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Modulation of mesenchymal stem cell immunogenicity through stable expression of human cytomegalovirus proteins
AuthorSoland, Melisa Andrea
AdvisorAlmeida-Porada, GraÃ§a D.
Agriculture, Veterinary and Rangeland Sciences
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Mesenchymal stem cells (MSC) have received considerable attention for their promising potential in cellular replacement therapies as a result of several characteristics they exhibit: 1) migration to sites of tissue injury; 2) engraftment into those damaged areas; 3) restoration of injured tissue through both differentiation into required cell types and trophic support of endogenous cellular pools; and 4) immunosuppressive capacity via modulation of both cellular and innate immune responses. Despite their documented immunomodulatory properties, however, MSC are not completely immune-inert and may elicit an immune response after in vivo transplantation in an allogeneic setting, leading to poor MSC allograft outcome and potential failure of cell-based regenerative therapies. In the present dissertation, we explored one strategy to potentially improve the immune evading abilities of MSC by exploiting the inherent immunomodulatory mechanisms of human cytomegalovirus (HCMV). To accomplish this, we genetically engineered MSC to express proteins coded for by the unique short (US) regions of HCMV: US2, US3, US6, and US11, which have been shown to be involved in down-regulation of HLA-I molecules and consequent inhibition of CTL proliferation. Study of immune responses to US-transduced MSC revealed a reduction in HLA-I surface expression, particularly for US6 and US11, and a resultant decrease in PBMNC stimulation and proliferation. Despite the decrease of HLA-I surface levels resulting from expression of US proteins on MSC, NK cell responses were also reduced due to surface upregulation of HLA-G1 molecule on MSC-US11 cells and CD155 protein on MSC-US6 cell line. Furthermore, study of US-transduced MSC ability to stimulate and activate the complement system cascade revealed that MSC expressing US2, US3 and US6 exhibited substantially elevated levels of the complement inhibitor, CD59. As a conclusion of the in vitro analysis of US-transduced MSC cell lines, cells expressing US6 or US11 proteins possessed significantly increased protection from CTL and NK killing, and, in the case of US6, an enhanced defense against complement attack. We thus concluded that these US-MSC lines should be less immunogenic and therefore exhibit increased engraftment potential in vivo. To test this experimentally, we transplanted MSC-US6 and MSC-US11 cell lines, together with an MSC-E control cell line into fetal sheep recipients and subsequently analyzed the levels of MSC engraftment within the liver and heart of the recipients. Analysis of fetal liver revealed that expression of either US6 or US11 significantly increased MSC engraftment capabilities compared to injection of the control cell line, but also decreased the ability of the engrafted US-MSC to differentiate into mature hepatocytes and hepatic stem/progenitor cells. In contrast, analysis of cardiac tissue indicated that expression of US6 or US11 proteins not only allowed engraftment within the heart, but also broadened the differentiative potential of these cells, enabling them to form mature cardiomyocytes and cardiac stem cells.In conclusion, expression of HCMV US6 or US11 proteins on MSC increased their ability to evade the immune system, both in vitro and in vivo. This increased immuno-invisibility led to enhanced engraftment capabilities within the liver and heart following transplantation in utero, and also broadened the differentiative capacity of these cells following engraftment within the heart. The observation that US-transduced MSC gave rise to not only phenotypically mature cells within the liver and heart, but also to stem/progenitor cells within these tissues suggests that MSC transduced to express HCMV US proteins could represent a clinically valuable source of cells for replenishing/repairing damaged/diseased cells within these organs.