The primary aim of the thesis is to contribute to demonstrating how established and emerging science in the regenerative medicine (RM) domain can be translated into profitable commercial practice, and generate clinically- and cost-effective therapies. It achieves this by exploring and assessing underlying economics, including investment readiness and economic assessment, exploring regulatory and reimbursement frameworks, developing stem cell culture systems and assessing fit with clinical practice. The thesis is the first public domain wide-ranging analysis of business trends in the production, manufacturing and supply segments of the RM industry. It analyses the clinical potential of the domain as well as the translational and commercial challenges facing the industry. The industry is at a turning point as big pharmaceutical companies engage with RM in order to explore technologies as potential therapeutics and discovery tools. This unlocks the industry by confirming an exit path for RM based small- and medium-sized enterprises. Translation has come to be recognised as a core issue in the overall space and translation of regenerative therapies into the clinic is presently challenging, high-risk and expensive. This research addresses the question what are the mechanisms required to enable translation of emerging scientific knowledge into commercially viable clinical RM products? These mechanisms are particularly important as their creation involves and requires major investment decisions, which can determine the success or failure of RM developments and indeed of the companies concerned. The lack of well-established business models and the complexity of the domain suggested a conceptual approach drawing upon relevant literature from product and process development, applied business and revenue models, technological evolution and capital market ingenuity. The research was carried out in two phases. The first phase was concerned with identification of key challenges and mapping the overall industry emergence including emergence of related regulations to provide a context and framework for understanding the domain. Based on the emergence mapping a timeline of key parallel factors was identified, and their inherent connections explored to identify transforming events affecting and influencing multiple factors on the journey to clinical success within a business environment. This creates the reference model. The second phase was concerned with manufacturing a stem cell based therapeutic and applying health economic principles to determine available headroom for investment, cost of goods and return on investment, taking hearing disorders as a case exemplar, and exploring the behaviour of the net present value curve to identify key parameters affecting the economic positioning of this novel regime. A key output of the research is the investment readiness reference model. It integrates key RM business issues against reducing uncertainty and increasing value. The model argues that the complex nature of RM products means that the issues affecting industry emergence and development go well beyond the primarily scientific and technological concerns on which much current research focuses. The performance of RM firms ultimately hinges upon the successful clinical application of their developed products, the key step for creating and realising value, and their ability to deal with the fundamental business issues specific to the area. The framework deals with these business issues, which are investment & technology readiness, business models, organisational challenges, public policy and industry emergence. This thesis explores ideas that may bridge the chasm between the promise and reality of RM i.e. mechanisms to enable late stage translation of RM products. It links technological capability and business models for firms in the domain. Furthermore, it offers a unique perspective on the nature and characteristics of investment readiness and financial assessment, specifically identifying key parameters affecting economic positioning. The key contributions are therefore: New insights into the key challenges involved in realising the commercial potential of cell based therapeutics. Technology road mapping to link fundamental enabling technological capability for developing RM products with robust business plans integrating strategy, technology development and the regulatory and reimbursement framework. A generic investment readiness reference model generated from the enabling technology, value and supply chain structures to identify key indicators and characteristics of industry readiness. A novel experimental programme demonstrating expansion, maintenance and differentiation of human embryonic stem cells by manual and automated methods. New insights into economic positioning by mapping net present value, and economic analysis by estimating available headroom, cost of goods and return on investment for a putative hearing therapeutic.

