Theses - Surgery
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Item Embargo The influence of ECM on mesenchymal stromal/stem cell activity and their interactions with monocytes/macrophagesAlkhrayef, MohammadOsteoarthritis is a prevalent degenerative condition affecting synovial joints, resulting in pain, stiffness, and difficulty with movement. Current treatments mainly alleviate symptoms without effectively regenerating the lost cartilage tissue. An early surgical intervention for focal cartilage damage, known as marrow stimulation or microfracture, aims to repair this damage by promoting endogenous healing. However, the resulting repair tissue often lacks the properties of native cartilage, raising questions about the long-term effectiveness of this approach. Therefore, to enhance the outcome of this osteochondral repair approach, it is necessary to understand the biological mechanisms involved in the healing process following microfracture. Osteochondral repair is a complex interplay of cellular interactions and changing extracellular matrix (ECM) environment. This thesis aimed to extend the current understanding of the biology underpinning this process, focusing on the interaction between mesenchymal stromal/stem cells (MSCs) and monocytes/macrophages within dynamically changing ECM proteins found at the injury site. We hypothesized that this interaction plays a pivotal role in determining the outcome of tissue repair. In our investigation, we first characterised the deposition and remodelling patterns of key ECM proteins, fibrin and collagen type 1, during osteochondral injury repair in C57BL/6 mice. Our findings revealed that fibrin was the predominant ECM protein in the initial seven days post-injury, which was subsequently gradually replaced by collagen type 1. At eight weeks post-injury, the tissue repair outcome at the articular surface was found to contain collagen type 1, perhaps representing an immature form of hyaline cartilage. Subsequent *in vitro* studies, using 3D ECM models constructed from fibrin and collagen type 1, demonstrated that these environments significantly modulate MSC morphology, proliferation, migration, and immunomodulatory activity. Notably, MSCs within these 3D environments exhibit differential responses to pro-inflammatory stimuli, with the ECM potentially acting as a reservoir for secreted cytokines and growth factors, orchestrating cellular activities over time. Three molecular mechanisms, the TNFα/NFkB pathway, TNFα/JNK/AP1 pathway, and the potential involvement of ROCK, were identified as specific effectors of MSC immunomodulatory activity within these environments. Further investigations into the crosstalk between MSCs and monocytes in controlled 3D ECM environments revealed a distinct M2 macrophage phenotype, characterized as CD206+ MerTK- CD163- CD209-. This subpopulation displayed enhanced expression of *IL10*, IL6, and IL8. Moreover, conditioned media from these co-cultures influenced chondrocyte migration and chondrogenesis, with TGFβ1 playing a pivotal role in these observations. In conclusion, this thesis underscores the profound influence of the ECM on cellular interactions during osteochondral repair. The insights gained pave the way for innovative therapeutic strategies, potentially enhancing tissue repair outcomes and offering improved treatments for conditions like osteoarthritis.Item Open Access Studies of normothermic machine perfusion in human kidney transplantationElliott, TegwenNormothermic machine perfusion (NMP) of human kidneys is a new preservation technique that allows for the assessment and treatment of kidneys prior to transplantation. However, there is currently no standard protocol for NMP of kidneys and limited understanding of the underlying mechanisms that occur during the perfusion period. My PhD set out to determine the effects of NMP and to identify areas of improvement of NMP for future protocols. Firstly, I used the Cambridge cohort of the NMP clinical trial to examine the effect of NMP on graft outcomes. NMP for one-hour was deemed to be safe and feasible in renal transplant. Kidneys with a prolonged cold ischaemic time prior to NMP, and extended second period of cold ischaemia after NMP resulted in prolonged delayed graft function and poorer early graft function. However, there was no effect on long term graft function and no significant benefit compared to those kidneys that received static cold storage (SCS). In the same cohort I assessed the relationship between urinary biomarkers and existing quality assessment scores (QAS) to evaluate the role of NMP as an assessment tool. Kidneys from older donors with longer cold ischaemia had poorer QAS and this resulted in worse 12 month follow-up outcomes compared to kidneys with better QAS score. Kidneys with higher QAS had increased urine concentration of biomarkers, but these biomarkers did not correlate with graft outcomes. I then sought to evaluate the role of heme in the NMP perfusate. I found that older units of red blood cells had high levels of heme and that levels increased significantly during one-hour of NMP. There was also an increase in the levels of inflammatory cytokines in the perfusate and expression of genes associated with apoptosis, oxidative stress and inflammation. However there appeared to be no association between the levels of heme and these inflammatory markers along with graft outcomes in the clinical series. High levels of interleukin-6 (IL-6) were consistently found in the perfusate after one-hour NMP in both clinical and discard series. Therefore I used a human organ culture model to investigate the potential of inhibiting IL-6. I found a significant decrease in the expression of inflammatory genes and markers of oxidative stress with IL-6 inhibition using an anti-IL-6 antibody. In summary, an end period of one-hour NMP is safe and feasible in clinical kidney transplantation. However, the negative effects of cold ischaemia are not ameliorated by one- hour NMP. Furthermore, it cannot protect against additional cold ischaemic injury after NMP. NMP can be used as a platform for assessment and urinary biomarkers are a potential marker of kidney quality and outcomes that could be used clinically Packed red blood cells from the blood bank are convenient for use during NMP. However, older units of red cells contain higher levels of heme which increases during NMP. Although this appeared to have no detrimental effect during one-hour NMP it may have consequences for longer durations of perfusion. Inhibition of IL-6 is a potential therapeutic target to reduce