Human mesenchymal stem cells maintain transgene expression during expansion and differentiation

Human adult bone marrow contains both hematopoietic stem cells that generat e cells of all hematopoietic lineages and human mesenchymal stem cells (hMS Cs), which support hematopoiesis and contribute to the regeneration of mult iple connective tissues. The goal of the current study was to demonstrate t hat transduced hMSCs maintain transgene expression after stem cell differen tiation in vitro and in vivo. We have introduced genes into cultured hMSCs by retroviral vector transfer and demonstrated long-term in vitro and in vi vo expression of human interleukin 3 (hIL-3) and green fluorescent protein (GFP). Protocols were developed to achieve transduction efficiencies of 80- 90% in these stem cells. In vitro expression of hIL-3 averaged 350 ng/10(6) cells/24 h over 17 passages (>6 months) and GFP expression was stable over the same time period. Transduced hMSCs were able to differentiate into oste ogenic, adipogenic, and chondrogenic lineages and maintained transgene expr ession after differentiation. Parallel studies were performed in vivo using NOD/SCID mice. Human MSCs expressing hIL-3 were cultured on several matric es and then delivered by subcutaneous, intravenous, and intraperitoneal rou tes. Sampling of peripheral blood demonstrated that systemic hIL-3 expressi on was maintained in the range of 100-800 pg/ml over a period of 3 months. These results illustrate the ability of hMSCs to express genes of therapeut ic potential and demonstrate their potential clinical utility as cellular v ehicles for systemic gene delivery.