A Novel Monoclonal Antibody Against Neuroepithelial and Ependymal Cells and Characteristics of Its Positive Cells in Neurospheres

Abstract

There are still few useful cell membrane surface antigens suitable for identification and isolation of neural stem cells (NSCs). We generated a novel monoclonal antibody (mAb), designated as mAb against immature neural cell antigens (INCA mAb), which reacted with the areas around a lateral ventricle of a fetal cerebrum. INCA mAb specifically reacted with neuroepithelial cells in fetal cerebrums and ependymal cells in adult cerebrums. The recognition molecules were O-linked 40 and 42 kDa glycoproteins on the cell membrane surface (gp40 INCA and gp42 INCA). Based on expression pattern analysis of the recognition molecules in developing cerebrums, it was concluded that gp42 INCA was a stage-specific antigen expressed on undifferentiated neuroepithelial cells, while gp40 INCA was a cell lineage-specific antigen expressed at the stages of differentiation from neuroepithelial cells to ependymal cells. A flow cytometric analysis showed that fetal and young adult neurospheres were divided into INCA mAb(-) CD133 polyclonal antibody (pAb)(-), INCA mAb(+) CD133 pAb(-), and INCA mAb(+) CD133 pAb(+) cell populations based on the reactivity against INCA mAb and CD133 pAb. The proportion of cells having the neurosphere formation capability in the INCA mAb(+) CD133 pAb(+) cell population was significantly larger than that of undivided neurospheres. Neurospheres formed by clonal expansion of INCA mAb(+) CD133 pAb(+) cells gave rise to neurons and glial cells. INCA mAb will be a useful immunological probe in the study of NSCs.

Two non-cytotoxic type 2 ribosome-inactivating proteins (Sambucus Sieboldiana lectin and Sambucus Nigra lectin) lead neurosphere cells to caspase-independent apoptosis

“…Cell membrane fraction lysates (10 µg/lane) prepared according to Kotani et al, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 12.5% acrylamide (ATTO, Tokyo, Japan), and then electro blotted onto polyvinylidene difluoride (PVDF) membranes (Immobilon; GE Healthcare, Buckinghamshire, UK) according to Towbin et al [4,17]. The PVDF membranes were blocked with 5% skim milk in PBS followed by 1 h incubation with bio-SSA lectin (1 µg/100 µl of PBS), bio-SNA lectin (0.7 µg/100 µl of PBS) and bio-MAM lectin (6 µg/100 µl of PBS) with or without 100 mM lactose.…”

“…In this paper, undifferentiated neural cells in neurospheres are collectively referred to as neurosphere cells. They have been used in a wide range of studies, such as neural stem cell (NSC) identification and isolation, proliferation and maintenance of NSCs, and differentiation of NSCs into neuronal and glial cells [2][3][4][5][6][7].…”