inflammation that warrants further investigation.Item Embargo Evaluating metabolism in human renal cancer using novel surgical modelsYong, Cissy; Yong, Cissy [0000-0002-8697-1494]Within the last two decades, there has been increasing recognition of the pivotal roles of metabolic reprogramming in cancers. Indeed, this evidence has cumulated in the establishment of dysregulated metabolism as a contemporary hallmark of cancer. Metabolomics has been vital to define the metabolic landscape of cancers. This technique allows the simultaneous analyses of hundreds of metabolites present within a system, capturing the downstream interaction between the genome and environment. Renal cell carcinomas (RCC) are increasingly recognised as metabolically-driven types of cancers. This reprogramming of metabolism in RCC has been shown to have a causal role in tumorigenesis, progression, and aggressive disease behaviour. However, the majority of studies in RCC metabolism use conventional metabolomics techniques that provide only a static ‘snapshot’ of cellular metabolism, with limited inferences possible on dysregulated pathway activity. To overcome these limitations, isotopic tracer studies have been recently employed to capture these dynamic processes and enable tracking of nutrient utilisation in cells, thereby permitting the identification of cancer-specific pathway activities, for translation into the clinical setting. These advanced techniques and approaches to study metabolism *in vivo*, in patients have yet to be widely established in RCC and furthermore, there is a lack of clinically relevant models of RCC metabolism locally. The aim of this thesis was to develop novel models of RCC to evaluate metabolism using isotopic tracer studies. In this thesis, I established isotopic tracer studies *in vivo* in surgical patients with RCC. Using a multiomics approach, I characterised the metabolic phenotypes of these tumours and for the first time, evidenced the suppression of gluconeogenesis *in vivo*. Secondly, by developing a novel *in situ* tissue sampling method, I demonstrated the profound impact of ischaemia, a variable commonly associated with conventional tissue sampling methods, on the metabolic characterisation of RCC. Lastly, in order to develop more clinically relevant models of RCC metabolism, I utilised our access to human tissues and explored the development of two patient-derived models, in the form of patient-derived xenografts and through the novel application of an *ex vivo* normothermic perfusion model. Whilst these models demonstrated the potential to recapitulate RCC metabolism, they highlighted the challenges in modelling this aspect of tumour biology. Overall, this work has yielded new insights into the metabolic reprogramming of RCC and the ischaemia-induced perturbations on characterising these tumours. Accurate tumour profiling is fundamental in driving clinically relevant research, and these findings have critical implications for future tissue sampling methods in research. Lastly, by laying the groundwork in this thesis for the development of more clinically translatable models, my goal is to expand the current experimental systems available to ultimately strengthen our research into RCC metabolism.Item Embargo INVESTING THE ROLES OF E26 TRANSFORMATION-SPECIFIC HOMOLOGOUS FACTOR IN CYSTIC FIBROSIS LUNG DISEASEPinte, LaetitiaCF (Cystic Fibrosis) is the most prevalent genetic disease among Caucasians. Here, a sodiumbicarbonate channel, the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), is impaired, dehydrating all epithelia. The principal cause of morbimortality of CF is CF lung disease (CFLD) where CF airways have impaired mucociliary clearance and undergo constant remodelling and chronic inflammation, ultimately leading to irreversible loss of lung function. Interestingly, within CF patients carrying the same CFTR mutation resides a high variability in terms of CFLD severity and response to treatments. These variabilities are thought to be due to differentially expressed non-CFTR genes. To locate these genes, Genome-Wide Association Studies (GWAS) collected and analysed data from large patient cohorts, identifying four loci (5p15, 3q29, 11p12-13, Xq22-23) associated with more severe clinical manifestations of CFLD. These loci could sit close by genes potentially impacting CFLD penetrance, and patient responsiveness to therapeutics. We aim to understand how genes outlined by GWAS influence CFLD. To this end, we first optimized a model system which relies on human induced pluripotent stem cells (hiPSC)-derived Airways Epithelial Cells (AECs). We first explored two differentiation protocols that generate lung AECs as organoids and Air-Liquid-Interface (ALI) cultures and outlined their respective advantages and limitations. Then, using the most robust differentiation protocol, we explored the expression of GWAS-outlined genes and observed that E26 Transformation-Specific Homologous Factor (EHF) was expressed at comparable levels to primary Human Bronchial Epithelial Cells. Thus, we decided to study EHF in our AECs in-vitro model. First, we knocked out EHF using CRISPR-Cas9 from two non-CF hiPSC lines and selected, within the pool of edited cells, homozygous and heterozygous knock-out (KO) clones alongside wild-type clones, then used as internal controls. The KO was confirmed by finding decreased or absent levels of EHF mRNA and protein in all clones. Second, the resulting hiPSCs lined were differentiated into AECs differentiation. The absence of EHF led to a decreased expression of MUC5B in organoids and alterations in the transcriptomic signature of Basal Cell (BCs) in ALI cultures. Finally, we assessed physiological functions (trans-epithelial electrical resistance (TEER), cilia motility, and cell migration) and functions known to be faulty in CFLD (CFTR function, survival to infection, and response to hypoxia). EHF-/- clones had significantly increased TEER values and forskolin-induced swelling while reducing the HIF1a-induced response to hypoxia, thereby modifying the pH of EHF-/- epithelia, compared to EHF+/+ clones. Taken together, our data outlined several mechanisms by which EHF could be linked to CFLD. In conclusion, we hypothesise that any polymorphisms increasing the expression of EHF could lead to more severe CFLD. Our study sheds light on how a single gene can impact the severity of disease through multiple mechanisms and draws perspectives onto broader targets for personalized therapies against CF.Item Open Access Developing novel prophylactic approaches against SARS-CoV-2 