[Refined adenoviral transduction for controlled gene transfer into human adult mesenchymal stem cells].
OBJECTIVE
Adenoviral gene transfer remains a powerful tool for basic research purposes. We hypothesize that adenoviral transduction of human mesenchymal stem cells (hMSC) in vitro can be improved by refined use of experimental parameters.


METHODS
hMSCs were transduced by adenoviral vectors encoding Luciferase or BMP-2 at a selection of multiplicities of infection (MOI) and exposure times. Transgene production and total protein content were measured. To determine practical relevance, expression of the bone marker genes Runx2 and Type I collagen was analyzed by quantitative PCR. As a phenotypic marker alkaline phosphatase was assessed. ANOVA and post hoc statistical analyses were used to determine differences among data (p < 0.05).


RESULTS
Prolonged exposure led to a decrease in transgene production and total protein content. Increasing MOI at exposure of up to 4 hours resulted in a higher production of the transgene. Transfer of the hBMP-2 gene promoted an enhanced lineage progression to the osteoblast phenotype indicating biological activity.


CONCLUSIONS
Time of exposure is of major importance for toxicity in vitro and should not exceed 4 hours for hMSC. While increase in exposure time leads to cell death, surviving cells, up to a certain limit, seem to compensate by increasing production of the transgene indicating that transduction efficiency cannot be positively measured in a binary yes-or-no scheme.
Koch
H
H
Klinik für Orthopädie und orthopädische Chirurgie, Universität Greifswald. hannjoer.koch@uni-greifswald.de
Jadlowiec
J A
JA
Whalen
J D
JD
Lattermann
C
C
Robbins
P
P
Fu
F H
FH
Merk
H
H
Hollinger
J O
JO
D000256:Adenoviridae / Q000235:genetics*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism* / Q000821:virology; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D023822:Tissue Engineering / Q000379:methods*; D014161:Transduction, Genetic / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta