Objective: To explore the possibility of using autologous bone marrow mesenchymal stem cells (BMSC) as a vehicle to deliver recombinant adeno-associated virus 2-mediated enhanced green fluorescent protein (rAAV-2-eGFP) in vitro, therefore to find an alternative solution for gene therapy of hematological malignancy.
Methods: BMSCs isolated from the bone marrow of patients with acute myelogenous leukemia (AML) at the onset of disease were infected by rAAV-2-eGFP at different multiplicity of infection (MOI=10(2), 10(3), 10(4), 10(5), 10(6), and 10(7), respectively). Phase-contrast fluorescent microscope and flow cytometry were employed to evaluate the expression of enhanced green fluorescent protein (eGFP).
Results: Ten to fourteen days after the transfection, eGFP expression began to be detected and the transfection efficiency ranged between 0.3% to 2%, which failed to be increased with the increase of MOI. The transduced eGFP could maintain a long-term stable expression in vitro in the 61 days of observation, and from 12 to 33 days after transfection, eGFP percentage underwent a decrease from the initial 1.16% to 0.5%-0.6% and maintained this expression level till 61 days after transfection.
Conclusion: rAAV can be used with BMSCs for in vitro gene therapy, but the poor transfection efficiency of these cells remains a significant obstacle for its further application.