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Induced Pluripotent Stem Cell (iPSC)-derived macrophages are a rapidly emerging model of tissue resident macrophages for investigating human disease. As potentially infinitely replicating cells, iPSC lend themselves for genetic manipulation by CRISPR-Cas9 editing before terminal differentiation into a vast array of cell types. As such, long standing challenges in exploration of macrophage gene functions during inflammatory diseases and infections, such as HIV-1, caused by donor variability and immortalised cell line deficiencies can finally be overcome. With over 30 different protocols for macrophage differentiation from iPSC now described, culturing of these cells is gradually becoming more accessible to researchers, and essential processes for differentiation better understood. However, with this progress comes the ever-greater need for clear definition of culturing conditions of these cells, so that we can compare results between methodologies, and eliminate undesirable sources of variability. This thesis sought to capitalise on this new system to explore the complicated and often contradictory role of TRIM5α in HIV-1 infection, and to explore with a precision only achievable through CRISPR-Cas9 genome editing the function of the cyclophilin family of peptidyl-prolyl isomerases. I demonstrate protection of HIV-1 from TRIM5α restriction by cyclophilin A, as well as regulation of the innate immune inflammatory response more generally by the cyclophilin family. However, prior to exploring this, this thesis sets out to address some of current issues in iPSC-derived macrophage culture and the ill-defined components of growth media and cellular environment. I describe a new macrophage differentiation medium using serum free, defined, and open-sourced components to bring needed transparency into the field. I also address the variability in macrophage phenotype and function brought about by the fundamental, but largely overlooked, impact of cell crowding in ex vivo culture. This revealed evidence for a potential plating density-dependent anti-inflammatory feedback loop relevant for human diseases like Alzheimer's disease.

Genome-wide association studies have identified over 400 loci associated with risk for type 2 diabetes (T2D), a large proportion are associated with pancreatic islet dysfunction. The parallel emergence of genome editing technology such as CRISPR/Cas9, alongside the ability to derive endocrine pancreatic cells from human stem cells, provides unprecedented opportunity to investigate and examine the mechanisms that underpin beta cell dysfunction. Given that several monogenic genes are also associated with T2D risk, investigating the mechanisms by which beta cell dysfunction occurs in monogenic diabetes also presents the opportunity to gain additional insight into T2D pathogenesis. The aim of my thesis was to model monogenic diabetes variants in INS and HNF1A in human beta cell models using both genome-edited human induced pluripotent stem cells (hiPSCs) and EndoC-βH1 cells. To investigate the role of INS mutations on beta cell function, I generated the first p.C43G INS hiPSC line, including a reporter line, using CRISPR/Cas9 editing and differentiated these cells along the pancreatic endocrine lineage. Gene expression analysis revealed no influence of the p.C43G mutation on either differentiation efficiency or ER stress, reflecting that differentiated hiPSCs may require further functional maturation in order to model INS-associated monogenic diabetes. Expression of the p.C43G INS mutant in the recently generated INS-KO EndoC-βH1 line was appropriately packaged into secretory vesicles. Subsequent gene expression and RNA-Seq analysis showed no significant differences between cells transfected with either the wildtype or p.C43G construct, echoing the findings from hiPSC differentiations. This would suggest that the negative effect of the mutation might translate through different cellular mechanisms. However, this study revealed various technical challenges, such as with transfection efficiency that considerably reduced the ability to fully characterise p.C43G expression in beta cells. Finally, I sought to investigate another gene involved in monogenic diabetes, HNF1A. As mouse models of HNF1A-diabetes do not fully recapitulate the human phenotype, I utilised a hiPSC line to characterise HNF1A knockout in parallel with a knockdown (KD) model of HNF1A within the EndoC-βH1 cell line. Loss of HNF1A led to decreased expression of insulin and glucagon at the final stage of differentiation. The HNF1A KO line, in comparison to the wildtype line, had an enrichment of differentially expressed genes involved in regulation of beta cell function, beta cell development and insulin secretion, recapitulating several features of the patient phenotype. Further comparisons with differentiated cells from a patient with an HNF1A-MODY causing variant and with my HNF1A KD EndoC-βH1 data, showed a significant overlap between the datasets demonstrating that many of the same gene signatures can be captured between different cellular models of HNF1A. My thesis illustrates the complementarity of utilising both iPSCs and EndoC-βH1 for disease modelling with each addressing different aspects of diabetes pathogenesis. This approach, although still being optimised, provides a better understanding of the cellular physiology that underlies genetic predisposition for diabetes.