infectionBrevini, TeresaPrevention of SARS-CoV-2 entry into cells through the modulation of viral host receptors, such as ACE2, could represent a new chemoprophylactic and therapeutic approach for COVID- 19 complementing vaccination. However, the mechanisms controlling ACE2 expression remain elusive, partly due to low ACE2 levels in experimental models. To address this challenge, I use biliary epithelial cell (cholangiocyte), one of the cell types with the highest ACE2 levels, to dissect the molecular mechanisms controlling ACE2 expression. I identify the farnesoid X receptor (FXR) as a direct regulator of ACE2 transcription in multiple COVID19- affected tissues, including the gastrointestinal and respiratory systems. I use this knowledge to optimise current experimental models for SARS-CoV-2 infection and I demonstrate that reduction of FXR signalling, with the over-the-counter compound z-guggulsterone (ZGG) and the off-patent drug ursodeoxycholic acid (UDCA), downregulates ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. I also show that UDCA-mediated ACE2 downregulation reduces susceptibility to SARS-CoV- 2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. I then illustrate that UDCA reduces ACE2 expression in the nasal epithelium in humans. Finally, I present a retrospective analysis using an independent patient cohort which identify a correlation between UDCA treatment and positive clinical outcomes following SARS-CoV-2 infection, including hospitalisation, ICU admission and death using retrospective COVID-19 registry data, and confirm these findings in a second independent cohort of liver transplant recipients. In conclusion, with the work described in this dissertation I identify a novel function of FXR in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, thereby paving the road for future clinical trials.Item Embargo Modelling pancreatic cancer in vivo with patient derived organoids & humanised mouse models for the investigation of immunotherapeutic treatment responseBareham, BethanyPancreatic ductal adenocarcinoma (PDAC) is the most prevalent form of pancreatic cancer, with a median survival time of <11 months. While incidences of PDAC continues to rise, treatment options remain limited and largely ineffective. The complex genomic, microenvironmental and immunophenotypic heterogeneity of PDAC tumours presents as a major obstacle in the assessment and development of novel therapeutic interventions. Immunotherapies aim to treat cancer through the activation and enhancement of anti-tumour immunity and have shown great success in the treatment of haematological cancers. However, they are limited in their ability to reach solid tumours such as PDAC. The discouraging success rate of immunotherapies in solid tumours may be due, at least in part, to the lack of representative preclinical models. Current preclinical models are heavily reliant on murine immune compartments or reconstitution with an allogeneic human immune system (HIS), and thus do not represent the immunophenotypic variations between patients. In this dissertation I outlined the development of a novel preclinical model of human pancreatic cancer (hPC) that incorporated patient derived organoids (PDOs) with autologous splenic mononuclear cell (SPMC)-derived HIS mice. I first assessed the immunophenotypic profile of lymphocytes, primary tumour tissue and adjacent tissue from patients with PDAC, intraductal tubular papillary neoplasm (ITPN) and ampullary carcinoma (AMP). I then outlined the derivation of PDAC, ITPN and AMP PDOs, and in vitro retention of parental morphology and key biomarkers. PDAC and ITPN PDOs were then implanted orthotopically into NSG-dKO mice to assess tumour establishment, recapitulation of parental tumour histology and gene expression. Mice bearing hPC PDO grafts were then humanised with either autologous or allogeneic SPMCs (hu-PDOs) for the assessment of graft survival, tumour immune infiltration, inflammation and upregulation of antigen presentation, pancreatic cancer, and immune pathways. Lastly, I treated autologous hu-PDOs with PD-1 blockade alone or in combination with CTLA-4 blockade to assess treatment linked inflammation, immune-mediated tumour killing and differential gene expression. Treatment response to immune checkpoint inhibition was further assessed with complementary in vitro autologous and allogeneic hPC PDO + SPMC derived T cell co-cultures. hPC PDOs in this study were established with a high level of success and formed in vivo grafts that were representative of the primary tumour. hPC PDO grafts appeared susceptible to both autologous and allogeneic immune attack, showing increased inflammation and graft rejection post allogeneic immune reconstitution. The autologous hu-PDO model showed increased immune-mediated tumour clearance in response to combined anti-PD-1 + anti-CTLA-4. Patient specific treatment responses to PD-1 blockade alone were observed in autologous hu-PDOs and appeared to correspond with in vitro co-cultures. Overall, the work presented within this dissertation demonstrates a novel in vivo model that is highly applicable to the field of immuno-oncology and shows promise in the modelling of individual treatment response to immunotherapies.Item Embargo Exogenous mesenchymal stromal cells in osteochondral repairSeah, Khuan Teck Matthew; Seah, Matthew [0000-0002-5850-125X]Despite osteoarthritis (OA) representing a large burden for health and social care systems, affecting approximately 10 million people in the UK alone, there remains no effective intervention capable of regenerating the damaged joint cartilage in OA and current clinical management for end-stage disease remains joint replacement surgery. There is therefore a need for a change in healthcare strategies to manage early disease, a shift that is likely to include cell-based regenerative therapies. Regenerative medicine and cell therapies are poised to have a tremendous impact on the future of medicine by delivering more effective and personalised treatment than are currently available today. Mesenchymal stromal cells (MSCs) are adult-derived, multipotent cells which have immunomodulatory effects, and can differentiate into a variety of cell types, including cartilage progenitor cells, making MSCs a candidate for musculoskeletal cell therapy. However, there has been several challenges to translating basic science research into new therapies and two pivotal questions remain unanswered: • Where does the MSC therapy go after administration? • What is its precise mechanism of action? To address these questions, the effects of an