Engineering Embryonic Stem Cell Allografts for Operational Tolerance
Stem cells, like all transplants not derived from an identical twin, are subject to scrutiny by the immune system and, without medical interventions that suppress the immune system, are usually killed after transplantation. However, rare exceptions to this rule exist because a small fraction of transplant patients has been able to maintain their transplant in the absence of immunosuppressive drug therapy and developed "operational tolerance" towards the foreign graft. Our team has extensively studied these patients and identified a number of genes that are characteristically overexpressed or silenced in these patients. Other instances of tolerance towards foreign cells also occur naturally, e.g. during pregnancy. While numerous genes that correlate with operational tolerance are known, it is less clear whether they actively contribute to tolerance and how they compare in their effectiveness. We will therefore transfer this collection of genes, one-by-one or as combinations, into mouse embryonic stem cells using gene therapy methods and identify those genes that can best protect the cells from rejection by the immune system.
To accurately monitor the survival of transplanted stem cells in mice over time we will use in vivo bioluminescence imaging (BLI). For this, we label the stem cells with luciferase, a protein from the firefly that emits light. These bioluminescent stem cells are transplanted into recipient mice (whose background luminescence is negligible) where the cells can be repeatedly and non-invasively visualized with a highly sensitive camera system. Thus, the cellular survival, growth and migration can be assessed over time and under various conditions, and we can determine whether the introduced genes affect the survival of stem cell transplants positively or negatively, and how they compare and, hopefully, cooperate.
Our preliminary data show that we can detect differences in survival of such engineered cells. This indicates that the proposed studies will succeed in prolonging the survival of mouse stem cell transplants, and that these studies are greatly accelerated by the use of BLI and of gene transfer methods developed over the past several years in our and other laboratories. The potential impact of this proposal is substantial, in that successful completion of the specific aims will both be an important step towards tissue replacement and regeneration using stem cells, and the first demonstration of a multiplexed gene screen in mice. If the genes found to modulate the immune system locally and to protect stem cell transplants in mice can be translated to the bedside, e.g. developed into small molecule drugs that are safe to administer, there is promise that we can reduce the untoward effects of systemically delivered drugs, and extend the lifespan of stem cell and organ transplants without the need for chronic immunosuppression. This would have a substantial impact of the management of a variety of medical conditions.
The California Institute of Regenerative Medicine is seeking to discover new therapeutic approaches that use stem cells for a wide range of diseases and to critically evaluate these for the citizens of California. Unfortunately, however, transplanted stem cells derived from genetically unrelated donors are recognized as foreign by the recipient's immune system and usually destroyed within a month. Currently, the only treatment option for stem cell and organ transplants consists of immunosuppressive drug therapy that is costly and due to its systemic and nonspecific effects carries substantial risks of infection and cancer.
Tolerance to foreign tissues, nevertheless, can develop naturally, for example in women during pregnancy where the partially mismatched fetus is tolerated, and in rare patients who are fortunate enough to maintain their mismatched grafts in the absence of immunosuppressive drug therapy. It is from these instances, which our group has studied extensively, that we will take our clues to develop a comprehensive understanding of transplant tolerance and its genetic basis. Because without this and without an effective means of transferring this tolerance to stem cells, the tremendous potential of the new stem cell therapies may not be realized. Even autologous stem cell therapies, in which patients receive induced pluripotent stem cells (iPSCs), which are derived from their own cells and therefore not mismatched, if they were to succeed otherwise, will not benefit patients with autoimmune disorders, like type 1 diabetes, who will still react to and destroy any transplanted tissue. Therefore, alternatives that modulate the immune response at the site of the engrafted cells or tissues are clearly needed.
Our plan is to select out of the mouse genome the quintessential set of genes whose up- or down-regulation is necessary and sufficient for the long-term protection of stem cell transplants in genetically mismatched mice. In this innovative and comprehensive genetic approach, we will modify the expression of individual genes or groups of genes in mouse stem cells, and observe the cells’ fate after transplantation with powerful imaging technologies that are non-invasive and highly sensitive. Thus, the effect of each gene on the survival of a stem cell transplant can be easily measured and comparatively evaluated, as well as each gene be examined for cooperativity with other genes in the induction of tolerance.
Acquiring this genetic knowledge and translating it into effective stem cell therapies for human patients will be the critical steps in a continuum of research that will clearly benefit citizens in California and elsewhere. Because making stem cell transplantation more efficient and better tolerated will not only advance the fields of stem cell biology and medicine but also that of organ transplantation in general so that less suffering and costs will be incurred in the future in terms of lost lives and funds.
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.
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.
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.