Gliotypic neural stem cells transiently adopt tumorigenic properties during normal differentiation

Abstract

An increasing body of evidence suggests that astrocytic gliomas of the central nervous system may be derived from gliotypic neural stem cells. To date, the study of these tumors, particularly the identification of originating cellular population(s), has been frustrated by technical difficulties in accessing the native niche of stem cells. To identify any hallmark signs of cancer in neural stem cells or their progeny, we cultured subventricular zone-derived tissue in a unique in vitro model that temporally and phenotypically recapitulates adult neurogenesis. Contrary to some reports, we found undifferentiated neural stem cells possess few characteristics, suggesting prototumorigenic potential. However, when induced to differentiate, neural stem cells give rise to intermediate progenitors that transiently exhibit multiple glioma characteristics, including aneuploidy, loss of growth-contact inhibition, alterations in cell cycle, and growth factor insensitivity. Further examination of progenitor populations revealed a subset of cells defined by the aberrant expression of (the pathological glioma marker) class III beta-tubulin that exhibit intrinsic parental properties of gliomas, including multilineage differentiation and continued proliferation in the absence of a complex cellular regulatory environment. As tumorigenic characteristics in progenitor cells normally disappear with the generation of mature progeny, this suggests that developmentally intermediate progenitor cells, rather than neural stem cells, may be the origin of so-called "stem cell-derived" tumors.

Figure 1

Transient alterations in proliferation, ploidy, and gene regulation characterize subventricular differentiation. (A): Subventricular-derived NSCs proliferated in response to removal of mitogenic stimuli. (B): Twenty-four-hr incremental BrDU pulse labeling revealed increases in thymidine analog incorporation 24–72 hr following mitogenic differentiation in subventricular cultures. Alteration in gene expression accompanied differentiation in SVZ tissues: increases in cell cycle promoting genes 12–36 hr following mitogenic withdrawal (C) were followed by increases in cell cycle checkpoint genes (D). Mitogenic deprivation signaled increases in cellular growth (E) and aneuploidic (hyperploidic) cells (F), which were resolved upon the generation of neuroblasts. Abbreviations: BrDU, 5′-bromodeoxyuridine; diff, differentiated; hr, hour; nSVZ, non-subventricular zone; SVZ, subventricular zone.

Figure 2

The absence of a complex multicellular environment yield enhances proliferation and multilineage potential in neural progenitors. (A): Quantified NS production from prol and differentiating (1, 4, or 7 d) subventricular zone (SVZ)-derived NSCs. (B): 3° sphere formation from 2° spheres generated in (A). (C): Progeny analysis of adherent individual cells expanded clonally for 3 d. (D): Proliferative capacity of individual SVZ cells placed in NS-forming conditions for 3 d. *, p < .05; one-way analysis of variance. Abbreviations: 2°, secondary; 3°, tertiary; d, day; GFAP, glial fibrillary acidic protein; NS, neurosphere; prol, proliferating.

Figure 3

Differentiation of astrotypic NSCs results in the upregulation of class III β-tubulin in intermediate progenitor cells. (A): Class III β-tubulin was appreciated in undiff cultures in cells generally possessing a protoplasmic astrocyte morphology (βIII-tubulin, red; glial fibrillary acidic protein [GFAP], blue; nestin-enhanced green fluorescent protein, green). (B): One d following induction of differentiation, compact single GFAP⁻/class III β-tubulin⁺ cells (left) were present, with a minor population of GFAP^+/−/class III β-tubulin⁺ cells (right) with an indeterminate morphology. (C): Compacted progenitors multiplied to form clusters following their appearance in culture. Progenitors at this developmental stage expressed epidermal growth factor receptor (red, inset) (class III β-tubulin, green). (D, E): Progenitors matured to form clusters of neuroblasts, which appeared in culture 4 d following differentiation (E) and formed defined neuronal phenotypes (class III β-tubulin, green). (G): Class III β-tubulin⁺ progenitors exhibited rapid expansion following their appearance in culture. Images show counterstaining with 4,6-diamidino-2-phenylindole. Scale bars = 50 µm (A–E), 100 µm (G). *, p < .05; **, p < .05; one-way analysis of variance. Abbreviations: avg, average; d, days; diff, differentiated; undiff, undifferentiated.

Figure 4

Class III β-tubulin/Tuj1 labels putative neural stem cell (NSC)/progenitor populations. Multiple antibody labeling in undifferentiated passage 3 subventricular NSCs revealed coexpression of A2B5 ([A], arrows), FGFR1 (B), and CD133/Prominin-1 (C). Tuj1 was expressed in the mitotic spindle of dividing cells ([A, C], insets). (D): Substantial fractions of Tuj1⁺ cells expressed NSC and progenitor markers. CD15 and NG2 were examined and were not expressed in undifferentiated cultures. (E–G): Tuj1 was expressed in multiple members of the Sox protein family, as well as in the supependymal cell layer of the lateral ventrical (H). Coronal section of the subventricular zone revealed that Tuj1⁺ cells were present in the subventricular layer adjacent to the LV (arrows). Scale bars = 50 µm (A, C, E–G), 25 µm (B), and 100 µm (H). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; FGFR1, fibroblast growth factor receptor 1; GFAP, glial fibrillary acidic protein; LV, lateral ventricle.