<p>Endothelial dysfunction is the predominant pathophysiological state prior to the onset of atherosclerosis. Currently, treatments for endothelial dysfunction are evaluated in vitro using two-dimensional (2D) cell culture assays or in vivo animal models. Microphysiological systems are small-scale three-dimensional (3D) tissue models that recapitulate the native tissue structure and function. An ideal microphysiological system is comprised of human cells embedded within a 3D matrix introduced to physiological fluid perfusion. Immune challenge in the form of cytokines or immune cells further recapitulates the native microenvironment.</p><p>A vascular microphysiological system was developed from a small-diameter tissue engineered blood vessel (TEBV) in a perfusion culture circuit. TEBVs were created from collagen gels embedded with human neonatal dermal fibroblasts and plastically compressed to yield collagen constructs with high fiber densities. TEBVs are rapidly producible and can be directly introduced into perfusion culture immediately after fabrication. Endothelium-independent vasoconstriction in response to phenylephrine and endothelium-dependent vasodilation in response to acetylcholine were used to analyze the health and function of the endothelium non-destructively over time.</p><p>Endothelial dysfunction was induced through introduction of the pro-inflammatory cytokine tumor necrosis factor – α (TNF-α). Late-outgrowth endothelial progenitor cells derived from the peripheral blood of coronary artery disease patients (CAD EPCs) were evaluated as a potential endothelial source for autologous implantation in both a two-dimensional (2D) direct co-culture model as well as a 3D model as an endothelial source for a tissue engineered blood vessel. CAD EPCs demonstrated similar adhesive properties to a confluent, quiescent layer of smooth muscle compared to human aortic endothelial cells. Within the TEBV system, CAD EPCs demonstrated the capacity to elicit endothelium-dependent vasodilation. CAD EPCs were compared to adult EPCs from young, healthy volunteers. Both CAD EPCs and healthy volunteer EPCs demonstrated similar endothelium-dependent vasoactivity in response to acetylcholine; however, in response to TNF-α, CAD EPCs demonstrated a reduced response to phenylephrine at high doses.</p><p>The treatment of TEBVs with statins was explored to model the drug response within the system. TEBVs were treated with lovastatin, atorvastatin, and rosuvastatin for three days prior to exposure to TNF-α. In all three cases, statins prevented TNF-α induced vasoconstriction in response to acetylcholine within the TEBVs, compared to TEBVs not treated with statins. Overall, this work characterizes and validates a novel vascular microphysiological system that can be tested in situ in order to determine the effects of various patient populations and drugs on endothelial health and function under healthy and inflammatory conditions.</p> / Dissertation

This dissertation is composed of two projects dedicated to the development of techniques and technologies for improving the quality of life for patients in both clinical and resource-limited settings. The purpose of the first project was to design a rapid diagnostic device to screen whole blood samples for the presence of infectious agents. Point-of-care (PoC) technologies are becoming increasingly important for the detection of infectious agents in resource-limited settings (RSLs) where state-of-the-art blood screening practices are not feasible for implementation. For this project, a rapid diagnostic device was developed to directly detect pathogen content within freshly drawn whole blood samples using a ligand-binding assay format. The assay is completely self-contained within a hermetically sealed device to minimize operational complexity and ensure operator safety. The diagnostic device is capable of processing complex sample matrices by selectively capturing, concentrating, and labeling infectious agents upon functionalized surfaces. Following sample processing, the assay is optically interrogated with a fluorescence-based reader to provide rapid feedback regarding sample purity. Designs of the rapid diagnostic platform evolved over several prototype generations corresponding to project milestones emphasizing ergonomic performance, military specification testing for environmental resilience, and manufacture to yield production-grade devices for future diagnostic performance data collection. The goal of the regenerative therapy-based portion of this research was to develop a novel technique for the selective enrichment of cells demonstrating enhanced regenerative capacity in tissue-extracted cell samples. Adherent cell cultures of stromal vascular fractions (SVFs) extracted from adipose tissues were exposed to nutrient deficient conditions' eliciting a bimodal cellular response between two dissimilar cell culture subpopulations. The regenerative capacity of these two distinct subpopulations was evaluated by assessing their characteristic morphology, metabolic activity, and ability to undergo multilineage differentiation. The SVF subpopulation which demonstrated sensitivity to the nutrient deficient conditions expressed typical morphological expression of adherent cell cultures, elevated metabolic activity, and the ability to differentiate along adipogenic, chondrogenic, and osteogenic lineages. The SVF subpopulation which demonstrated resistance to the nutrient deficient conditions, however, expressed atypical morphologies, impaired metabolic activity, and did not survive culture with differentiation growth media. Based on the data, the 'treatment-sensitive' SVF subpopulation demonstrated a greater regenerative capacity than the‘treatment-resistant' subpopulation. Furthermore, the treatment-resistant subpopulation of the SVF may be representative of the damaged, senescent, and otherwise less-functional cells that comprise a significant portion of tissue-extracted cell samples and pose a significant risk to therapeutic efficacy and reproducibility. Ultimately, this expedient and inexpensive bioprocessing technique may serve to improve cell-based regenerative therapies by eliminating undesirable cells from culture.

