Successful survival of large volume engineered tissues depends on the development of a vasculature to support the metabolic demands of donor tissue in vivo. Pancreatic islet transplantation is a cell therapy procedure to treat Type 1 diabetes that can potentially benefit from such a vascularization strategy. The treatment is limited as the majority of transplanted islets (60%) fail to engraft due to insufficient revascularization in the host(1, 2). Modular tissue engineering is a means of designing large volume functional tissues using micron sized tissues with an intrinsic vascularization. In this thesis, we explored the potential of endothelialized modules to drive vascularization in vivo and promote islet engraftment. Human endothelial cells (EC) covered modules were transplanted in the omental pouch of athymic rats and human EC formed vessels near implanted modules until 7 days when host macrophages were depleted. Rat endothelial cells covered modules were similarly transplanted in the omental pouch of allogeneic rats with and without immunosuppressants. When the drugs were administered, endothelialized modules significantly increased the vessel density. Moreover, donor GFP labelled EC formed vessels that integrated with the host vasculature and were perfusable until 60 days; this key result demonstrate for the first time that unmodified primary endothelial cells form stable vessels in an allograft model. Transplantation of islets in such endothelialized modules significantly improved the vessel density around transplanted islets. Donor endothelial cells formed vessels near transplanted islets in allogeneic immunesuppressed recipients. Meanwhile, there was an increase in islet viability with transplantation of endothelialized modules in syngeneic recipients but this difference was not significant. In summary, endothelialized modules were effective in promoting stable vascularization and improving transplanted islet vascularisation. Future work should promote faster maturity of donor vessels and modulate the host immune and inflammatory responses to significantly improve transplanted islet engraftment.

Liver transplantation is the only life-saving treatment for patients with end-stage liver disease; however, organ availability is insufficient to meet demands. Steatotic livers are extended criteria donor (ECD) organs that could be used for transplantation if not for an increased susceptibility ischemia reperfusion injury (IRI). Heme oxygenase-1 is a gene, that when upregulated has be shown to reduce IRI in animal models of transplantation. Increasing HO-1 activity in steatotic livers by delivery of a functional cell-penetrating HO-1 protein (through the use of cell-penetrating peptides) may provide protection against IRI, making these organs useful for transplantation. The purpose of this thesis was the generation and testing of a cell-penetrating HO-1 protein. HO-1 and EGFP gene sequences were cloned into the pET-28B(+) vector in frame with a CPP or TAT sequence. Resulting plasmids were cloned into E. coli, and protein expression was induced using IPTG. Proteins were purified using Ni-NTA affinity chromatography under denaturing and non-denaturing conditions. Non-denatured proteins were tested for HO-1 activity and the ability of both denatured and non-denatured proteins to transduce cells in vitro was tested by fluorescence microscopy. The cell-penetrating ability of nondenatured proteins was further tested in J774, HepG2 and HUVEC cells using immunofluorescence. Five HO-1 and two EGFP cell-penetrating proteins were generated expressed and purified successfully. Purified non-denatured HO-1 retains its enzymatic activity. Non-denatured CPP-EGFP and CPP-HO1 penetrated cells more effectively than their denatured counterparts. CPP-EGFP and CPP-HO1 proteins are able to penetrate multiple cell types in vitro. Successful generation and testing of a cell-penetrating HO-1 protein, for use in an animal model of steatotic liver transplantation. This protein demonstrates promise for use as a potential therapeutic agent in the field of liver transplantation.

Cardiac progenitor cells (CPCs) are abundant in the embryonic heart and have hallmark features which include a rapid rate of cell division and the ability to differentiate into mature heart muscle cells (cardiomyocytes). Based on these features, CPCs are considered an attractive candidate cell type for transplantation therapies which aim to replenish the diseased heart muscle tissue (myocardium) with new muscle forming cells. A better understanding of how pharmacological drugs and endogenous hormones/signalling molecules modulate the balance between proliferation and differentiation of CPCs could be used to develop more effective cell based therapies for myocardial repair. Furthermore, this information could provide valuable new insight into molecular mechanisms regulating normal cardiogenesis during the embryonic period. The specific aims of the present study were to characterize the effects of the Ca2+ channel blocking drug nifedipine and the endogenous hormone/paracrine factor atrial natriuretic peptide (ANP) on CPC proliferation and differentiation. Results showed that primary cultured CPCs, isolated from the ventricles of embryonic day (E) 11.5 mouse embryos, underwent a reduction in cell cycle activity following exposure to nifedipine. Furthermore, systemic administration of nifedipine to adult mice receiving transplanted E11.5 ventricular cells (containing CPCs) was associated with smaller graft sizes compared to control animals that did not receive the drug. Results from the present study also demonstrated that ANP receptor mediated signalling systems are biologically active in E11.5 ventricular cells and have an antiproliferative effect on cultured E11.5 CPCs. Moreover, preliminary data provided evidence that genetic ablation of the ANP high affinity receptor (NPRA) may be associated with impaired development of the ventricular cardiac conduction system. Collectively, work from this thesis provides evidence that interactions between transplanted cells and pharmacological drugs could have a significant impact on the effectiveness of cell based therapies and that ANP signalling systems may play a critical role in cardiac ontogeny by regulating the balance between CPC proliferation and differentiation.