Cellular mechanosensing is the process of converting mechanical signals into biological responses. Stem cells are self-renewing cells with the potential to transform into specialized cell types - this differentiation process is influenced by cellular mechanosensing. Cells sense material stiffness, and stiffer environments result in increased cellular mechanosensing and preferential differentiation into bone-producing osteoblasts. Cell shape also plays an important role due to its influence on cytoskeletal contractility, and photopatterning can be used to study the effects of cell shape on cellular mechanosensing. Although the effects of material stiffness and cell shape have been studied, little is known about the joint effects of these factors on stem cell mechanosensing. Taken together, the goal of this research is to develop a biomaterial system to study the combinatorial effects of shape and stiffness on mesenchymal stem cell (MSC) mechanosensing. Hydrogels of three stiffness (5 kPa, 10 kPa, 20 kPa) were photopatterned with shapes (circle, square, octagon) that cause a range of contractile forces in cells. These shapes were made into patterns on a glass photomask, allowing hydrogels placed under the photomask to be photopatterned. Photopatterns were found to over 90% accurate. Highly angular shapes, such as the octagon, and increased stiffness were both seen to influence an increased nuclear localization of mechanosensing protein YAP, with stiffness having a greater influence than shape.

This study focused on stem cells research to better understand strategic issues of business models involved in technology commercialisation of novel life science technology. The research questions of this study can thus be defined as: (l) what strategy or business model is appropriate for stem cells research commercialisation? and (2) which theoretical issues impact on the selection of strategies which are appropriate for the commercialisation of novel technologies, specifically novel life science technology such as stem cells research. Three objectives were derived from the important question of how to commercialise technology and of particular interest to this study, novel technology such as stem cells research, to: (1) understand the extent and nature of uncertainties affecting stem cells research into routine therapeutic practice; (2) examine the effect of economic, political and ethical issues on the adoption and implementation of commercialisation strategy for stem cells research; and (3) determine possible strategies for survival and growth of biotechnology firms focusing on stem cells research. Fieldwork as carried out in Scotland where purposive sampling used led to the selection of five interviewees for the first round of interviews. Snowballing technique increased the number of interviewees to 12 in the second round of interviewees. The interviewees were essentially key actors in the Scottish stem cells community who possessed the relevant scientific, ethics, regulatory as well business and industry knowledge of stem cells research. The interviews were open-ended. A review of major broadsheet newspapers from the US and UK were also conducted as an exploratory study to address the balance of this study by incorporating an element of how policy issues related to science and technology controversy are defmed, symbolised and resolved. Interview data were transcribed and analysed using content analsysis software QSR NvivoFindings from both rounds of interviews indicated the importance of collaborative strategy such as strategic alliances in overcoming strategic issues of stem cells commercialisation. More government support for stem cells research appeared to be needed to overcome funding issues. The inclusion of newspaper review in the study indicated the positive reporting of stem cells in the UK compared to us. The contribution of findings from the newspaper article analysis establishes a link between public understandings of new technology that were not captured through the two rounds of interviews It is suggested that strategic alliances should be given consideration in future stem cells business model and commercialisation strategy research efforts. Ethical risk too should be part of this suggested model. The implications of these results for theoretical along with practical implications of the fmdings were discussed and opportunities for future research provided

Cellulose binding domains (CBDs) are found in cellulolytic microorganisms as discrete domains in free cellulases or as cellulosomes, which are extracellular multi-enzyme complexes. CBDs bind to cellulose and help the catalytic domains to access cellulose substrates. CBDs are used as affinity tags for immobilizing cells, proteins or molecules on cellulose matrices. They can also be used in protein engineering to alter protein expression and solubilities. Cohesins and dockerins are domains exclusive to cellulosomes. They interact with high affinity and the interactions are Ca2+-dependent. Chelation of Ca2+ causes irreversible conformational change to dockerins thus disrupting the interactions. The first aim of this project was to validate a putative CBD from endoglucanase EngD of C. cellulovorans, to test its effect on solubility as a fusion in chimeras. The second aim was to use chimeric proteins containing CBD, cohesin, dockerin and LG to establish a system for efficient cell immobilization, expansion and harvest in hollow cellulose fibres. A putative CBD from an enzyme with its natural linker (PTCBDengD), a CBD from a scaffoldin (CBDcbpA), three cohesin domains from different strains (Cip7, Coh6 and CipC1) and an antibody binding protein (LG) were used to construct various chimeric and fusion proteins. The two CBDs were fused to different cohesins and LG respectively and the chimeras? solubility was analyzed. The results showed that fusing with CBDcbpA did not significantly help to increase the solubility of the insoluble domain Coh6 and it even greatly reduced the solubility of the soluble domain CipC1. In contrast, PTCBDengD fusion increased the solubility of Coh6 by three fold and it did not alter the solubility of soluble protein/ domains. These results suggested that PTCBDengD may be a better domain to use as a fusion tag for expression and other biotechnology applications. Cellulose binding specificity of PTCBDengD and its chimeric proteins were tested and SDS-PAGE analysis results clearly demonstrated that PTCBDengD and its chimeras specifically bound to crystalline cellulose Avicel and non-crystalline cellulose Cuprophan. The results confirmed that PTCBDengD is a true CBD. Chimeric protein pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc were used to build the scaffold on Cuprophan hollow cellulose fibre for reversible cell immobilization studies. Cell adhesion assay results showed that the double-chimera systems efficiently immobilize cells onto Cuprophan hollow fibre. Dissociation of LG-Doc from amorphous cellulose Cuprophan-bound CBDcbpA-Cip7 by EDTA treatment resulted in decrease of cell binding by almost 90%; however, re-association of LG-Doc after EDTA dissociation only achieved 50% efficiency of cell immobilization. Dot blot and SDS-PAGE analysis showed that dissociation/ re-association of LG-Doc to Cip7-PTCBDengD could be decreased in was interfered by the presence of cellulose. Preliminary results indicated that crystalline cellulose Avicel may improve dissociation/ re-association efficiency. In conclusion, studies on recombinant proteins validated CBDengD's specific affinity to cellulose and its solubilizing effect on its fusion partner. Chimera pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc are effective in cell immobilization. However, optimization is required to develop recyclable protein scaffolding and complexes on cellulosic matrices.