To repair, replace, or regenerate damaged or diseased tissue has been a long-standing, albeit elusive, goal in medical research. Here, we characterize patient-derivable mesenchymal stem cell types, termed adipose-derived stem cells (ASCs). These cells, which can be derived from liposuction fat and lipoaspirate saline, are sources for patient-derivable extracellular matrix (ECM), fibrinogen (Fg) and adipose tissue extracellular matrix, and may prove useful for synthesizing new bone tissue analogues in vitro. Traditionally and rapidly isolated ASCs were thoroughly characterized as multipotent, having osteogenic, adipogenic, and chondrogenic differentiation potential, and they exhibited comparable proliferative lifespans. These ASCs also shared an indistinguishable immunophenotype when compared to bone marrow-derived mesenchymal stem cells, suggesting that these cells are an excellent source for bone following tissue engineering experimentation. In order to synthesize bone ex-vivo, electrospun scaffolds of fibrinogen (Fg), polydioxanone (PDO), and Fg:PDO blends were seeded with early passage ASCs, fibroblasts, or osteosarcoma cells and were maintained for 21 days in osteogenic or regular growth media. Constructs were analyzed both histologically and molecularly for evidence of osteoblastogenesis. Using SEM, the appearance of regular, mineralized-appearing structures were found in osteogenic-induced ASC seeded scaffolds beyond 14 days, only in the scaffolds containing Fg. Further, at 21 days of culture, Fg scaffolds with ASCs in osteogenic media became hard and brittle. Robust new collagen synthesis and matrix remodeling were observed on all Fg scaffolds, the levels of which were elevated over time. Pronounced mineralization was found throughout bone-induced ASC scaffolds, while control scaffolds (BJ foreskin fibroblasts) showed no mineral deposition (although they did demonstrate excellent cellularity). Analysis of gene expression (qRT-PCR) indicated that electrospun Fg supported osteoblastogenesis through the upregulation of alkaline phosphatase and osteocalcin gene expression. To confirm our gene expression results, osteogenic-induced ASCs on Fg scaffolds were also shown to secrete osteocalcin in the extracellular matrix, a key marker in osteoblastogenesis. Thus, electrospun Fg is an excellent material for ASC growth, proliferation, and osteogenic differentiation, providing an ideal system for furthering basic bone model-based research and for advancing regenerative medicine. In addition to establishing Fg as a source of scaffolding, we developed and characterized a novel method for isolating and subsequently electrospinning adipose tissue matrix. Because adipose ECM contains many primordial matrix proteins important for embryonic development and regeneration (such as laminin, type IV collagen, and fibronectin), adipose ECM may prove to be an autologous tissue engineering matrix and stem cell culture substrate. We show here that adipose tissue ECM can, in fact, be electrospun into a nanofiberous mesh, histologically shown to contain connective tissue, collagens, elastic fibers/elastin, proteoglycans, and glycoproteins in the newly synthesized matrix. We also show that this novel electrospun adipose tissue scaffold is capable of supporting stem cell growth. Taken together, experiments using ASCs cultured on extracellular matrices of electrospun Fg or adipose ECM present an excellent framework for future advances in regenerative medicine therapeutics and research.

