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


CONCLUSIONS
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.
Cenciarelli
Carlo
C
Istituto di Neurochirurgia, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, Roma 00168, Italy. cencia65@hotmail.com
Budoni
Manuela
M
Mercanti
Delio
D
Fernandez
Eduardo
E
Pallini
Roberto
R
Aloe
Luigi
L
Cimino
Vincenzo
V
Maira
Giulio
G
Casalbore
Patrizia
P
D000818:Animals; D001253:Astrocytes / Q000166:cytology; D002454:Cell Differentiation; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008856:Microscopy, Fluorescence; D020932:Nerve Growth Factor / Q000235:genetics* / Q000378:metabolism; D009474:Neurons / Q000166:cytology*; D009836:Oligodendroglia / Q000166:cytology; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology; D014161:Transduction, Genetic; D014162:Transfection
D020932:Nerve Growth Factor