Transcription factors GATA-1, Fli-1 and FOG-1 are essential proteins for normal megakaryopoiesis, however, the detailed analyses of their functions within developmental stages of megakaryopoiesis are lacking. In my thesis, over expression of gene in target cells was adopted as the main strategy to study the biological functions of these proteins, therefore, an efficient gene delivery method was first developed by using retrovirus.This approach was then utilized to over express GATA-1, Fli-1 and FOG-1 in murine leukemia M1 cells and mouse hematopoietic stem cells (HSCs), and their effects on different developmental stages of megakaryopoiesis were investigated. In the transduced M1 cells, enforced expression of GATA-1 and Fli-1 was found to induce the megakaryocytic development, which was associated with the formation of megakaryocyte (Mk) and the increased expression of Mk specific genes c-Mpl and GPIX. In the transduced mouse HSCs, it was found that the expression of endogenous GATA-1, Fli-1 and FOG-1 was up-regulated throughout Mk differentiation; enforced expression of these transcription factors led to the significantly enhanced Mk development. Megakaryocytes over expressing GATA-1, Fli-1 and FOG-1 were characterized by the increased expression of various Mk-specific genes including GPIX, c-Mpl, platelet factor 4 (PF4), acetylcholinesterase (AChE) and NF-E2, an important transcription factor for terminal megakaryopoiesis; however, GATA-1, Fli-1 and FOG-1 displayed the different abilities in promoting the proliferation of hematopoietic cells and MK differentiation, as well as regulating other transcription factors involved in hematopoiesis. To further elucidate the role of the functional domains of Fli-1, various mutants of Fli-1 were also over expressed in mouse HSCs. The results demonstrated that first, the combination of the activation domain of Fli-1 and its Ets domain is required for early megakaryopoiesis but not sufficient for terminal megakaryopoiesis; second, DNA binding of Fli-1 was not the only requirement for early Mk enhancement, moreover, the interaction between Fli-1 and GATA-1 through the Ets domain and the resultant transcriptional synergy was the essential determinant for Fli?1 ability in Mk development. Taken together, the studies presented in this thesis provided strong in vitro evidence that GATA-1, Fli-1 and FOG-1 indeed play the critical roles in normal megakaryopoiesis.

Stem cells play a vital role in the turn over of permanently self-renewing tissues (e.g. corneal epithelium, epidermis, bone marrow). Stratified cornea epithelia serve, as ideal tissue models for studying ‘stemness’, as the site of cells in the different stages of differentiation are anatomically easily identifiable. Studies targeting limbal stem cell maintenance, in-vitro gene expression during storage and differentiation will benefit future therapeutic applications (e.g. corneal transplantation). Knowledge of stem cell behaviour will help explain the pathophysiology of corneal related ocular surface disorders (e.g. idiopathic limbal stem cell deficiency, pterygium and limbal dermoids), establish new treatment modalities (e.g. allogenic cellular transplants) and help promote invivo expansion of residual stem cell populations in deficiency states. The major obstacle in the progress of research on limbal stem cells is the lack of knowledge of phenotypic markers of limbal epithelial stem cells. This project first studied the limbal expression of phenotypic markers that were discovered in other human adult and embryonic stem cell populations using microarray differential expression studies. Gene expression profiles of the relatively primitive human foetal limbus were compared with that of the central cornea. Microarray was also performed to identify the differential gene expression profile of cultured primary human limbal epithelial cells, when compared to the limbal epithelial cell population isolated after 5 serial cultures. 33 genes were upregulated in the human foetal limbus and primary cultured human limbal epithelium, when compared respectively to the central foetal cornea (first experiment) and the limbal epithelial cell population after the 5th trypsin passaged culture (second experiment). Four foetal limbal and primary limbal epithelial upregulated genes (CK15, CK14, Cdh3, and Wnt4) were confirmed to be upregulated by semi quantitative RT-PCR and immunohistochemical experiments on the human foetal cornea, adult human cadaveric cornea and cultured human limbal epithelial cells. The microarray defined phenotypic profile of both the foetal limbus and cultured adult limbal epithelial cells will help identify these cells in in-vitro and in-vivo states. The expression of these 4 selected markers in the limbal dermoid and pterygium suggests that limbal epithelial cells containing a stem cell population are involved in the pathogenesis of these two disorders.