Células-tronco humanas derivadas da polpa dental (hDPSCs) e células-tronco humanas derivadas de tecido adiposo (AhSCs) são células multipotentes capazes de diferenciação osteogênica in vitro e in vivo, e promissoras fontes de células para a engenharia de tecido ósseo, dada a sua facilidade de expansão, isolamento e diferenciação. É de grande interesse compreender qual é o melhor tipo celular para diferenciação osteogênica, assim, o objetivo deste estudo foi comparar o potencial de diferenciação osteogênica in vitro e in vivo entre hDPSCs e hASCs. Foram isoladas e estabelecidas seis populações de células-tronco de hDPSCs (entre 7-12 anos) e seis da hASC (de indivíduos com idade entre 30-49 anos). Após a indução in vitro, a diferenciação osteogênica foi comprovado através das colorações de fosfatase alcalina (9 dias) e vermelho de alizarina (14 e 21 dias). A quantificação da mineralização da matriz após 21 dias de diferenciação osteogênica revelou 2,24 mais ossificação das hDPSCs em relação às hASCs. Para realizar o experimento in vivo, foram triados seis biomateriais para verificar qual melhor biomaterial para o nosso modelo, defeito crítico em calvária de Ratos Wistar não imunossuprimidos, com três amostras de hDPSCs. Após 45 dias, CellCeram(TM) exibiu a melhor neoformação óssea in vivo, e foi selecionado para comparar os potenciais osteogênicos in vivo entre hDPSCs e hASCs. Células (10e6) foram associadas a discos de 4,5 mm CellCeram(TM), grupo controle foi realizado através do transplante do biomaterial livre de células. Neoformação óssea foi mensurada 45 dias após a cirurgia através da coloração histológica de hematoxilina / eosina. A formação óssea total foi quantificada através da análise de imagens de todas as ilhas de ossificação. A associação entre hDPSCs e CellCeram(TM) promoveu 7,24 vezes mais neoformação óssea quando comparado com a associação entre esse mesmo material e hASCs (p <0,0001). A utilização de células-tronco adultas para regeneração óssea é uma ótima abordagem para uso terapêutico, e calcular ou predizer o potencial osteogênico das células utilizadas é extremamente importante e necessário para futura aplicação em novas estratégias de bioengenharia de tecido ósseo / Human dental pulp stem cells (hDPSCs) and human adipose-derived stem cells (AhSCs) are multipotent cells capable of undergoing osteogenesis in vitro and in vivo, and promising cell-source populations for bone tissue engineering given their easiness of isolation, expansion and differentiation. It is of great interest to understand which is the best cell type for osteogenic differentiation, thus the aim of this study is to compare the in vitro and the in vivo osteogenic differentiation potentials between DPSCs and ASCs. We isolated six stem cell populations from DPSCs (aged 7-12 years) and six from ASCs (from subjects aged 30-49 years) and cell culture was established. After in vitro induction the populations were able to undergo osteogenic differentiation, as evidenced by alkaline phosphatase (9 days) and alizarin red S (14 and 21 days) stainings. Quantification of matrix mineralization after 21 days of osteogenic differentiation revealed an enhancement of 2.24-fold increase between hDPSCs and hASCs differentiation. To perform the in vivo experiment, we promoted a screening of six scaffolds to find out which would be best scaffold to our model, a calvarial critical-sized defect in Wistar non-immunosuppressed rats, with three different culture samples of hDPSCs. After 45 days, CellCeram(TM) displayed the best in vivo bone neoformation, and was used to compare the in vivo osteogenic potentials between hDPSCs and hASCs. Cells (10e6) were associated to 4.5 mm CellCeram(TM) discs, and control groups were performed transplanting the biomaterial free of cells. Bone healing was measured through histological hematoxylin/eosin staining 45 days after surgery. Newly formed bone was also evaluated by total bone island surface quantification through image analysis. The association between hDPSCs and CellCeram(TM) induced a mean of 7.24 times more bone formation when compared to the association between this same material and hASCs (p<0.0001). The use of adult stem cells for bone regeneration is a robust therapeutic option, and calculate or predicts the osteogenic potential of the cell used are extremely important and necessary to future application, and translation to new strategies in bone tissue engineering

