Our objective is to protect embryonic (ES) and induced pluripotent stem (iPS) cells, and their derivatives, from allograft rejection by genetic modification. For this, we are seeking to identify a minimal set of immunomodulatory genes that can protect such cells after allotransplantation. Our search employs a rapid gene transfer and in vivo analysis platform, with which we are overexpressing, or silencing, single, multiple, or libraries of genes in stem cells and other cells to be transplantatied. Upon transfer of the cells into immunogenetically mismatched mice, we can determine the cells’ in vivo phenotype by in vivo bioluminescence imaging (BLI), i.e. whether or not they are protected from immune rejection for a sustained period of time.
In Year 01, we had tested four immunomodulatory murine genes individually (IL-4, TGF-β1, SDF1, and IDO-1), and found no significant protective effect. In Year 02, we have examined combinations of two genes per cell using a set of now eight genes, i.e. adding IL-10, IL-1βRA, CCL21, and CD47. For this, we have cloned their cDNAs into two transposon vectors with distinct antibiotic resistance markers. These vectors were transferred into NIH3T3 fibroblast cells, which serve as a transplant surrogate, and gene expression was confirmed by qRT-PCR. When the modified fibroblast cells expressing dual gene combinations of all eight genes, or all but one gene, were injected subcutaneously into allogeneic mice, we observed one gene, IL-4, to have a small, but statistically significant effect on the survival of cells. Fibroblasts transformed with control oncogenes exhibited prolonged bioluminescence due to their initially increased proliferation, however, ultimately, they too were rejected.
In Year 03 we will advance this screen to higher complexity, expand the number of cDNAs to be analyzed, and test three, and four gene combinations. At the same time, to identify particular immune cell subpopulations critical for rejection we will challenge partially immunodeficient mice with cells containing two and three gene combinations. We will also test libraries of shRNAs and/or miRNAs, and a knockdown transposon for a loss-of-function gene screen. Finally, once we arrive at a minimal set of immunoprotective genes we will analyze their effect of on cellular gene expression by microarray analysis and luminex, and their effect on immune cells in the microenvironment by flow cytometry.