Lentiviral vector-mediated transduction of neural progenitor cells before implantation into injured spinal cord and brain to detect their migration, deliver neurotrophic factors and repair tissue.
OBJECTIVE
Stem cells represent an attractive source for cell replacement therapy in neurological disorders due to their self-renewal and multi-potency. Genetic manipulation of these cells may allow controlled release of therapeutic proteins, suppress immune rejection, or produce essential neurotransmitters. Furthermore, when the expression cassette is incorporated into the host genome ex vivo, this technique also may be used as a method to trace cells following implantation into tissues of interest.


METHODS
We explored the possibility of transducing pluripotent fetal rat cortical neural progenitor cells (NPCs) using lentiviral vectors encoding the green fluorescent protein (GFP) or neurotrophic factors (BDNF, CNTF, D15A, GDNF, MNT and NT-3) prior to implanting these cells into the contused spinal cord or injured brain.


RESULTS
In vitro staining of these cells for neural markers (such as nestin, GFAP, Tuj-1 and RIP) after transduction did not reveal any significant difference from non-transduced cells. When they were transduced with a vector encoding CNTF or MNT, however, cells started expressing GFAP in vitro. Following delayed (1 week) implantation into the lesion site of the moderately contused rat spinal cord or the injured brain, transduced cells survived up to 12 weeks post-implantation (the longest time point examined) and most of the NPCs turned into an astrocytic phenotype in the spinal cord, but not in the brain. Nestin and GFP positive cells were detected in the brain, but not in the spinal cord lesion. GFP positive cells in the spinal cord migrated rostrally and caudally from the lesion/implantation site towards uninjured tissue.


CONCLUSIONS
Novel findings in this study are the longterm expression of a foreign gene in NPCs using lentiviral vectors; this enabled tracking of the cells following implantation. This expression also allowed the observation that NPCs developed differently in the injured spinal cord and brain. Moreover, NPCs could be transduced to overexpress neurotrophic factors. In sum, NPC survival and the long-term transgene expression that allows easy tracking of migrating cells make NPCs promising candidates for implantation into the injured spinal cord or brain and a potentially powerful tool to enhance regeneration when transduced ex vivo to produce therapeutic molecules.
Blits
Bas
B
The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, USA.
Kitay
Brandon M
BM
Farahvar
Arash
A
Caperton
Caroline V
CV
Dietrich
W Dalton
WD
Bunge
Mary Bartlett
MB
D000818:Animals; D001930:Brain Injuries / Q000628:therapy*; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004622:Embryo, Mammalian; D005260:Female; D005822:Genetic Vectors / Q000502:physiology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D007381:Intermediate Filament Proteins / Q000378:metabolism; D016086:Lentivirus / Q000502:physiology; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D009474:Neurons / Q000502:physiology* / Q000821:virology; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D013119:Spinal Cord Injuries / Q000628:therapy*; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000502:physiology* / Q000821:virology; D014161:Transduction, Genetic / Q000379:methods*; D014404:Tubulin / Q000378:metabolism
D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; C577486:Nes protein, rat; D064231:Nestin; D014404:Tubulin; D049452:Green Fluorescent Proteins