In this report, we used a sox2 enhancer-based selection strategy to isolate and profile uncommitted neural precursor cells from the second trimester human fetal ventricular and subventricular zones. These cells expressed sox2 protein, were multilineage competent, and proved capable of sustained self-renewal in vitro, indicating their inclusion of competent neural stem cells. In addition, these E/sox2:EGFP-defined isolates sustained both telomerase transcription and enzymatic activity, in contrast to their matched E/sox2:GFP-depleted populations, which failed to do so. Genomic analysis of the E/sox2:EGFP+ cell population, and comparison to the matched unsorted and sox2-depleted populations, revealed that E/sox2:EGFP+ cells differentially expressed a discrete set of transcripts suggestive of active wnt, EGF and notch pathway signaling.
Neural stem cells are defined as both multilineage competent for neurons and glia, as well as self-renewing. In humans, self-renewal competence is attended by sustained telomerase activity, which prevents telomeric erosion, thereby maintaining mitotic competence (Wright et al., 1996
; Wright and Shay, 2005
). Stem cell-derived daughter cells - which may be defined as transit amplifying progenitors, based upon their mitotic expansion concurrent with departure from the ventricular zone - include restricted neuronal and glial progenitors, as well as still-multipotential progenitors, all of which may share a loss of self-renewal competence (Goldman, 2005b
; Goldman, 2005a
). At least in development, this loss in self-renewal is associated with a fall in the telomerase activity of neural progenitors (Ostenfeld et al., 2000
). As a result, whereas a sox2−
pool devoid of self-renewing stem and progenitor cells would be expected to manifest little telomerase enzymatic activity, one might anticipate that a sox2+
pool, comprised of both neural stem cells and their derived transit-amplifying progenitor cells, would exhibit sustained telomerase activity. We observed this to be the case, as telomerase enzymatic activity was robustly higher in the E/sox2:EGFP+
cells, relative to their depleted remainder of E/sox2−
This maintenance of telomerase activity by E/sox2+
cells was associated with their corresponding differential over-expression of telomerase-dependent transcripts, among others. Indeed, E/sox2:EGFP+
cells expressed several categories of transcripts associated with both mitogenic expansion and undifferentiated self-renewal. Likely mitogenic pathways were represented largely by genes associated with the MAP kinase pathways, activators of which were significantly up-regulated in E/sox2:EGFP+
cells. In particular, E/sox2:EGFP+
cells differentially expressed a number of receptor tyrosine kinases, that included the class 1, 4 and 6 receptor tyrosine kinases EGFR, FGF1/2R, and MET, respectively, each of which has been previously implicated in the expansion of neural stem and progenitor cells. Yet perhaps most striking in their degree of over-expression by E/sox2:EGFP+
cells were components of the wnt signaling pathway. Wnt ligands have been noted to comprise self-renewal signals for murine neural stem cells (Kalani et al., 2008
), and may serve a similar function in human neural precursors as well. Nonetheless, we were surprised to note that in human fetal VZ/SVZ-derived E/sox2:EGFP+
cells, the most specifically and differentially over-expressed wnt ligand proved to be wnt16, a functionally obscure molecule that may signal through the canonical pathway (Mazieres et al., 2005
). In addition, the wnt receptors FZD-6, FZD-8, and FZD-10 were all highly over-expressed, as were a number of wnt target genes. These over-expressed wnt targets included dickkopf3 (DKK3), follistatin (FST), Krupple-like factor 5 (KLF5), TWIST1, jagged1 (JAG1), NrCAM (NRCAM), RUNX2, autotaxin (ENPP2), and cyclin D (CCND1), among others. Several of these are of especial interest. The simultaneous over-expression of the wnt 16 and fzd-6, -8 and -10 together with DKK3, an antagonist of LRP5/6 and hence of wnt signaling, suggests the possibility of autocrine wnt signaling with concurrent lateral inhibition of neighboring wnt-regulated neural progenitors.
E/sox2:EGFP-defined cells were noted to differentially express a number of other wnt target genes, a number of which may serve to modulate wnt pathway activity. For instance, the E/sox2:EGFP-overexpressed transcript NrCAM has been shown to bind and regulate the receptor tyrosine phosphatase PTPRZ, which is involved in both radial cell maintenance and ß-catenin-dependent progenitor cell expansion, introducing yet another mechanism by which the undifferentiated self-renewal of E/sox2:EGFP+ cells might be regulated in trans. The differential up-regulation of these wnt-regulated genes in E/sox2:EGFP-defined progenitors is consistent with their distinct roles in both maintaining the undifferentiated self-renewal of early neuroepithelial stem cells, and in potentiating the phenotypic diversification of both neighbors and daughter cells into differentiated derivatives.
In regards to the latter, E/sox2:EGFP+ cells were found to over-express a number of serially activated components of the notch pathway, suggesting active notch signaling and concurrent repression of genes associated with the assumption of a differentiated fate. Notch2, notch3, hes1 and hes5, all positive activators and/or effectors of notch signaling, were all differentially over-expressed by E/sox2:EGFP+ cells (). Conversely, genes typically suppressed by notch receptor activation, such as numb and the bHLH transcription factors mash1, ngn1/2, and neuroD1/2, were all relatively under-expressed in E/sox2:EGFP+ cells, again suggesting active notch signaling. In this regard, it is interesting to note that E/sox2:EGFP+ cells differentially expressed high levels of jagged1 (JAG), a trans-activating positive stimulus for notch signaling. The high level expression of JAG by E/sox2:EGFP+ cells suggests again that E/sox2:EGFP+ progenitors may exert a strong influence upon the turnover and fate of their neighbors.
cells also manifested high differential over-expression of noggin (NOG), a soluble antagonist of the bone morphogenetic proteins, and hence of BMP signaling. Given the pro-gliogenic actions of the BMPs (Mabie et al., 1997
; Lim et al., 2000
), the over-expression of noggin by sox2+
NSCs might serve to prevent their premature glial differentiation, and hence preserve their undifferentiated expansion competence (Kondo and Raff, 2004
). Together, these data suggest the concurrence of wnt and notch signaling in E/sox2:EGFP-defined neural stem and progenitor cells, occurring in the context of a noggin-mediated minimization of concurrent BMP receptor-dependent signaling. Acting in concert, these signals act to ensure the cell-autonomous, active repression of terminal differentiation by these phenotypically plastic neural progenitor cells.
Thus, sox2 enhancer-based FACS permits the prospective and selective enrichment of a population of multipotential, self-renewing, and telomerase-expressing neural precursor cells from the fetal human brain. These E/sox2:EGFP+
cells included an hTERT+
fraction with active telomerase enzymatic activity, that likely defined the self-renewing fraction of neural stem cells within the larger pool of E/sox2:EGFP-defined neural progenitors. Most importantly, the E/sox2-based isolation of human neural stem and progenitor cells has permitted the definition of both the transcriptome and dominant signaling pathways of these cells. Assessed in the context of regional gene expression within the second trimester human forebrain (Johnson et al., 2009
), these progenitor-selective pathways should permit the prediction of ligand-receptor interactions with both neural and non-neural cells within the host germinal matrix. Indeed, by defining their selective engagement of distinct receptor tyrosine kinase, wnt, notch and BMP signaling pathways, and by identifying the specific receptors employed by human neural progenitors, this analysis provides substantial molecular insight into how the expansion and fate of human neural stem and progenitor cells may be modulated for therapeutic benefit.