Nos últimos anos, as células-tronco, devido a sua capacidade de originar diversos tecidos corporais e pelo poder de auto-renovação, impulsionaram os estudos de engenharia tecidual, sobretudo em medicina regenerativa. Nesse aspecto, o tecido adiposo vem se mostrando como fonte ideal para obtenção de tais células, devido à facilidade de captação, à baixa morbidade associada ao procedimento e ao elevado rendimento celular. Com o objetivo de estabelecer um modelo experimental versátil e que satisfizesse várias áreas de interesse, propôs-se o coelho Oryctolagus cuniculus como fonte de tecido adiposo. Esse animal apresenta bolsa adiposa interescapular com peso médio de 17,2g, o que corresponde a cerca de 6,6 g/Kg em machos adultos (peso corporal médio de 2590g). A coleta do material foi por meio de lipoaspiração a seco, com cânula de 3,5mm; levou-se em média, 11 minutos para o procedimento, obtendo-se aproximadamente 10 ml de gordura. Após o processamento pela técnica enzimática, em cada mililitro de gordura encontrou-se em média 1x105 células-tronco. O estudo constatou ainda que, por meio da criopreservação em nitrogênio líquido, as células mantinham suas características citométricas após períodos de congelamento que variaram de uma semana a 13 meses. As células apresentaram características de sua indiferenciação, como a expressão dos marcadores de superfície: CD90, 80,6%; HLA-DR, 2,8% e caspase 3, 10,5%. A análise do ciclo celular com 100% de confluência mostrou que 70,8% das células encontravamse quiescentes; 22,1% apoptóticas. As células com alta capacidade replicativa, que corresponde à fase S do ciclo celular, 1,4% e 0,9% encontravam-se em replicação, mostrando que as células-tronco do tecido adiposo, em cultura, não apresentam uma proliferação descontrolada, tendendo a se estabilizar, principalmente quando atingem confluência máxima em monocamada. Todas essas vantagens fazem com que o modelo proposto possa ser facilmente reprodutível, contribuindo para o estudo das células-tronco do tecido adiposo. / In the latest years, the study on tissue engineering, mainly in the area of regenerative medicine, has advanced because the medical community is highly interested in stem cells. This is due to both the potential of these cells to originate any body tissue and their power of self-renewal. Adipose tissue has been used as an ideal source of such cells, due to the simplicity of their collection, high cellular yield, and low morbidity associated with the procedure. In order to establish a versatile experimental model, which could meet the needs of researchers from various areas, the rabbit Oryctolagus cuniculus was proposed as a source of adipose tissue. This animal has an adipose pad in the interscapular region with an average weight of 17.2g, which corresponds to about 6.6g of fat material per kilogram of an adult male animal (mean body weight = 2.6kg). The material was collected by means of a liposuction procedure. Using a 3.5- mm diameter tube, a volume of nearly 10ml of fat material was obtained in a mean time of 11min. After processing the fat tissue by enzymatic technique, about 1x105 stem cells were found per milliliter of fat material. Using cryopreservation of the cells by freezing them in liquid nitrogen, it was observed that the cytometric characteristics were maintained after a period of time ranging from 1 week to 13 months. The cells presented evident characteristics of undifferentiation, such as expression of the surface markers CD90, HLA-DR, and Caspase-3 (80.6, 2.8, and 10.5 %, respectively). Analysis of the cellular cycle with 100% confluence allowed us to show that 70.8% of the cells were quiescent, 22.1% were apoptotic, 1.4% had high replication capacity (phase S of the cellular cycle) and 0.9% were already in replication (phase G2/M), indicating that stem cells from adipose tissue did not show uncontrolled proliferation, tending to stabilize, mainly when they reach maximal confluence in monolayer. These advantages make this model easily reproducible, facilitating the study of adipose tissue stem cells.