intra-articular injection of human bone marrow derived MSCs into a mouse knee osteochondral injury model were investigated in young C57Bl/6 mice, aged C57Bl/6 mice and GNL3 heterozygote mice. MSC treatment was associated with improved osteochondral tissue repair in young C57Bl/6 mice but this was more limited in aged C57BL/6 mice and GNL3 heterozygote mice. Cage activity monitoring was used to assess mouse recovery following surgery, and the administration of human MSCs was associated with significantly improved recovery following injury when compared to untreated controls. To better understand the mechanisms of action of an MSC therapy, a novel gene reporter system which utilises the organic anion transporting protein, Oatp1a1, was used for cell tracking. Using a lentiviral vector system, transfected MSCs expressing this cell surface protein can transport several imageable small molecules across the cell membrane, including the MRI contrast agent, gadolinium-ethoxybenzyl-DTPA (Gd-EOB-DTPA, PrimovistTM) which is licensed for clinical use. Cells were successfully imaged in vitro and in vivo as a proof-of-concept for this approach in the musculoskeletal system. Whilst imaging provided circumstantial evidence of MSC therapy fate, single cell RNAseq of retrieved cells (following injection into the mouse after osteochondral injury) was performed to elucidate what transcriptomic changes were important to driving tissue repair over time. Here, the data demonstrated that the exogenous human MSC therapy can be retrieved from both the repair tissue (in the epiphysis) and synovial tissue of the damaged knee joint at both 1 and 4 weeks after intra-articular injection. The retrieved human MSC therapy from the mouse joint tissue showed spatial and temporal transcriptional heterogeneity which was pronounced between the two tissue sources. Supported by mass cytometry data, the paracrine role of MSC treatment was further emphasises, as MSC therapy was associated with the induction of regulatory T cells (Tregs) in the mouse knee joint tissues. As Tregs are involved in skeletal homeostasis, more work needs to be performed to investigate the crosstalk that exists between MSCs and Tregs. Overall, the presented work shows that exogenous human MSC therapy in the mouse may improve both histological and clinical outcomes following osteochondral injury. Following intra-articular injection of human MSCs, the transcriptomes of the retrieved human cells were studied for the first time and their heterogeneity described. Subpopulations with different functional roles may be implicated in the different phases of osteochondral repair. As tissue digestion and isolation protocols are refined, it is likely that rarer populations may be retrieved from the mouse osteochondral model, but the data presented here offers insights into the interaction between the MSC therapy and the host cells, opening new avenues for the role which MSCs can play as a cell therapy in osteochondral repair.Item Open Access Generation and characterisation of primary and pluripotent stem cell-derived human hepatoblast organoidsZacharis, EkateriniUse of human pluripotent stem cells (hPSCs) has an unique potential for cell therapy since they could allow the production of almost any cell type at a large scale. Accordingly, many protocols have been developed to differentiate hPSCs into cells of clinical interest, including hepatocytes. However, these protocols are neither efficient nor robust and result in immature hepatocytes that can perform only some functions of adult liver cells. To optimise such protocols, it is imperative to understand the signalling pathways directing liver development. However, such knowledge is difficult to capture especially in human for obvious ethical reasons. As an alternative to primary tissues, our lab recently established a method to derive and grow primary human hepatoblast organoids (HBOs) isolated from human foetal liver tissues. These cells accurately and reproducibly recapitulate the hepatoblast identity in vitro, as they possess self-renewal and differentiation capacities, like the in vivo counterpart. Importantly, hepatoblasts are the first stem cell type of the liver and thus, they provide a new platform to study liver organogenesis. In this dissertation, the aim is two-fold. Firstly, to exploit the properties of HBOs for dissecting the signalling pathways that contribute to hepatoblast identity and secondly, to use this information to optimise a protocol for the generation of bona fide hepatoblast organoids in vitro from hPSCs. For the first part, I describe the effect of Wnt signalling pathway on hepatoblasts, showing that absence of Wnt cues results in diminished self-renewal capacity without directly inducing differentiation. Also, I demonstrate that Wnt presence can reactivate proliferation during the hepatoblast-hepatocyte transition. On the second aim, I develop an alternative differentiation protocol for the generation of an hPSC-derived hepatoblast cell population. To that end, I successfully generated a self-renewing pool of hepatoblast-like cells that display markers and characteristics similar to primary HBOs. Overall, the research presented in this dissertation has advanced the understanding of human liver development. Moreover, the generation of hepatoblast cells using our optimised protocol could provide a new source of liver cells for disease modelling and regenerative medicine.Item Open Access SMAD2/3 - FOXH1 regulation during cell cycle progression upon differentiationPerrin, MarionThroughout development, balance between differentiation and proliferation is key to ensure the proper induction of the initial layout of the future body plan and to later produce the correct amounts of each cell type. While the characteristics of cell cycle regulation in pluripotent and differentiated cells have been well described, the way they coordinate cell fate acquisition is only beginning to be elucidated. Indeed, the activity of TGFβ signalling via its downstream effector SMAD2/3 is directed by cell cycle regulators and especially cyclin Ds. Indeed, cyclin Ds direct cell fate propensity either by activating CDK4/6 activity, which results in blocking SMAD2/3 nuclear entry or by directly binding to developmental genes and co-recruiting epigenetic modifiers. Despite the importance of these interplay, the molecular mechanisms orchestrating transcriptional networks during progression of the cell cycle upon differentiation remain to be fully uncovered. In this dissertation, I combined cell cycle synchronisation mediated by nocodazole with our