Year 3 + NCE
The objective of our grant has been to identify a minimal set of immunomodulatory genes that can protect embryonic (ES) and induced pluripotent stem (iPS) cells, and their derivatives, from allograft rejection. To meet this objective we have developed a rapid gene transfer and analysis platform using engineered transposons, with which we have overexpressed individual and multiple genes in murine cells. Upon adoptive transfer into immunogenetically mismatched mice, we have determined the effect of these genes on the survival of the mismatched cells over time using in vivo bioluminescence imaging (BLI).
In Year 1, we found no protective effect from four immunomodulatory murine genes transferred individually (IL-4, TGF-β1, SDF1, and IDO-1) into mismatched cells. In Year 02, we tested two-fold gene combinations of an enlarged set of genes, i.e. IL-4, IL-10, TGF-β1, IL-1βRA, SDF1, CCL21, IDO-1, and CD47 transferred by two transposons with distinct antibiotic resistance markers into a bioluminescent fibroblast cell line that serves as a transplant surrogate. When fibroblast cells expressing dual gene combinations of all genes, or all but one gene, were injected s.c. into allogeneic mice, we observed, again, no significant effect on the survival of cells. Only fibroblasts transformed with control oncogenes remained for a prolonged bioluminescence, because of increased proliferation, but they were ultimately also rejected.
In Year 03 we have further expanded the number of cDNAs cloned in this system to twenty genes (adding Timp3, FasL, B7-H1, B7-H4, Galectin-1). In vivo, we tested a combination of 14 genes delivered by three transposons. This set prolonged the survival of transplanted fibroblasts significantly (p=0.0068), however, the cells were still rejected within two to three weeks. While this suggests an inhibitory effect on the innate immune response to mismatched grafts, more research is needed to arrive at findings that have an impact on the clinical application of mismatched stem cells and their derivatives.