Abstract
The purpose of this study was to generate quadruple fluorescent protein (QFP) transgenic mice as a source for QFP-expressing neural stem and progenitor cells (NSCs/NPCs) that could be utilized as a tool for transplantation research. When undifferentiated, these NSCs only express cyan fluorescent protein (CFP); however, upon neuronal differentiation, the cells express yellow fluorescent protein (YFP). During astrocytic differentiation, the cells express green fluorescent protein (GFP), and during oligodendrocytic differentiation, the cells express red fluorescent protein (DsRed). Using immunocytochemistry, immunoblotting, flow cytometry and electrophysiology, quadruple transgenic NPCs (Q-NPCs) and GFP-sorted NPCs were comprehensively characterized in vitro. Overall, the various transgenes did not significantly affect proliferation and differentiation of transgenic NPCs in comparison to wild-type NPCs. In contrast to a strong CFP and GFP expression in vitro, NPCs did not express YFP and dsRed either during proliferation or after differentiation in vitro. GFP-positive sorted NPCs, expressing GFP under the control of the human GFAP promoter, demonstrated a significant improvement in astroglial differentiation in comparison to GFP-negative sorted NPCs. In contrast to non-sorted and GFP-positive sorted NPCs, GFP-negative sorted NPCs demonstrated a high proportion of neuronal differentiation and proved to be functional in vitro. At 6 weeks after the intracerebroventricular transplantation of Q-NPCs into neonatal wild-type mice, CFP/DCX (doublecortin) double-positive transplanted cells were observed. The Q-NPCs did not express any other fluorescent proteins and did not mature into neuronal or glial cells. Although this model failed to visualize NPC differentiation in vivo, we determined that activation of the NPC glial fibrillary acid protein (GFAP) promoter, as indicated by GFP expression, can be used to separate neuronal and glial progenitors as a valuable tool for transplantation studies.