well-established endoderm differentiation protocol to study the coordination between cell cycle and differentiation. This approach first revealed that the transition from pluripotency to definitive endoderm requires two cell cycles. I then focused on the forkhead transcription factor FOXH1, a well-known SMAD2/3 binding partner during endoderm formation. Attempts to generate a human FOXH1 knockout stem cell line revealed that FOXH1 is essential for selfrenewal. Alternatively, its essential role in endoderm but not mesendoderm formation was confirmed with an inducible knockdown system against FOXH1. Going further, I showed that FOXH1 has a unique role in the first cell cycle which is required to form definitive endoderm in the second cycle. Finally, I investigated whether this unique feature could be explained by FOXH1 chromatin binding pattern by performing ChIP-Seq or by its dynamic interactions with co-partners by performing co-immunoprecipitation. Altogether, this work uncovers that FOXH1 orchestrates different cellular states in coordination with cell cycle progression and thus suggests that its role is modulated by the molecular context. Altogether, these data confirm that studying key factors of differentiation in the context of cell cycle progression can lead to unravelling new mechanisms involved in the temporal acquisition of cell identity.Item Open Access Characterisation of Alloantibody-Human Leukocyte Antigen Interactions to Improve Immunological Risk Assessment in Solid Organ TransplantationPriddey, AshleyAntibody mediated rejection remains a major challenge in solid organ transplantation, where the development of immunoassays for characterisation a recipient’s donor HLA-specific antibodies has revolutionised the field of solid organ transplantation. These immunoassays are essential for patient evaluation, immune monitoring, and antibody-related immunological risk assessment, however uncertainties concerning data interpretation often prevents determination of an antibody’s clinical significance. The initial aim of this research was to expand the knowledge on the properties of HLA-specific antibodies that govern their pathogenic potential. Luminex single antigen beads (SABs), SAB-C1q, flow cytometry (FC) and complement dependent cytotoxicity (CDC) assays were used to determine the reactivity and complement fixing capacities of human monoclonal antibodies, whilst biolayer interferometry (BLI) was used for real-time quantification of alloantibody-HLA kinetics. Outputs from each immunoassay were found to be dependent on antibody concentration, [Ab], where the degree of CDC was proportional to the antibody’s affinity, KD. The strongest affinity for each antibody was measured with the sensitising antigen. Solid-phase assays offered a higher sensitivity of antibody detection, however stronger interactions could not be distinguished from one another. In attempt to further assess the relationship between antibody-HLA interaction affinity and effector function, the ability to establish an in vitro model of alloantibody-mediated endothelial cell activation and tissue injury was explored. Lastly, using the principles of microfluidic diffusional sizing and Bayesian inference, microfluidic antibody affinity profiling (MAAP) was developed to enable in-solution, simultaneous determination of the KD and [Ab] of antibodies directly in patient serum samples. Quantification of purified monoclonal antibodies spiked into both PBS and blank serum provided a proof-of-principle before moving into HLA antibody-incompatible transplant sera. Here, MAAP was able to quantify the affinity and concentration of antibodies in real-life patient sera, whilst also providing examples of how this information may be used as a tool in clinic to improve transplant-related decision-making processes. Overall, this work provides evidence for the importance of antibody abundance and affinity in clinically relevant humoral alloresponses and, through development of MAAP, outlines a path towards in depth profiling of antibody responses in patient sera.Item Open Access MSC DERIVED EV IN REGENERATIVE MEDICINEHotham, WilliamOne of the greatest requirements of modern medicine is the ability to treat patients suffering from osteoarthritis (OA) and bone fractures. Currently, there is no long-term therapy for OA; symptoms can be managed with anti-inflammatories and analgesics until they worsen to the extent that the damage becomes debilitating, and joint arthroplasty, is necessitated. However, these replacements are not perfect; firstly, there is the need for surgery and secondly, if the patient is young, the prosthetic can deteriorate, engendering further surgery. Bone fractures are regularly seen in orthopaedic clinics and are commonly repaired using fixation techniques or biomaterials. After any intervention, the fracture site can remain compromised, potentially engendering re-fracture and/or further surgical involvement. Regenerative strategies for both OA and bone fracture aim to alleviate pain, whilst maintaining or restoring damaged tissues to healthy states. Mesenchymal stem/stromal cells (MSC) are thought to facilitate tissue repair via either progenitor or secreaome functions. BM-MSC have, in previous work, been investigated as a therapy for OA via either their direct application or through their secreted Extracellular Vesicles (EV). In this study, MSC have been successfully isolated from bone marrow, and from these isolated cells, EV have been captured and characterised. The isolated EV have been shown to be readily internalised by chondrocytes and, in order to determine the method of EV internalisation by chondrocytes, in vitro drug inhibition studies were performed on labelled EV. Via inhibition of the caveolin dependent endocytosis pathway, EV uptake was prevented, thus indicating that this method of endocytosis is the method of EV internalisation. In regenerative medicine for knee OA, it is likely that MSC and EV would be injected into the knee. In order to determine if the MSC and EV would reside in the joint, both were labelled with gold nanostars and Supra Magnetic Iron Oxide Nanoparticles (SPION). These labelled cells and EV were then injected into a sheep stifle 1 week post creation of an OA model (meniscal transection model). These labelled cells and EV could then be seen within the knee for up to 4 weeks post injection, as ascertained via Magnetic Resonance Imaging (MRI) and MultiSpectral Optoacoustic Tomography (MSOT). Upon evaluating the regenerative effects of the MSC and EV, no difference in cartilage damage could be seen. During