 5′ to the sox2 coding region (), was subcloned into pENTR/D and subsequently into pAd/PL-DEST, so as to generate both plasmid and adenoviral vectors containing E/sox2:P/β-globin:EGFP:pA. C, D, E/sox2:EGFP⁺ cells, 3 d after transfection of an unsorted dissociate with plasmid cDNA encoding E/sox2:P/βglobin:EGFP. E–H, Expression of E/sox2:EGFP in cultured VZ/SVZ neural progenitor cells (NPCs) after infection with AdE/sox2-0.4:P/β-globin:EGFP. E, Virtually all E/sox2:EGFP⁺ cells (green) express sox2 immunoreactivity (red), indicating that the 0.4 kb sox2 neural enhancer drives EGFP expression in these cells. F, G, Essentially all E/sox2:EGFP⁺ cells (green) coexpress the neural progenitor markers nestin (F) and musashi1 (G) (red). H, Approximately one-third of E/sox2:EGFP⁺ cells (green) expressed immunodetectable GFAP (red); these sox2⁺/GFAP⁺ cells typically exhibited a radial glial morphology in vitro. Scale bars: D (for C, D), E (for E–H), 25 μm.