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The successful and safe application of mesenchymal stem cells (MSCs) as therapies for a wide variety of indications requires an accurate means of monitoring their differentiation into target tissue after transplantation in vivo. Here we describe a novel suicide gene vector, GS-E6, which can enable specific incorporation of the neomycin phosphotransferase II (nptII) gene into the genome of MSCs and pluripotent stem cells, without altering their stem cell properties or altering their normal differentiation potential. We have validated the efficiency and specificity of the gene transfer system in vitro and in vivo by transducing murine embryonic stem cells, embryoid bodies, and neural stem cells with the suicide gene and by monitoring nptII expression and neomycin resistance in vitro and in vivo. We were able to show that the vector is able to transduce MSCs and pluripotent stem cells in vitro with efficiencies in the range of 5-10%, respectively. To evaluate the vector’s potential for genetic marking of cells during in vitro differentiation or engraftment in vivo, we sorted fluorescent-activated cell sorter (FACS)-enriched cultures of MSCs with or without suicide gene transduction and transplanted them into both male and female immune-deficient mice. We showed that the cells tolerated transplantation at efficiencies as low as 50-70%, which can be achieved by vector delivery into placenta or bone marrow. Vector-transduced cells displayed the neomycin-resistant phenotype, which could be detected over time during long-term cultures or in vivo. Importantly, the frequency of cochlear progenitor cell and endolymphatic sac cell differentiation in the vector-transduced cultures was comparable to that of non-transduced controls. We conclude that GS-E6 is a safe and effective suicide gene vector for stem cell genetic marking and that it may be applied to improve the efficiency of cell-based therapies.The present invention relates to the field of surgery and more particularly to a fluid drainage system for use during the surgical repair of heart valve defects.
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