bone fracture, MSC and osteoblasts are recruited to the site of injury. Bioglasses have been used previously as a material to improve bone repair through the release of ions and conditioning the local environment. Our work has shown that conditioned media from bioglasses can influence both MSC and osteoblasts to augment the bone repair process. Through screening bioglasses on MSC and osteoblasts, the potential for bioglasses to alter MSC derived EV to promote osteogenesis has been shown. As a conclusion, this study has shown that the BM-MSC are a source of EV, and that both the MSC and EV can potentially be used in a musculoskeletal scenario of regenerative medicine.Item Open Access Lymphocyte subset variability between human lymphoid tissuesSiu, Jacqueline; Siu, Jacqueline [0000-0001-5181-407X]Most adaptive immune lymphocytes are in the lymphoid tissues. However, studies of human lymphocytes to date have primarily investigated cells from the blood, and lymphoid tissues remain poorly studied. This thesis explores the hypothesis that there is local variation of B cell subsets between the tissues that are micro-anatomically different and receive antigenic stimulation via different routes. Matched samples of appendix, mesenteric lymph nodes (mLNs), and spleen from human cadaver were analysed by mass cytometry (cytometry by time of flight [CyTOF]) for deep phenotypic profiling and tissue-specific subset variation. Populations of B and T cells that had significant abundance differences between the three tissues were identified in an unsupervised manner. The subsets that varied significantly in abundance between tissues included: germinal centre (GC) B cells, marginal zone (MZ) B cells, IgM-only B cells, IgA+ B cells, follicular helper T cells and regulatory T cells, as well as CD4+ and CD8+ effector memory cells. These differentially abundant subsets were further characterised by their different marker expression profiles between tissues, and results were confirmed using a second supervised analysis method. Most notably from the unsupervised analysis and subsequently the supervised analysis, MZ B cells were further divided into two populations based on their tissue-specific abundance and distinct phenotype including differences in CCR7 and BAFF-R expression. IgM-only B cells also included two populations with different phenotype and tissue-specific abundances. Single-cell RNA sequencing was used to further investigate the differentiation, functional, and dissemination differences of these subsets between lymphoid tissues. The two MZ B cell populations were transcriptomically distinct and had significant tissue-specific differential abundances. Within each tissue, B cell clones from the CCR7+ MZ B cell subset were significantly associated with IgM-only subset whereas the other CCR7- MZ B cell subset had a greater association with activated naive and double negative B cells. Between the paired tissues, members of the same B cell clone were observed in multiple tissues. Clones of CCR7+ MZ B cells and IgM-only B cells were more disseminated between tissues than other subsets including the CCR7- MZ B cells. On the other hand, transitional and naive B cells only had significant correlation within the same tissue and not between tissues implying local maturation in tissues. Overall work in this thesis identifies regional variation in B cell subset abundance, tissue based local B cell differentiation and also clonal links between B cell subsets in distant human tissues sites.Item Open Access Long-term expansion, genomic stability, in vivo safety and immunogenicity of adult human pancreas organoidsGeorgakopoulos, Nikitas; Georgakopoulos, Nikitas [0000-0002-1879-6583]Pancreatic organoid systems have recently been described for the in vitro culture of pancreatic ductal cells from mouse and human. Mouse pancreatic organoids exhibit unlimited expansion potential, while previously reported human pancreas organoid (hPO) cultures do not expand efficiently long-term in a chemically defined, serum-free medium. The aim of this project was to generate a 3D culture system for long-term expansion of human pancreas ductal cells as hPOs to serve as the basis for studies of human pancreas ductal epithelium, exocrine pancreatic diseases and the future development of a genomically stable replacement cell therapy for diabetes mellitus. hPOs can be generated and expanded in a chemically defined, serum-free, human pancreas organoid culture medium with high efficiency from both fresh and cryopreserved primary tissue. Crucially, the hPO culture system also supports the establishment and expansion of these organoids in a chemically defined, modifiable and scalable, biomimetic hydrogel thus facilitating their translation into the clinic. Moreover, hPOs expanded over months in culture maintain their ductal morphology and biomarker expression of the primary tissue while they can also be expanded from tissue with underlying disease such as type 2 diabetes (T2D). This project further demonstrates that hPOs maintain stable chromosomal numbers following long-term in vitro culture, especially when compared to an established positive tumour organoid control. When clonal hPOs were subjected to whole genome sequencing (WGS), they maintain genomic integrity following culture and acquire less mutations in culture than iPSC-derived cultures, resembling other reported organoid systems. Xenografts of hPOs survive long-term in vivo when transplanted into the pancreas of immunodeficient mice. Notably, mouse orthotopic transplants show no signs of tumorigenicity. To further assess their clinical applicability, hPOs were assessed for the expression of antigenic molecules, demonstrating that under in vitro conditions that mimic the inflammatory milieu, hPOs can upregulate HLA Class II. Moreover, this project utilises recently established human immune system (HIS) mice to interrogate in vivo hPO immune rejection under autologous and allogeneic conditions. Lastly, preliminary experiments show that hPOs can be genetically manipulated to express GFP and Luciferase which can be used for in vivo survival and immune rejection tracking.Item Open Access Mapping Human Liver Development using Single-Cell Transcriptomics to Reveal Pathways Directing Stem Cell FatesWesley, Brandon; Wesley, Brandon [0000-0003-0530-329X]The liver is an organ with a diversity of essential functions including xenobiotic removal, bile acid production, storage of iron and vitamins, and metabolism of glucose and fatty acids. Hepatocytes, comprising the liver parenchyma, fulfil these functions and form