Abstract 13158: Development of Reporter Gene Techniques for Long-Term Assessment of Transplanted Human Circulating Progenitor Cells with Positron Emission Tomography
Background: PET with direct cell radiolabeling can only track transplanted cells for several hours. To further investigate cell fate in vivo, we transduced human circulating progenitor cells (CPCs) with reporter genes for long-term assessment of cell therapy.
Methods: A lentiviral vector, carrying a triple-fusion reporter (TFR) construct consisting of PET (herpes simplex virus type 1 truncated thymidine kinase; HSV1-sr39tk), fluorescence (monomeric red fluorescence protein; mRFP), and bioluminescence (firefly luciferase) reporter genes, was produced by a transient transfection in 293FT cells. Human CPCs were transduced with the created viral particle. The transduction efficiency was determined by flow cytometry analysis of RFP expression. The expression of HSV1-tk was examined by Western blot. Transduced CPC morphology, viability, migration and angiogenic capacities were assessed. Transduced cells were isolated by FACS and transduction stability was measured.
Results: A dose-response effect was achieved by transducing cells at increasing multiplicities of infection (MOIs); with a mean transduction efficiency of 13% and 16% at MOIs of 50 and 100, respectively. Western blot analysis confirmed HSV1-tk expression in transduced CPCs. Both transduced and control CPCs exhibited similar rounded or spindle shapes. No significant difference was observed in cell viability between the transduced and the control CPCs at MOIs of 50 or below; however, there was a reduction in transduced CPC viability at a MOI of 100 (66.2±9.5%) compared to the control (82.8±10.3%, p<0.05). The migration potential and capillary tube length of cells were not adversely affected by the transduction process. 4 weeks after transduction, 80.3±8.4% of the sorted cells continued to express RFP.
Conclusions: Primary human CPCs transduced with a lentiviral vector show stable expression of reporter genes. The TFR gene approach can be developed for tracking transplanted CPCs, while preserving the cells’ morphology, viability, and function. This technique can be used for the development of reporter gene PET imaging, to monitor the fate of transplanted cells longitudinally and quantitatively, and thereby help elucidate the mechanisms and effectiveness of cell-based therapies.
- © 2011 by American Heart Association, Inc.