Haematopoietic stem cell transplant (HSCT) is a treatment for some malignant conditions and is considered to be a demanding experience both physically and psychologically. This study aimed to explore (a) the experience of participants preparing for and undergoing a HSCT and (b) how they experienced viewing a film of survivor testimonies as part of this process. The survivor testimonies were designed to be used as a clinical intervention for people preparing for a HSCT. Seven participants were recruited and a multiple interview design was employed to explore their experience at three different stages of treatment. The interview data was analysed using Interpretative Phenomenological Analysis (IPA). The data cohered around a central theme of vulnerability, which changed over time, from being overwhelming, to being resisted, to being recalibrated. It emerged that the effect of the testimonies was overwhelmingly positive. Participants found the resource a hopeful and optimistic counterbalance to the fear and uncertainty they experienced being diagnosed with a life threatening condition and undergoing a potentially curative but also life threatening treatment procedure. The knowledge acquired from using a film as a stimulus in research interviews is discussed, along with the contribution to the existing literature on the experience of HSCT patients, the clinical implications of how the care of transplant patients can be improved and how the resource of survivor testimonies can be used to support patients going forward.

Induced pluripotent stem (iPS) cells have the potential to generate a wide array of cell types from multiple lineages that enable us to explore the mechanisms that are involved in the conversion of cell states. The reprogramming process that generates iPS cells is complex, but since its discovery, technical advancements and improvements in the methodology have improved the speed and efficiency of generating integration-free, clinically relevant iPS cells. However, despite improvements, the mechanisms of reprogramming are not fully understood and so the process remains largely inefficient and slow. It has been reported that reprogramming mediated through the delivery of exogenous mRNAs encoding OCT4, SOX2, KLF4 and cMYC is a fast and efficient method for generating integration-free iPS cells. Here we show that mRNA reprogramming can be enhanced further by employing an mRNA dose-ramping approach that provides greater control of the dose of mRNA that is introduced into the target cells. This improvement upon existing methods promotes the viability of the target cells during reprogramming which in turn improves the efficiency, speed and success of generating iPS cells. We also show that an optimisation to the reprogramming factor cocktail, replacing OCT4 with a fusion between OCT4 and the transcriptional activation domain of MYOD1 – called M3O, further improves the kinetics of reprogramming. Reprogramming disease cells is also possible in that several iPS cell-disease models have been established that have successfully modelled aspects of disease development in vitro. Here we show the applicability of using the mRNA approach we have developed, on neuroblastoma cells and the characterisation of iPS cells reprogrammed from neuroblastoma cells using OCT4, SOX2, KLF4 and cMYC delivered using Sendai viral vectors. Finally, we demonstrate how human fibroblasts introduced to a vector encoding MYOD1 causes them to transdifferentiate in to myoblast-like cells without a genomic footprint. Together this data demonstrates how integration-free mRNA can be used to control gene expression to direct cell fate through reprogramming and transdifferentiation. This mRNA approach provides proof of concept that warrants the testing of other genes to explore their function in reprogramming and other pathways that govern cell fate.

