Expression of GD2 and GD3 gangliosides in human embryonic neural stem cells

Abstract

NSCs (neural stem cells) are undifferentiated neural cells endowed with a high potential for proliferation and a capacity for self-renewal with retention of multipotency to differentiate into neurons and glial cells. It has been recently reported that GD3, a b-series ganglioside, is a marker molecule for identifying and isolating mouse NSCs. However, the expression of gangliosides in human NSCs is largely unknown. In the present study, we analysed the expression of gangliosides, GD2 and GD3, in human NSCs that were isolated from human brains at gestational week 17 in the form of neurospheres, which are floating clonal aggregates formed by NSCs in vitro. Employing immunocytochemistry, we found that human NSCs were strongly reactive to anti-GD2 antibody and relatively weakly reactive to anti-GD3 antibody. Treatment of these cells with an organic solvent such as 100% methanol, which selectively removes glycolipids from plasma membrane, abolished the immunoreactivity with those antibodies, indicating that the reactivity was due to GD2 and GD3, but not to GD2-/GD3-like glycoproteins or proteoglycans. The immunoreactivity of human NSCs to antibody against SSEA-1 (stage-specific embryonic antigen-1), a well-known carbohydrate antigen of NSCs, was not decreased by the treatment with 100% methanol, indicating that SSEA-1 is mainly carried by glycoproteins and/or proteoglycans in human NSCs. Our study suggests that GD2 and GD3 can be marker gangliosides for identifying human NSCs.

Figure 1. Marker expression of human NSCs

Human NSCs, Poetic Normal Human Neural Progenitors, prepared from human brains at gestational week 17 in the form of neurospheres (A) were immunostained with anti-human nestin antibody (mouse IgG) and anti-SSEA-1 antibody (mouse IgM) (B). Human NSCs were positive for nestin (green) and SSEA-1 (green), marker molecules of NSCs. coIgG and coIgM indicate human NSCs stained with isotype control mouse IgG and IgM respectively. Nuclei were stained with Hoechst 33258 (blue).

Figure 2. Effect of methanol on GD2, SSEA-1 and GD3 immunoreactivity in human NSCs

Human NSCs treated with or without methanol (MeOH) were immunostained with antibodies against GD2 (A), SSEA-1 (B) and GD3 (C). Human NSCs were positive for GD2 (green; A) and relatively weakly positive for GD3 (green; C). No immunoreactivity of human NSCs to antibodies against GD2 (A) and GD3 (C) after treatment with 100% methanol for 10 min to wash out lipids of plasma membranes indicates that the immunoreactivity is attributed to GD2 and GD3 gangliosides, but not to glycoproteins or proteoglycans. (B) Human NSCs treated with methanol were still positive for SSEA-1 (green), indicating that SSEA-1 is mainly carried by glycoproteins and/or proteoglycans in these cells. (D) Mouse embryonic NSCs prepared from mouse brains at embryonic day 14 in the form of neurospheres were stained with anti-GD3 antibody (green) as a positive control. Nuclei were stained with Hoechst 33258 (blue).

Figure 3. Effect of methanol on GD2 and GD3 immunoreactivity in mouse embryonic NSCs

Mouse embryonic NSCs were stained with anti-GD3 antibody (green) and anti-GD2 antibody (green). No immunoreactivity to antibodies against GD2 and GD3 in cells treated with 100% methanol (MeOH) for 10 min indicates that the GD3 and GD2 immunoreactivity is attributed to GD3 and GD2 gangliosides, but not to glycoproteins or proteoglycans. Nuclei were stained with Hoechst 33258 (blue).