Pre-clinical studies are a necessary step in the process of material and drug testing. For this, high-throughput monolayer cell cultures are conducted followed by in vivo animal experiments. However, animal use is ethically questionable and in many cases yields misleading results. In vitro three dimensional (3D) tissue engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer cultures and are less ethically questionable than animal models. Therefore, an in vitro biomimetic musculoskeletal junction (MSKjct) is required as a more relevant pre-clinical testbed. This thesis describes the steps taken to co-culture 3D TE skeletal muscle and bone models as a material testbed and towards an in vitro MSKjct.

Cell Therapies are promising clinical instruments with significant therapeutic potential and commercial promise. However, the industry engaged in their commercial and clinical development faces significant financial, technical, regulatory and market challenges. These challenges are compounded by an understanding gap in the cell therapy industry. Commercial failures and financial difficulties have forced the industry to address the need to provide value and estimate and control costs early in the development timeline. The problem is that this issue is not being systematically or thoroughly addressed in the academic community while they pursue potential future treatments. Articles that highlight the need to understand costs and value are appearing with increasing frequency highlighting a growing consensus that work needs to be carried out in this area. However examples of models and tools to predict or estimate or even calculate costs in developing and producing a product do not exist in the literature. This work consists of three parts. Part one entails a new model of the characteristics observed in cell therapy new product development. This model is an evolution of an activity based dependency structure matrix (DSM). Result from the model suggests that some favoured development strategies (such as applying for an orphan indication status) provide less financial benefit than is commonly expected. The ability to scale manufacturing levels between clinical trial phases is also a pressing problem. 3 Part two presents a model to predict the cost of manufacturing and delivering a cell therapy product. This cost of good supplied (COGS) model combines both rules and predictive activity based costing across multiple manufacturing platforms, cell types and supply chain configurations. This model highlights the significant cost burden of validating both single and, more markedly, multiple sites of manufacture. The model also examines the potential for economies of scale when using different production technology in the manufacture of human Mesenchymal Stem Cells. Based in part on the results and knowledge gleaned in parts one and two, part three outlines the development of a novel, scalable expansion system developed to enable lower cost, controlled manufacture of adherent cell populations. While still at an early stage of development the technology has demonstrated the ability to maintain cells in a high rate of growth for a longer period than traditional culture techniques. This allows for the creation of a manufacturing technology with a higher expansion ratio than manufacturing systems on the market today.

Nos últimos anos, as células-tronco, devido a sua capacidade de originar diversos tecidos corporais e pelo poder de auto-renovação, impulsionaram os estudos de engenharia tecidual, sobretudo em medicina regenerativa. Nesse aspecto, o tecido adiposo vem se mostrando como fonte ideal para obtenção de tais células, devido à facilidade de captação, à baixa morbidade associada ao procedimento e ao elevado rendimento celular. Com o objetivo de estabelecer um modelo experimental versátil e que satisfizesse várias áreas de interesse, propôs-se o coelho Oryctolagus cuniculus como fonte de tecido adiposo. Esse animal apresenta bolsa adiposa interescapular com peso médio de 17,2g, o que corresponde a cerca de 6,6 g/Kg em machos adultos (peso corporal médio de 2590g). A coleta do material foi por meio de lipoaspiração a seco, com cânula de 3,5mm; levou-se em média, 11 minutos para o procedimento, obtendo-se aproximadamente 10 ml de gordura. Após o processamento pela técnica enzimática, em cada mililitro de gordura encontrou-se em média 1x105 células-tronco. O estudo constatou ainda que, por meio da criopreservação em nitrogênio líquido, as células mantinham suas características citométricas após períodos de congelamento que variaram de uma semana a 13 meses. As células apresentaram características de sua indiferenciação, como a expressão