In vitro analysis of mouse neural stem cells genetically modified to stably express human NGF by a novel multigenic viral expression system.

OBJECTIVES

The purpose of this study was to characterize mouse neural stem cells (NSC) transduced by a multigenic lentiviral vector (LV) and stably express recombinant human nerve growth factor (rhNGF). We obtained NSC-derived cell lines which express human NGF in relevant amount to exploit their ability for therapeutic applications.

METHODS

We constructed advanced multigenic LV vectors which contain a tricistronic cassette to express simultaneously up to three independent genes: (1) rhNGF (beta subunit); (2) EGFP (enhanced green fluorescent protein) and (3) Neo(R) (neomycin antibiotic resistance gene). Lentiviruses were obtained by transfecting LV constructs plus helper plasmids in human embryonic kidney (HEK)-293T packaging cells. Lentiviral virions were released in culture media and subsequent used to infect mouse NSC. Genetycin 418-resistant NSC were obtained after 1 month of selection in the presence of antibiotic (G418). Levels of human NGF secreted by rhNGF-NSC were determined by ELISA (enzyme-linked immunosorbent assay). Features of multipotentiality of engineered NSC-derived cell lines versus naive cells (control-NSC) were assessed by immunocytochemical analysis in differentiation conditions. Self-renewal of NSC was tested by neurospheres assay (NSA).

RESULTS

Levels of secreted human NGF, from conditioned media obtained by rhNGF-NSC cultures, were found to be elevated in either proliferation or differentiation conditions as compared with control cells. Moreover, released hNGF demonstrated biologic activity on PC12 cells by a functional test of neurite outgrowth. Immunocytochemical analysis revealed that engineered NSC showed to be all positives for EGFP. After thirty passages in vitro in the presence of G418, engineered cells versus naive NSC cultures maintained their multipotentiality to differentiate into neurons, astrocytes and oligodendrocytes. Furthermore, it was found that rhNGF-NSC-derived neurons expressed choline acetyltransferase (ChAT) and displayed an enhanced axonal growth. NSA showed an altered sphere forming frequency either in rhNGF-NSC or both group of control NSC.

DISCUSSION

Lentivirus-mediated rhNGF gene transfer into NSC was achieved using a new version of LV vectors. We obtained rhNGF-NSC-derived cell lines which released hNGF to high levels in the culture medium. The expression of neural differentiation markers, like microtubule associated protein 2 (MAP2) (a/b), glial fibrillary acidic protein (GFAP) and chondroitin sulphate proteoglycan (NG2), was not enhanced in rhNGF-NSC compared with control cells. Secreted hNGF increased axonal sprouting by rhNGF-NSC-derived neurons which was associated with ChAT expression. rhNGF-NSC may prospectively be good candidates for the treatment of either neurodegenerative diseases such as Alzheimer disease or central nervous system injuries.