Abstract

We hypothesized that following irradiation the esophageal microenvironment would have increased reactive oxygen species (ROS) production and that antioxidant gene therapy would decrease ROS resulting in increased engraftment of esophageal stem cells. Decreased ROS production following irradiation has been shown to result from expression of Manganese Superoxide Dismutase (MnSOD) transgene product delivered by plasmid/liposome complexes. We tested whether increased expression of MnSOD following intraesophageal administration of Manganese Superoxide Dismutase Plasmid/Liposomes (MnSOD-PL) increased engraftment of esophageal stem cells. Female C57BL/6NHsd mice which had received MnSOD-PL (100 μg plasmid DNA) intraesophageally 24 hr earlier were irradiated along with control mice to 29 Gy to the esophagus. The mice were injected intravenously with bone marrow from male ROSA (Lac-Z, G418r transgenic mice) five days later (time of greatest esophageal basal cell apoptosis). Fourteen days later the mice (first generation) were sacrificed, esophagus removed, single cell suspensions prepared, stained with Hoescht dye and sorted by flow cytometer into side population (SP) and nonside population (NSP) cells. Half of the SP and NSP cells isolated from the MnSOD-PL treated mice were injected into a second generation of recipient MnSOD-PL treated as well as control mice. The remaining cells were grown in either methylcellulose or in 96 well plates in G418. Second generation recipient mice were sacrificed 14 days after transplant and the SP and NSP cells isolated and grown in either methylcellulose or in 96 well plates as described above. Tissue culture grown G418r colonies were stained for donor (Lac-Z+) cells. In first generation recipient mice, those pre-treated with MnSOD-PL had increased expression of Lac-Z+ SP and NSP cells per esophagus (38.4 ± 4.2% and 21.8 ± 5.5%, respectively) compared to the control irradiated mice (21.4 ± 4.4%, p=0.0105, and 5.2 ± 2.4%, p=0.0277, respectively). Second generation MnSOD-PL treated recipient mice, transplanted with esophageal SP cells from MnSOD-PL treated mice had more Lac-Z+ SP cells per esophagus (47.9 + 3.5%) than that detected in mice injected with NSP cells from the same donors (22.3 ± 2.4%, p < 0.0001), control irradiation only donor SP cells (22.3 ± 2.0, p = 0.0001) or control irradiation only donor NSP cells (9.6 ± 2.5%, p < 0.0001). To further demonstrate that MnSOD-PL pretreatment resulted in better engraftment of donor cells, the first and second generation sorted cells were analyzed for the presence of the Y chromosome. In the first generation, MnSOD-PL pretreatment resulted in an increased percent of Y+ SP and NSP cells (23.9 ± 4.6, p = 0.0318, and 11.4 ± 4.0, respectively) compared to the control SP and NSP (10.5 ± 3.5 or 7.9 ± 3.2, respectively). In the second generation recipient mice, MnSOD-PL pre-treatment prior to first generation MnSOD-PL SP cells resulted in more Y+ cells in the SP fraction compared to control irradiated recipients (15.2 ± 4.4 or 4.5 ± 2.3, respectively, p = 0.0467). Therefore, swallowed MnSOD-PL pretreatment improves esophageal engraftment of serially transferable marrow progenitors of esophageal stem cells.

Author notes

Disclosure: No relevant conflicts of interest to declare.