Homologous transplantation of neural stem cells to the injured spinal cord of mice.
OBJECTIVE
Murine neural stem cells (NSCs) were homografted onto the injured spinal cord (SC) to assess their potential to improve motor behavior, to differentiate as neurons, and to establish synapse-like contacts with the descending axonal paths of the host. In addition, we investigated whether transduced NSCs over-expressing vascular endothelial growth factor might exert any angiogenetic effect in the injured SC.


METHODS
NSCs derived from mouse embryos were transduced to express either green fluorescent protein or vascular endothelial growth factor. The cells were engrafted in mice where an extended dorsal funiculotomy had been performed at the T8-T9 level. At intervals from 4 to 12 weeks after grafting, motor behavior was assessed using an open field locomotor scale and footprint analysis. At the same time points, the SC was studied by conventional histology, immunohistochemistry, and fluorescence microscopy. The interactions between the grafted NSCs and descending axonal paths were investigated using anterogradely transported fluorescent axonal tracers.


RESULTS
By the 12-week time point, mice engrafted with NSCs significantly improved both their locomotor score on open field test and their base of support on footprint analysis. Histological studies showed that green fluorescent protein-positive NSCs survived as long as 12 weeks after grafting, migrated from the grafting site with a tropism toward the lesion, and either remained undifferentiated or differentiated into the astrocytic phenotype without neuronal or oligodendrocytic differentiation. Interestingly, the NSC-derived astrocytes expressed vimentin, suggesting that these cells differentiated as immature astrocytes. The tips of severed descending axonal paths came adjacent to grafted NSCs without forming synapse-like structures. When genetically engineered to over-express vascular endothelial growth factor, the grafted NSCs significantly increased vessel density in the injured area.


CONCLUSIONS
In the traumatically injured mice SC, NSC grafting improves motor recovery. Although differentiation of engrafted NSCs is restricted exclusively toward the astrocytic phenotype, the NSC-derived astrocytes show features that are typical of the early phase after SC injury when the glial scar is still permissive to regenerating axons. The immature phenotype of the NSC-derived astrocytes suggests that these cells might support neurite outgrowth by the host neurons. Thus, modifying the glial scar with NSCs might enhance axonal regeneration in the injured area. The use of genetically engineered NSCs that express trophic factors appears to be an attractive tool in SC transplantation research.
Pallini
Roberto
R
Department of Neurosurgery, Laboratory for Neural Stem Cells, Center for Research on Regeneration of the Nervous System, Catholic University School of Medicine, Rome, Italy. pallini@rm.unicatt.it
Vitiani
Lucia Ricci
LR
Bez
Alessandra
A
Casalbore
Patrizia
P
Facchiano
Francesco
F
Di Giorgi Gerevini
Valeria
V
Falchetti
Maria Laura
ML
Fernandez
Eduardo
E
Maira
Giulio
G
Peschle
Cesare
C
Parati
Eugenio
E
D000818:Animals; D001522:Behavior, Animal; D015152:Blotting, Northern / Q000379:methods; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004622:Embryo, Mammalian; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D007211:Indoles; D007381:Intermediate Filament Proteins / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D051379:Mice; D009043:Motor Activity / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D016900:Neurofilament Proteins / Q000378:metabolism; D009474:Neurons / Q000637:transplantation*; D011597:Psychomotor Performance / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013119:Spinal Cord Injuries / Q000503:physiopathology / Q000601:surgery*; D033581:Stem Cell Transplantation / Q000379:methods; D014162:Transfection / Q000379:methods; D014184:Transplantation, Homologous / Q000379:methods; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D005904:Glial Fibrillary Acidic Protein; D007211:Indoles; D007381:Intermediate Filament Proteins; D019394:Ki-67 Antigen; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D016900:Neurofilament Proteins; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D014746:Vimentin; D049452:Green Fluorescent Proteins; C007293:DAPI