Human leukocyte antigen-G (HLA-G), a non-classical MHC I protein, plays an essential role in immune tolerance and is associated with a lower incidence of graft rejection and cardiac allograft vasculopathy (CAV). To examine the pattern of HLA-G expression post-transplantation we determined that HLA-G can be up-regulated in smooth muscle cells (SMCs) following exposure to everolimus. We also determined that HLA-G at 500 and 1000 ng/ml reduces SMC proliferation. In further studies, treatment with HLA-G inhibited TNFα-stimulated neutrophil adhesion to endothelial cells (ECs) at all concentrations tested (0.1-1 ng/ml), suggesting a role in inflammation. The expression of HLA-G is influenced by a polymorphism in the HLA-G gene. We sought to determine if the 14bp insertion/deletion polymorphism can predict the development of CAV. There was no association between this polymorphism and CAV; however, this study had a small number of patients; therefore further investigations are needed to confirm these findings.

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Cellular Cardiomyoplasty (cell therapy for myocardial regeneration) targets the basic pathophysiology of heart failure and represents a novel technique for augmenting the function of the failing heart. Previous studies have demonstrated massive mechanical losses in the first few minutes. Thus, efforts to reduce mechanical losses may prove more beneficial than those directed against biological losses alone. We believe that "Wash-out" into the disrupted blood vessels is responsible for these early losses. / In the first part of this study we hypothesized that by increasing the size of the injectate, the amount of immediate losses can be reduced achieving better retention. Using Alginate-poly-L-lysine-Alginate (APA) miscrocapsules of two different sizes (200mum&400mum) and comparing retention with bare microspheres (10mum) of similar size to MSCs, we demonstrated that immediate retention rate increased by four folds. The retention rate for group 1 (microspheres only) was 4.28+/-3.46% which was significantly lower than that for groups 2 (microspheres in 200mum microcapsules) at 16.45+/-12.66% and group 3 (microspheres in 400mum microcapsules) at 12.93+/-6.28% for Group (p<0.05). There was no difference between group 2 and 3. / In the second part, we investigated the potential of gradually increasing the cell load on functional improvement and engraftment using conventional intramuscular delivery. Five groups of rats received escalating doses of MSCs after surgically induced ischemia (gp1 no cells, gp2 0.5x 10 6, gp3 1.5x106, gp4 3x106,gp5 5x106 MSCs). At 7 weeks, we observed significant improvement in cardiac function in groups 3 to 5 compared to post-infarction baseline. This was not observed in groups 1 & 2. However, in groups 3 to 5, we observed no functional advantage for increasing the cell load beyond a minimal therapeutic dose. This is consistent with our hypothesis that small cells are washed out into the circulation. / We also showed the ability of Alginate-Poly-l-lysine-Alginate (APA) microcapsules to sustain the viability of encapsulated MSCs in-vitro. Finally, the ability of encapsulated MSCs to improve the function of the heart in-vivo was tested.

A qualitative phenomenological study using van Manen’s human science method was conducted to gain insight into the lived experience of patients and their family members transitioning through one hundred days post haematopoietic stem cell transplantation (HSCT). Three families between zero and five years post HSCT were recruited from a bone marrow transplant unit in central Canada. Multiple in-depth open-ended interviews and field notes were employed to arrive at a detailed description of the lived experience of patients and family members. Awakening from the cocoon emerged as the main essence of patient’s and family members’ experiences, supported by three themes: the disruptions, the chrysalis, and new beginnings. The results from this study provide evidence that the families viewed the HSCT in a positive perspective and highlight the importance of supporting families throughout the acute phase of transplantation.

The long-term success of transplantation is limited by the need for immunosuppression; thus, tolerance induction is an important therapeutic goal. A 16-day treatment with rapamycin in mice led to indefinite graft survival of fully mismatched cardiac allografts, whereas untreated hearts were rejected after 8-10 days. Specific tolerance was confirmed through subsequent skin grafts and in vitro lymphocyte assays that showed recipient mice remained immunocompetent towards 3rd party antigens but were impaired in responding to donor antigens. Mechanisms that account for this tolerant state were then investigated. Splenic CD8+CD44+ memory T-cells were reduced in tolerant mice but had increased frequencies of the CD62LLO population. CD4+CD25+Foxp3+ regulatory T-cells were increased in tolerant mice. Through multiplex PCR, 4 regulatory T-cell related genes were found up-regulated and 2 proinflammatory genes were down-regulated in accepted hearts. This expression pattern may serve as a putative biomarker of tolerance in patients undergoing transplantation.