dos marcadores de superfície: CD90, 80,6%; HLA-DR, 2,8% e caspase 3, 10,5%. A análise do ciclo celular com 100% de confluência mostrou que 70,8% das células encontravamse quiescentes; 22,1% apoptóticas. As células com alta capacidade replicativa, que corresponde à fase S do ciclo celular, 1,4% e 0,9% encontravam-se em replicação, mostrando que as células-tronco do tecido adiposo, em cultura, não apresentam uma proliferação descontrolada, tendendo a se estabilizar, principalmente quando atingem confluência máxima em monocamada. Todas essas vantagens fazem com que o modelo proposto possa ser facilmente reprodutível, contribuindo para o estudo das células-tronco do tecido adiposo. / In the latest years, the study on tissue engineering, mainly in the area of regenerative medicine, has advanced because the medical community is highly interested in stem cells. This is due to both the potential of these cells to originate any body tissue and their power of self-renewal. Adipose tissue has been used as an ideal source of such cells, due to the simplicity of their collection, high cellular yield, and low morbidity associated with the procedure. In order to establish a versatile experimental model, which could meet the needs of researchers from various areas, the rabbit Oryctolagus cuniculus was proposed as a source of adipose tissue. This animal has an adipose pad in the interscapular region with an average weight of 17.2g, which corresponds to about 6.6g of fat material per kilogram of an adult male animal (mean body weight = 2.6kg). The material was collected by means of a liposuction procedure. Using a 3.5- mm diameter tube, a volume of nearly 10ml of fat material was obtained in a mean time of 11min. After processing the fat tissue by enzymatic technique, about 1x105 stem cells were found per milliliter of fat material. Using cryopreservation of the cells by freezing them in liquid nitrogen, it was observed that the cytometric characteristics were maintained after a period of time ranging from 1 week to 13 months. The cells presented evident characteristics of undifferentiation, such as expression of the surface markers CD90, HLA-DR, and Caspase-3 (80.6, 2.8, and 10.5 %, respectively). Analysis of the cellular cycle with 100% confluence allowed us to show that 70.8% of the cells were quiescent, 22.1% were apoptotic, 1.4% had high replication capacity (phase S of the cellular cycle) and 0.9% were already in replication (phase G2/M), indicating that stem cells from adipose tissue did not show uncontrolled proliferation, tending to stabilize, mainly when they reach maximal confluence in monolayer. These advantages make this model easily reproducible, facilitating the study of adipose tissue stem cells.

Type 1 diabetes affects one in every 400-600 children and adolescents in the US. Standard therapy with exogenous insulin is burdensome, associated with a significant risk of dangerous hypoglycemia, and only partially efficacious in preventing the long term complications of diabetes. Pancreatic islet transplantation has emerged as a promising therapy for type 1 diabetes. However, this cell-based therapy is significantly limited by inadequate islet supply (more than one donor pancreas is needed per recipient), instant blood-mediated inflammatory reaction, and loss of islet viability/function during isolation and following implantation. In particular, inadequate revascularization of transplanted islets results in reduced islet viability, function, and engraftment. Delivery of pro-vascularization factors has been shown to improve vascularization and islet function, but these strategies are hindered by insufficient and/or complex release pharmacokinetics and inadequate delivery matrices as well as technical and safety considerations. We hypothesized that controlled presentation of angiogenic cues within a bioartificial matrix could enhance the vascularization, viability, and function of transplanted islets. The primary objective of this dissertation was to enhance allogenic islet engraftment, survival and function by utilizing synthetic hydrogels as engineered delivery matrices. Polyethylene glycol (PEG)-maleimide hydrogels presenting cell adhesive motifs and vascular endothelial growth factor (VEGF) were designed to support islet activities and promote vascularization in vivo. We analyzed the material properties and cyto-compatibility of these engineered materials, islet engraftment in a transplantation model, and glycemic control in diabetic subjects. The rationale for this project is to establish novel biomaterial strategies for islet delivery that support islet viability and function via the induction of local vascularization.