The synthetic replacement of full thickness skin is suboptimal both aesthetically and functionally. One approach to improve existing dermal substitutes is to pre-vascularise them to facilitate incorporation. In so doing, the aim is to improve the trajectory of wound healing. More expeditious maturation has been suggested to improve outcomes. Endothelial colony forming cells (ECFCs), specialised progenitor cells required for vasculogenesis, were isolated from cord (CBECFC) and peripheral (PBECFC) blood. Mesenchymal stromal cells were separated from adipose tissue (AdMSCs). Using a proprietary device (μ-chemotaxis 3D), human umbilical vein endothelial cells, and CBECFC- and PBECFC-derived cells were compared for chemokinetic and chemotactic movement within collagen I gels, with or without fibronectin. PBECFC-derived cells migrated further than CBECFC-derived cells and HUVECs towards the chemoattractant. These data informed the fabrication of collagen I gels containing co-cultures of ECFC-derived cells with MSCs. An attempt was made to compress these gels to facilitate handling, but vascular tubule formation was not amenable to compression. HUVECs seeded as a monoculture within compressed gels also had a 100% mortality rate, although 62.5% AdMSCs and 66.4% human dermal fibroblasts survived the compression process. Since pre-formed tubules did not survive the compression process, various concentrations of ECFC-derived cells and MSCs were seeded within uncompressed collagen I gels in order to obtain an optimised vascular network. AdMSCs were compared to BMMSCs. PBECFC-derived cells were compared to CBECFC-derived cells and HUVECs. Optimised gels containing tubules formed by adult derived PBECFC-derived cells and AdMSCs were then scaled up and implanted into an in vivo immunodeficient mouse model. Host incorporation of the construct within this pre-vascularised gel was significantly improved compared to an empty gel control (p=0.04). In summary, it was possible to fabricate a pre-vascularised collagen I scaffold, using adult-derived stem/progenitor cells, increasing the rate of host incorporation in an in vivo murine model.

In preimplantation mouse embryos, signalling and gene regulatory networks cooperate to determine lineage segregation, and modulating signalling in vitro allows for stem cell populations to be established from these lineages. Fibroblast growth factor (FGF) signalling triggers the differentiation of primitive endoderm (PrE) cells fated to contribute to the yolk sac, while cells unreceptive to FGF form the epiblast (Epi) that subsequently contributes to the embryo proper. In vitro, FGF signalling is required for preimplantation Epi-derived mouse ES cells to exit self-renewal. Conversely, in human ES cells and postimplantation Epi-derived mouse epiblast stem cells, FGF signalling is instead required for pluripotency maintenance. It remains unclear how these divergent outcomes arise, especially as these cells rely on a similar core pluripotency gene network. This study demonstrates that ectopic expression of the PrE transcription factor Gata6 destabilises mouse ES cell pluripotency in vitro and upregulates PrE-associated genes independently of FGF signalling. As previous studies show that PrE specification is compromised in Fgf4-/- embryos, despite initiation of Gata6, this suggests FGF signalling and Gata6 cooperatively drive PrE specification in vivo. Characterising Gata6 function determines that it directly binds to both up- and downregulated gene targets and potently initiates reprogramming in multiple cell types, including human ES cells, suggesting it may also antagonise pluripotency in vivo. Surprisingly, FGF stimulation negatively affects establishment of the pluripotent human Epi. Characterising alternative signalling pathways in the human embryo finds that modulating IGF signalling promotes proliferation of the human ICM, and similar to human ES cells, intact TGFβ/Nodal signalling is required for pluripotent gene expression in the Epi. Consequently, as signalling requirements in the human Epi appear somewhat distinct from both the mouse Epi and existing human ES cells, modulating embryo-specific signalling pathways may permit derivation of human ES cells that more accurately reflect the pluripotent Epi compartment.

Peptide posttranslational modifications have been shown to regulate multiple aspects of cell signalling, thereby influencing cellular functions. The addition of O-linked Nacetylglucosamine to serine or threonine residues (O-GlcNAcylation) of proteins has only recently been characterized and its overall role in cell signalling remains elusive to date. Recent studies suggest an essential role of O-GlcNAcylation on the viability and pluripotency of mouse and human embryonic stem (ES) cells. Here we show that increased levels of O-GlcNAcylation in response to specific inhibition of O-GlcNAc hydrolase (Oga) hinder mouse ES cell differentiation. In addition to these findings, I could also demonstrate that increased O-GlcNAcylation leads to expression of a gene set normally epigenetically repressed in mouse ES cells and associated with a subpopulation resembling cells in the 2-cell-stage embryo. I also extended our lab's investigations to human induced pluripotent stem (iPS) cells. While mesendodermal differentiation remains unaffected by high O-GlcNAc levels, neural differentiation is severely disrupted in these cells. Human iPS cells with elevated O-GlcNAcylation are unable to commit to the ectodermal lineage and fail to organize in neural tube-like structures, so-called neural rosettes. Following these observations we performed mRNA sequencing analysis on human iPS cells with high O-GlcNAc levels and found gene expression to be significantly altered. Genes affected by increased O-GlcNAcylation include modulators of key neural developmental processes, for example components of the bone morphogenic protein signalling cascade.

