Characterization of GD3 ganglioside as a novel biomarker of mouse neural stem cells

Abstract

Neural stem cells (NSCs) are undifferentiated neural cells characterized by their high proliferative potential and the capacity for self-renewal with retention of multipotency. Over the past two decades, there has been a huge effort to identify NSCs morphologically, genetically, and molecular biologically. It is still controversial, however, what bona fide NSCs are. To define and characterize NSCs more systematically, it is crucial to explore novel cell-surface marker molecules of NSCs. In this study, we focused on GD3, a b-series ganglioside that is enriched in the immature brain and the subventricular zone (SVZ) of the postnatal and adult brain, and evaluated the usefulness of GD3 as a cell-surface biomarker for identifying NSCs. We demonstrated that GD3 was expressed in more than 80% of NSCs prepared from embryonic, postnatal, and adult mouse brain tissue by the neurosphere culture method. The percentage of GD3-expressing NSCs in neurospheres was nearly the same as it was in neurospheres derived from embryonic, postnatal, and adult brains but decreased drastically to about 40% after differentiation. GD3(+) cells isolated from embryonic mouse striata, postnatal, and adult mouse SVZs by fluorescence-activated cell sorting with an R24 anti-GD3 monoclonal antibody efficiently generated neurospheres compared with GD3(-) cells. These cells possessed multipotency to differentiate into neurons, astrocytes, and oligodendrocytes. These data indicate that GD3 is a unique and powerful cell-surface biomarker to identify and isolate NSCs.

Fig. 1

GD3 expression in mouse brains. (A and B) Cryosections of adult mouse brains were stained with the R24 anti-GD3 antibody and AK97 anti-SSEA-1 antibody. Panel (B) is higher magnification view of panel (A). Nuclei were stained with Hoechst 33258 (H33258). “LV” indicates lateral ventricles.

Fig. 2

NSCs prepared from mouse striata (E14) and SVZs (P2, P10, P30, and adult) in the form of neurospheres. (A) Primary, secondary, tertiary, quaternary, and quinary neurospheres prepared from mouse striata (E14) or SVZs (P2, P10, P30, and adult). Scale bar represents 100 μm. (B) The proliferation rates of neurosphere-forming cells estimated by WST-8 assay at 0, 1, 3, and 5 days in vitro (n = 3). (C) The expression of NSC-specific genes in neurosphere-forming cells prepared from mouse striata (E14) or SVZs (P2, P10, P30, and adult) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). “Brain” indicates the control cDNA prepared from the total RNA of adult mouse brains. “–RT” indicates negative controls without reverse transcription. β-Actin was detected as the control. (D) Expressions of nestin (NSC marker), β-III tubulin (neuron marker), GFAP (astrocyte marker), and GalC (oligodendrocyte marker) in neurosphere-forming cells and the differentiated cells cultured in the presence of 1% fetal bovine serum and the absence of FGF-2 and EGF. Nuclei were stained with H33258. Scale bar represents 100 μm. (E) The percentages of nestin⁺ cells in neurosphere-forming cells (n = 10).

Fig. 3

Expression of GD3 in NSCs. (A) Expression of GD3 and GM1 in NSCs and the differentiated cells. (B) The percentages of GD3⁺ cells in NSCs and the differentiated cells (n = 8–11). (C) TLC of total lipids extracted from embryonic, postnatal, and adult neurospheres. (D) TLC of total lipids extracted from primary, secondary, tertiary, quaternary, and quinary neurospheres. (E) TLC-immunostaining of total lipids extracted from neurospheres with an R24 anti-GD3 monoclonal antibody. (F) TLC of total lipids extracted from differentiated cells derived from neurospheres. In panels (C)–(F), bovine brain gangliosides (Std; GM1, GD1a, GD1b, and GT1b) and authentic GD3 were used as standard gangliosides. In panels (C), (D), and (F), gangliosides were visualized with the resorcinol-HCl reagent. Gangliosides prepared from the same number of cells were applied to each lane. (G) Double-immunostaining of NSCs prepared from embryonic, postnatal, and adult neurospheres with the R24 anti-GD3 monoclonal antibody and AK97 anti-SSEA-1 monoclonal antibody. (H) The percentage of SSEA-1⁺ cells in GD3⁺ NSCs prepared from embryonic, postnatal, and adult neurospheres (n = 10). (I) Double-immunostaining of a neurosphere from adult mouse brain tissue with the anti-GD3 antibody and anti-SSEA-1 antibody. In panels (A), (G), and (I), scale bars represent 100 μm. Nuclei were stained with H33258.

Fig. 4

Isolation of GD3⁺ cells from mouse brains by FACS with an R24 anti-GD3 monoclonal antibody. (A) Dot plots of E14 mouse brain cells stained with the R24 anti-GD3 monoclonal antibody (anti-GD3 Mab) or subclass control mouse IgG. (B) The percentages of GD3⁺ and GD3⁻ cells in the living cells prepared from striata (E14) or SVZs (P2, P10, P30, and adult). (C) Immunostaining of GD3⁺ and GD3⁻ cell populations from E14 striata with antibodies to nestin, SSEA-1, MAP2, and β-III tubulin at 3 h after cell sorting. Nuclei were stained with H33258. Scale bar represents 100 μm. (D) Expression of NSC-specific genes (Sox2, nestin, and Musashi-1) and neuron-specific genes (tau and MAP2) in GD3⁺ and GD3⁻ cells from E14 striata analyzed by RT-PCR. “–RT” indicates negative controls without reverse transcription. β-Actin was detected as the control. (E) Phase views of neurospheres generated by GD3⁺ and GD3⁻ cell populations that were sorted from E14 striata and P2, P10, P30, and adult SVZs. Scale bar represents 1 mm. (F) The number of neurospheres formed in GD3⁺ and GD3⁻ cell populations (n = 5–10). The number of neurospheres was counted at 5 days after cell sorting. (G) Cell staining of NSCs and differentiated cells derived from neurosphere formed by GD3⁺ cells. Nuclei were stained with H33258. Scale bar represents 100 μm.