an extensive network with non-parenchymal cholangiocytes, endothelial cells, Kupffer cells, and hepatic stellate cells. Disorders affecting hepatocytes are life threatening and increasing in prevalence12, with end-stage treatment relying upon a limited supply of liver donations for transplant. Consequently, the production of hepatocyte-like cells (HLCs) from human pluripotent stem cells (hPSCs) for clinical applications such as cell-based therapies and toxicology screens has become a critical research focus. Therapeutic advances, especially in regenerative medicine, are currently hampered by the lack of knowledge concerning how human hepatic cells develop. Here, I addressed this limitation by describing the developmental trajectories of different cell types comprising the human fetal liver using single-cell transcriptomics (scRNA-seq). These analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing critical, supportive roles during organogenesis. We also uncovered a novel progenitor of sinusoidal endothelial cells and hepatic stellate cells in the early liver, thus providing an explanation for the similar, complementary roles these cells place in liver extracellular matrix formation and vascularisation in support of hepatoblast growth and differentiation during development. We utilised this information to derive bipotential hepatoblast organoids and used this novel model system to validate the importance of key signalling pathways and developmental cues. Currently, hPSC-derived hepatocytes do not directly resemble their primary tissue counterparts and have limited clinical applicability. We used scRNA-seq to compare HLCs with primary development to identify important regulators involved in late-stage maturation of HLCs and allow us to improve the derivation of these cells in vitro. This marks an important application of development biology to inform stem cell differentiation for the production of clinically-relevant cells.Item Controlled Access Creation of organoid systems to model human endodermal organ development in vitroRoss, AlexanderOrganoid systems have revolutionised the study of adult stem cells and organ homeostasis. However, there have been limited attempts to use the technology to study human organ development. The work of this thesis established two novel hepatic organoid systems; one based upon the archetypal liver stem cell, the hepatoblast, whilst the other was based upon a hitherto undescribed biliary progenitor cell. The work continued with exploration of the development of the human intestine, characterising how intestinal organoids develop in vitro, and going on to assess human intestinal maturation in vivo at single cell resolution. First, the developing human liver was characterised during the first trimester and the cellular composition at this crucial developmental timepoint was investigated at single cell resolution. The hepatoblast organoids (HBO) were then demonstrated to retain their hepatoblast profile in culture and demonstrated the functional capacity of this fetal cell-type. Exploiting this new model, the molecular processes of differentiation were then explored as the HBO were demonstrated to have bipotential capacity to generate hepatocytes and cholangiocytes. Furthermore, these organoids were then transplanted into mice and were able to differentiate into complex functional tissues. Next, a newly discovered stem cell was characterised and termed the ‘biliary progenitor cell’. This new cell type was able to generate three-dimensional organoids, fetal biliary organoids (FBO), that could be transplanted into mice and provide insight into biliary development. Next induced pluripotent stem cell (iPSC)-derived and primary fetal intestinal derived organoids were characterised against purified primary intestinal epithelial tissue, demonstrating a dynamic transcriptome of fetal organoids in vitro and a lack of tissue specificity of iPSC-derived organoids. The fetal and paediatric intestine were assessed at single cell level to assess the cell types and developmental pathways involved in the process of development, and this information was then utilised to try to model development in vitro using small molecule stimulation of fetal intestinal organoids. The work described in this thesis develops our understanding of human endodermal organ development. The newly described organoid models present an exciting opportunity to model human liver development, generate cells for drug-screening, and offer the potential for cell-based therapies, whilst the exploration of the human intestine during development may aide our understanding of how such pathways may be abrogated in developmental defects, or reverted to during disease states.Item Open Access Examining the regional physiology of the transplant kidney during normothermic machine perfusion.(2021-07-24) Adams, Thomas David; Adams, Thomas [0000-0003-0693-3023]Ischaemia-reperfusion injury (IRI) is an unavoidable consequence of deceased-donor kidney transplantation that has a profound effect upon both immediate and long-term graft function. Disruption to microvascular perfusion (MVP) and tissue oxygenation (PtO2) are central to the development of IRI, but detailed regional pathophysiology remains unresolved in the context of renal transplantation. Normothermic machine perfusion (NMP) is a novel preservation technology that aims to improve pre-implantation kidney quality, but may also be used experimentally to investigate transplant reperfusion. My PhD set out to determine the optimal perfusion conditions to improve kidney quality, and to investigate the underlying heterogeneity in pathophysiology within regions of the kidney which may impact kidney quality. Firstly, we used a porcine model of donor kidney retrieval, NMP, and simulated reperfusion to test the hypothesis that reducing current NMP perfusate oxygenation (PPO2) from superoxic levels would improve renal function and reduce reperfusion injury. In kidneys exposed to either short or long cold ischaemic times, reducing PPO2 from the clinical standard to normoxic or hypoxic PPO2 altered oxygen kinetics during NMP but did not influence tubular function, clearance, urine output, or biomarkers of renal injury during simulated reperfusion. Secondly, we used porcine and human models of transplant reperfusion to test the hypothesis that the renal medulla would be disproportionately affected by tissue hypoperfusion, hypoxia and acute inflammation. In a porcine reperfusion series, PtO2 and MVP were significantly altered following IRI when compared to pre-ischaemic baselines, with greater