Surface-anchoring of spontaneously dimerized epidermal growth factor for highly selective expansion of neural stem cells.

To develop culture substrates for use in selective expansion of neural stem cells (NSCs), epidermal growth factor (EGF)-containing chimeric proteins were designed and synthesized by means of recombinant DNA technology. The chimeric proteins consisted of three components including an EGF domain, an alpha-helical oligopeptide, and a hexahistidine sequence. Two different alpha-helical oligopeptides were separately incorporated into chimeric proteins. Structural analyses by native gel electrophoresis and circular dichroism spectroscopy revealed that the heterodimer of these proteins was spontaneously formed through coiled-coil association of the alpha-helical oligopeptides. The monomeric and dimeric forms of these chimeric proteins were immobilized to the glass-based substrate via coordinate bonding between the hexahistidine and Ni(II) ions fixed on a substrate. The results of cell culture assays with NSCs showed that cells proliferated most rapidly and selectively on a substrate with the surface-anchored EGF dimer. The rate of cell proliferation on the surface with dimeric EGF was 1.3-2.0 times higher on the surfaces with monomeric EGF. In addition, the content of stem cells, determined 96 h after cell seeding, was highest on the surface with dimeric EGF (98%) among the surfaces studied (90-97% on surfaces with monomeric EGF). The observed growth rate and the stem cell content on the surface with EGF dimer were far beyond those in the standard neurosphere culture. The effect of surface-anchored dimeric EGF may be attributed to the enhanced dimerization of EGF-EGF receptor complexes leading to efficient signaling for mitogenic activity. We conclude that surface-anchoring of the EGF dimer provides an excellent substrate that allows the highly efficient expansion of NSCs.