Single cell probing has found a number of applications in different areas of research. It can help us to better understand cell-to-cell interactions; it has found numerous applications in immunology, cancer research, detection of pathogenic infections and genetic abnormalities. The single cell analysis is very important in stem cells research and development of cells. The main obstacle in the single cell analysis is the small amount of analyte that a single cell could provide. Another difficulty is connected to the cell-to-cell variability inside the uniform population due to the differences of single cells in size, activity, mitotic stage, and functions. To overcome these problems several methods of single cell analysis including utilization of fluorescence and microfluidic platforms were developed. However, existing methods for cell isolation and analysis are laborious, costly, taking long time and often lack sensitivity, which is necessary for a single cell level probing. Therefore there is a need for novel high throughput and convenient experimental techniques for analysis of gene expression in individual cells. Fiber-based devices with high surface area that are modified with mRNA capturing element could be an attractive candidate for this purpose. We proposed to develop microfluidic electrospun fiber yarns based biosensors for applications in detection of small amounts of targeted analytes, such as mRNA material of a single cell or specific secondary antibodies molecules. In order to achieve this goal of improving single cell analysis we produced highly sensitive microfiber probes for PCR analysis of mRNA present in concentrations corresponding to those in a single cell. The bundle of microfibers with high specific surface area was modified with oligo dT25 which specifically binds the poly-A tail of mRNA. Now that RNA is captured by the fiber, relative expression of β-actin by contractile and synthetic vascular smooth muscle cells, as well as by adult and neonatal myocytes, was used as a model to evaluate performance of the fiber-based devices for mRNA extraction. Application of fiber yarns as the mRNA isolation probes for PCR analysis improves usability during the sample collection and processing and allows for focused probing from a desired area. The sensitivity of an enzyme-linked immunosorbent assay (ELISA) performed directly on the fiber-based devices was also evaluated. Electrospun fiber yarns were covalently coated with a model capture antibody and then used to evaluate presence of the specific secondary antibodies in solutions. The colorimetric ELISA procedure was performed with ABTS substrate to investigate sensitivity of prepared sensors in the ELISA application. Overall, these fibers provide a new flexible platform for a number of analysis types that can considerably improve its convenience and availability for various applications. This approach is universal and could be utilized to develop fiber-based devices that could be potentially used in point-of-care devices and for forensic analysis.

The construction of tissue-engineered blood vessels (TEBV) is a promising solution for treating cardiovascular diseases. In order to attain the proper organization and mechanical properties found in native vessels, vascular smooth muscle cells (VSMC) areusedasacrucialcomponentofTEBVs. With the growing interest in stem cells, research has shown that marrow stromal cells (MSC) are a potential cell source for VSMCs due to their easier and less invasive isolation procedure, fast proliferation rates, and most importantly, the ability to differentiate into VSMCs. While current studies have provided evidence that mechanical stimulation can induce MSC alignment and differentiation into VSMCs, a definitive set of conditions must be determined in order to successfully control the differentiation. The goal of this dissertation is to further define the effects of mechanical stimuli on the differentiation of MSCs. To do so, we created a uniaxial cell stretcher capable of delivering tunable mechanical conditions to cells seeded on a substrate. With this device, we applied a range of controlled stretch frequencies to MSCs and evaluated the final cell type for each condition using quantitative RT-PCR and immunocytochemistry. We found that at 10% strain, a 1 Hz frequency induced differentiation into the VSMC lineage as well as a highly aligned organization while a 0.1 Hz frequency induced osteogenesis. Using these results, we proceeded to investigate the potential of MSC transdifferentiation through mechanical stimulation by applying the two distinct lineage-inducing conditions sequentially and evaluating the final cell type. We found that while mechanical stimulation was capable of inducing MSC transdifferentiation, it could only proceed in the direction toward the VSMC phenotype under the applied conditions. Our findings revealed the potential use ofmechanical stimulation as a tool for not only controlling but also maintaining MSC differentiation. We expect the methods and specific mechanical conditioning developed in this project to become a useful and novel approach to TEBV construction.