Stem cells hold enormous potential for the treatment of injuries and degenerative diseases. In the pursuit of stem cell therapies, a plethora of biomaterials have been developed to induce lineage-specific differentiation or support cell propagation for research and clinical applications. However, the use of stem cells is hindered by the cost of scale-up and risk of zoonotic transmissions from animalderived culture components. Here, we utilise a recombinant protein scaffold composed of self-assembling nanofibres, termed ZT, and assess the systems adaptability for in vitro applications. Protein-based scaffolds offer distinct advantages over conventional materials such as the display of peptidic motifs with near-native stoichiometries and control of the spatial density and nanotopographical distribution of genetically-encoded bioactivities. Herein, the functionalisation potential of the ZT system is explored via the generation of chimeric protein building blocks that exhibit the integrin-binding RGD motif. Specific sites within the building blocks were found to tolerate diversification, in the form of exogenous peptides or a fused protein domain, without structural perturbation or inhibition of assembly. The bioactivity of functionalised ZT nanofibres was assessed using murine mesenchymal stem cells (mMSCs), which recognised the integrin-binding moieties. The ability of one ZT nanofibre variant to induce mMSC chondrogenesis was investigated, which proved unsuccessful in the current context. A second generation of ZT variants were generated by the incorporation of chondroinductive motifs for future applications in cartilage tissue engineering. Additionally, the capacity of functionalised ZT nanofibres to act as culture substrates for human embryonic stem cell (hESC) self-renewal was explored.

Human mesenchymal stem cells have attracted significant attention during the last decade as a versatile tool for cell therapy, gene therapy and tissue engineering. The existence of mesenchymal stem cells in adult tissues and their capacity for differentiation into multiple lineages are major benefits for clinical applications, circumventing the ethical and safety concerns surrounding the use of embryonic stem cells. It has been long established that mesenchymal stem cells have the potential to differentiate into mesenchymal lineages such as bone, cartilage and adipose tissue. Recent studies have uncovered the potential of mesenchymal stem cells to differentiate into endodermal and ectodermal derivatives, suggesting a greater plasticity than originally envisaged. In the current study, a novel approach using the chick embryo was developed to investigate the differentiation potential of bone marrow-derived human mesenchymal stem cells when exposed to developmental signals in vivo. In order to investigate the suitability of the chick embryo as a host, mesenchymal stem cells were first transplanted into fore- and hind limb of stage 17 chick embryos. Expression of differentiation markers were subsequently analysed using immunocytochemistry and molecular analysis. Expression of osteogenic-specific genes such as alkaline phosphatase, RUNX2 and osteocalcin was observed in human mesenchymal stem cells grafted into wing and limb buds of the chick embryo. To investigate the extra-mesodermal differentiation potential of mesenchymal stem cells, expression of lineage-specific genes was subsequently analysed after grafting mesenchymal stem cells into the chick neural crest. Mesenchymal stem cells showed extensive migration through head mesenchyme after injection into the chick neural crest. Injected cells also significantly up-regulated neural crest specific markers such as SLUG, FOXD3 and MITF, suggesting differentiation toward neural crest cell lineages.

