Abstract
Aim: 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. Conclusion: 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.
Translated title of the contribution | Refined adenoviral transduction for controlled gene transfer into human adult mesenchymal stem cells |
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Original language | German |
Pages (from-to) | 677-683 |
Number of pages | 7 |
Journal | Zeitschrift fur Orthopadie und Ihre Grenzgebiete |
Volume | 143 |
Issue number | 6 |
DOIs | |
State | Published - Nov 2005 |
Externally published | Yes |
Keywords
- Adenoviral transduction
- BMP-2
- Human mesenchymal stem cells
- Osteoblastic differentiation
- Quantitative real-time PCR