Abstract
MAPC are non-hematopoietic stem cells with the capacity to form most, if not all, cell types of the body. To date, the observations of homing of the MAPC have been limited to post mortem analyses. As MAPC may be useful in cellular therapies, our goal was to map their biodistribution in live organisms. To determine the real-time organ-specific homing pattern of donor MAPC, MAPC (from BM of C57BL/6J-rosa26 mice) were co-nucleofected with cDNAs encoding the red fluorescent protein DsRed2 and luciferase, using the Sleeping Beauty (SB) transposon system. Non-viral gene transfer mediated by SB is potentially advantageous to viral gene transfer because transposons may be less immunogenic since no viral proteins are present, and they are relatively easy to produce. DsRed2 and luciferase genes were cloned into plasmid vectors containing the transposase recognition sequences flanking the reporter genes (pT/CAGGS-DsRed2; pT/CAGGS-Luciferase). MAPC (106) were co-nucleofected (Amaxa, setting T-20, buffer T) with 5mcg of each marker plasmid and the SB transposase plasmid (p/CMV-HSB2) at a 1:50 ratio. 19% of MAPC expressed DsRed2 7 days after nucleofection. The MAPC were FACS sorted (1 cell per well) for cells with the highest DsRed2 expression. All MAPC tested expressed both DsRed2 and luciferase, suggesting that co-nucleofection is an efficient means of delivery of two plasmids. Two transgenic MAPC clones selected for further analysis were confirmed to be euploid by cytogenetic analysis, and maintained differentiation potential into the three germ layers. To verify transgene integration by transposition, the genomic sites of transposon integration were determined using splinkerette PCR. In the genome of MAPC clone 1, DsRed2 transposed in two sites on chromosome 5. One integration site (5qA3) was in the 3′ untranslated region of activin receptor interacting protein 1 (Acvrinp1). In clone 2 DsRed2 transposed into a single site on chromosome 10, in an intron of a gene termed SHPRH, which encodes a putative protein with SNF2/helicase and PHD-finger domains. To investigate the real time kinetics of MAPC population after infusion, 5 x 106 DsRed2 and luciferase positive MAPC (clone 2) were infused via tail vein into 8-week-old Rag2/IL-2Rgc−/− mice (T-, B- and NK-immunodeficient mice were used as a recipient to minimize the likelihood that the host would reject donor MAPC). Using whole body imaging (Xenogen) we were able to follow the distribution of the luciferase-marked MAPC over a period of 10 weeks. In addition, using DsRed2 expression the donor MAPC-derived cells in whole lung and in lung cryosections were identified. In summary, we show for the first time stable gene expression in adult stem cells using Sleeping Beauty transposon mediated non-viral gene transfer. These results show that MAPC-based cellular therapies can be monitored in vivo and suggest that transposon-based technology may be an attractive alternative to viral based gene delivery and therapy.
317. Secreted Gaussia Luciferase Is a More Sensitive Reporter Than Firefly Luciferase for Non-Viral Gene Transfer to Airway Epithelium Ex Vivo and In Vivo
