The potential of stem cell therapies is extraordinary, however, successful implementation of such therapies will require an understanding of immune recognition of regenerated tissue, and identification of therapeutic agents that can modulate the immune system and prevent rejection. We have set out to identify the gene networks in mammalian cells that can protect immunologically mismatched embryonic (ES) and induced pluripotent stem (iPS) cells from graft rejection to advance efforts to induce operational tolerance towards transplanted cells and tissues.
Our rapid genetic analysis platform allows surveying the mouse genome in vivo by overexpressing or silencing single, multiple, or libraries of genes in stem cells and their derivatives, and analyzing their resulting in vivo phenotype, i.e. their sustained protection from immune rejection. For this, we stably transfer genes of interest (for positive selection), or regulatory RNAs (for negative selection), as well as a luciferase-expressing cassette, into ES cells or their derivatives, or into a tumor cell line that serves as surrogate transplants. The luciferase gene provides an immediate readout for following the modified cells after transplantation into mismatched recipient mice as the cells’ survival can be assessed in real time using in vivo bioluminescence imaging (BLI).
The first set of immunomodulatory genes we have now tested contains key immunomodulators: interleukin 4, Transforming Growth Factor beta 1, Stromal Derived Factor 1, and Indoleamine Deoxygenase -1. We have transferred murine cDNAs encoding these proteins into a bioluminescent fibrosarcoma cell line, which we use as a surrogate for differentiated stem cell derivatives. When the modified fibrosarcoma cells overexpressing one of each of these genes were injected into the muscle of allogeneic mice, we observed a small extension of the survival time in cells expressing either TGF-β or murine IL-4. We conclude that while this is promising, in order to achieve long-term protection from allograft rejection a combination of several genes will be necessary.
In Year 02 we will therefore a) expand the number of genes to be analyzed in this system, and test gene combinations. In Year 03, we will test libraries of immunomodulatory genes, and regulatory RNAs to arrive at a minimal set of immunomodulatory genes that can successfully protect stem cells and their derivatives from immune rejection over extended periods of time.