Stem cells have the ability to repair, replace or regenerate tissues. As a result their potential for regenerative medicine is vast. The processing of cells for therapeutic use and clinical diagnostics will rely on cell sorting steps to ensure a homogeneous population is obtained. Existing cell sorting technologies, either rely on the physical properties of cells, which tend to provide poor specificity' or require cells to be labelled with antibodies. Many of these techniques also tend to be expensive. Consequently there is a need for a fully synthetic, inexpensive, label-free separation system, capable of sorting cells with minimum manipulation. Initially, the generation of suitable substrates was investigated for high throughput polymer functionalisation in order to generate chemically heterogeneous surfaces through microarrays with the potential to induce differential cell adhesion. There was a need to develop immobilisation systems that were compatible with the high-throughput production of microarrays. The grafted-from and grafting-to methodologies were explored to find a suitable and versatile system, for high-throughput fabrication and immobilisation. The grafting-from approach relied on the generation of polymer brushes from a suitable low-fouling substrate. Despite the success of this technique, compatibility with high-throughput systems was limited. Therefore a grafting to approach was devised, in which specialist RAFT polymers could be immobilised to low fouling substrates using mid conditions, compatible with high-throughput systems. However the ability to produce a library of specialised polymers was limited. Consequently, a method was developed to immobilize thiol-functionalised materials to a low-fouling polymer substrate using thiol-ene click chemistry in a high-throughput format to fabricate immobilised microarrays. A heterogeneous population of cells derived from mouse embryoid bodies was subsequently seeded onto the arrays. Immunohistochemistry techniques were employed to track the differentiation of cells into different lineages. This technique allowed for the high-throughput quantification and identification of lineage specific cells by the means of automated fluorescence imaging and analysis. Lineage specific cell density was shown to vary according to material combination. Combinatorial chemistry was shown to be paramount in the adhesive selectivity of cells derived from mesoderm, endoderm and ectoderm lineages. Although glucose was shown to be the major component for each best-performing material, the addition of a minor component altered the surface chemistry and the specific adhesive properties for each material. In the future, successful materials are investigated further to generate structure-activity relationships. Ultimately we seek the generation of new surface-based devices that have the capacity to be fully synthetic, selective, inexpensive and disposable.

Objective: To explore the potential benefit of non-invasive cardio-haemodynamic variables in the management of sepsis in the Emergency Department (ED) by measuring their changes with normal treatment and their relationship to outcome. Methods: Study 1: Prospective cohort study of a convenience sample of adult ED patients with uncomplicated sepsis. Cardio-haemodynamic parameters were obtained using a Thoracic Electrical Bioimpedance (TEB) device. Study 2: Prospective cohort study of a convenience sample of adult ED patients with severe sepsis / septic shock. Cardiohaemodynamic parameters were obtained using a TEB device, transcutaneous Doppler ultrasound and Near-Infrared Spectroscopy. Measurements for both studies were taken on ED arrival, ED departure and after 24 hours, whilst patients received normal treatment. All patients were followed up for 30 days. Results: 50 patients were enrolled in study 1 and 73 patients in study 2. Septic patients had a significantly higher cardiac output and significantly lower stroke volume and systemic vascular resistance than non-septic ED controls. After 24 hours of normal treatment cardio-haemodynamic parameters of patients with uncomplicated sepsis and survivors from severe sepsis / septic shock began to normalise. In addition, patients with severe sepsis/septic shock had abnormal tissue oxygen saturation on ED arrival, which, in survivors normalised with treatment. Conclusion: This is the first description of cardio-haemodynamic parameters in septic patients at their entry to hospital (ED). Septic patients have initially abnormal haemodynamics and the ability to normalise haemodynamics and tissue oxygen saturation is associated with good outcome. This thesis has identified a number of parameters, which warrant validation to define their role as diagnostic or co-diagnostic biomarkers for sepsis and sepsis outcome.

Cartilage injuries and joint diseases such as osteoarthritis are a leading cause of pain and disability worldwide. Stem cell based tissue engineering applications have the potential to further our understanding of cartilage formation during development and degradation with disease progression. The regeneration of damaged articular cartilage relies on the identification of an appropriate cell source with a high proliferative propensity and consistent chondrogenic capacity. Mesenchymal stem cells (MSCs) are multi potent progenitors that can be stimulated to differentiate into musculoskeletal lineages including chondrocytes and are therefore particularly suited for cartilage repair strategies. Human first trimester foetal bone marrow derived MSCs have enhanced expansion potential compared to adult bone marrow derived MSCs and may represent an alternative cell source for cartilage development and disease modelling.