Effect of hydroxyurea and etoposide on transduction of human bone marrow mesenchymal stem and progenitor cell by adeno-associated virus vectors
Abstract
AIM:
To study the effect of hydroxyurea and etoposide on transduction of human marrow mesenchymal and progenitor stem cells by adeno-associated virus (AAV).
METHODS:
Isolated human bone marrow mesenchymal stem and progenitor cells (hMSCs) were cultured in DMEM containing 10% FBS or 5% FBS and dexamethasone 1 micromol/L respectively. After being treated with hydroxyurea and etoposide, hMSCs were transduced by AAV-LUC. After two days luciferase activity (relative light unites per second or RLU/s) were tested, which indirectly reflected the relative transduction efficiency of different groups, and virus DNA was isolated by Hirt extraction for Southern hybridization.
RESULTS:
Transduction luciferase activity and transduction efficiency in cultures treated with hydroxyurea and etoposide were significantly higher than that in control cultures. Dividing cells had about 20-fold higher transduction efficiency compared with control cells. Transduction efficiency in stationary cells was about 50 times higher than that in control cells. Southern analysis showed that hydroxyurea and etoposide enhanced second-strand DNA synthesis by rAAV.
CONCLUSION:
Hydroxyurea and etoposide could increase transduction efficiency of hMSCs by AAV vectors, and stationary cells were more sensitive to these drugs than dividing cells.
Keywords:
To study the effect of hydroxyurea and etoposide on transduction of human marrow mesenchymal and progenitor stem cells by adeno-associated virus (AAV).
METHODS:
Isolated human bone marrow mesenchymal stem and progenitor cells (hMSCs) were cultured in DMEM containing 10% FBS or 5% FBS and dexamethasone 1 micromol/L respectively. After being treated with hydroxyurea and etoposide, hMSCs were transduced by AAV-LUC. After two days luciferase activity (relative light unites per second or RLU/s) were tested, which indirectly reflected the relative transduction efficiency of different groups, and virus DNA was isolated by Hirt extraction for Southern hybridization.
RESULTS:
Transduction luciferase activity and transduction efficiency in cultures treated with hydroxyurea and etoposide were significantly higher than that in control cultures. Dividing cells had about 20-fold higher transduction efficiency compared with control cells. Transduction efficiency in stationary cells was about 50 times higher than that in control cells. Southern analysis showed that hydroxyurea and etoposide enhanced second-strand DNA synthesis by rAAV.
CONCLUSION:
Hydroxyurea and etoposide could increase transduction efficiency of hMSCs by AAV vectors, and stationary cells were more sensitive to these drugs than dividing cells.