variation and heterogeneity seen in the medulla than in the cortex. In a human reperfusion series, there was widespread initial microcirculatory disruption, persistent lower medullary PtO2 and a distinct medullary inflammatory environment. In summary we have described novel porcine and human renal medullary physiology and inflammation during transplant reperfusion that highlight the need for medulla-specific strategies to ameliorate IRI. We have further determined changes to renal physiology and injury in response to perfusate oxygenation in a novel preservation technology that may guide clinical implementation.Item Open Access The Immunogenicity of Cholangiocyte Cellular TherapiesTysoe, OliviaCholangiocytes are the epithelial cells of the biliary system, responsible for the transport and modification of bile. Cholangiocyte disorders, known as cholangiopathies, are a diverse group of life-threatening conditions characterised by cholestasis, ductopenia and eventual liver failure. There are currently no curative treatments for cholangiopathies aside from liver transplantation and while individual cholangiopathies are rare, together they account for a third of adult and 70% of paediatric liver transplants. There is a scarcity of suitable organs for transplantation, however, so development of cholangiocyte cellular therapies capable of replacing or repairing damaged bile ducts would have significant therapeutic value. The safety of such therapies must be assessed before they are suitable for clinical translation and an essential component of that is an evaluation of immunogenicity. In this dissertation I investigated the immunogenicity of cholangiocyte organoids (COs) from an established system developed within the Vallier lab. I first assessed the survival of COs in vivo and generate CO lines expressing luciferase to allow for real-time bioluminescent imaging in vivo. I characterised the expression of HLA molecules on COs compared to primary cholangiocytes and after exposure to a series of pro-inflammatory environments and demonstrated that expression of HLA molecules is reduced in COs compared to primary cholangiocytes but that exposure to pro-inflammatory cytokines restored high levels of HLA expression. I also showed that physiologically-relevant concentrations of pro-inflammatory cytokines were sufficient to upregulate HLA class II expression on COs. To assess the immunogenicity of COs in a transplant environment I used two humanised mouse models. I demonstrated that these models can induce an immune response against allogeneic COs and compared the response to allogeneic and autologous COs in both models. I identified HLA-matched CO lines and immune cell donors and investigated the impact of HLA-matching on the immunogenicity of CO allografts in a humanised mouse model. Overall, the work presented in this dissertation advances the understanding of the immunogenicity of cholangiocyte organoids, both in terms of their expression of immunogenic antigens and the response towards COs within both an allogeneic and autologous transplantation setting.Item Open Access Germinal centre autoantibody responses following heart transplantation(2020-10-24) Qureshi, MuhammadThe development of humoral autoimmunity following organ transplantation is increasingly recognised, but of uncertain significance. I address the key question whether autoimmunity contributes independently to chronic graft rejection. In a MHC class II-mismatched murine model, I show that antinuclear autoantibody responses were initiated upon graft-versus-host allorecognition of recipient B cells by donor CD4 T cells transferred within the heart allograft. Rejected allografts displayed features of chronic humoral vascular rejection, with rejection mediated by long-lasting germinal centre (GC) responses, whose maintenance was dependent upon additional, cognate help from recipient T follicular helper (TFH) cells, and which diversified to encompass responses against vimentin autoantigen. Heart grafts transplanted into stable donor/ recipient mixed haematopoietic chimeras also provoked GC autoimmunity and were rejected with similar tempo, indicating that autoantibody can mediate graft damage despite host tolerance to alloantigen. An autonomous effector role for autoantibody was further suggested by the demonstration that F1 (BALB/c x C57BL/6) recipients reject parental BALB/c heart allografts, with rejection again dependent upon GC autoimmunity and help from recipient TFH cells. Thus GC autoantibody responses contribute to graft rejection independent of host adaptive alloimmunity, through a mechanism by which donor CD4 T cells initiate an auto reactive GC reaction that is then maintained and propagated by host TFH cells. The demonstration that one set of CD4 T cells triggers autoimmunity, but that a second subset of TFH cell is responsible for maintaining the response as a germinal centre reaction, has wider implications for our understanding of the pathogenesis of autoimmune disease.Item Open Access UNDERSTANDING ANATOMICAL PERFUSION AND STRATEGIES TO OPTIMIZE VASCULARITY IN FREE TISSUE TRANSFER FOR AUTOLOGOUS BREAST RECONSTRUCTION USING THE DEEP INFERIOR EPIGASTRIC ARTERY PERFORATOR (DIEP) FLAPMohan, Anita; Mohan, Anita [0000-0001-9131-1047]Breast cancer is the commonest cancer that affects women in the United Kingdom (UK). Autologous free tissue transfer using abdominal tissue remains an excellent option for breast reconstruction following mastectomy, given greater availability of tissue and lower donor site morbidity associated with muscle-sparing approaches (perforator-based). This research evaluated microvascular anatomy of Deep Inferior Epigastric Artery Perforator (DIEP) flaps, the role of linking vessels on dynamic perfusion in bilateral breast reconstruction and strategies to augment flap vascularity. For the ex-vivo anatomical studies, three and four-dimensional computed tomographic angiography (CTA) were used to evaluate patterns of the microvascular blood supply of individual perforators and corresponding perfusion patterns in the hemi-abdomen. This was combined with an in-vivo clinical study of women undergoing bilateral DIEP breast reconstruction following mastectomy, where both preoperative CTA and intra-operative Laser-Assisted Indocyanine Green Fluorescence Angiography (LA-ICGFA) were used to evaluate perforator anatomy and dynamic perfusion zones of individual perforators. Finally, an experimental in-vivo animal model was used to investigate strategies of pretreatment of perforator flaps with negative pressure wound therapy to augment vascularity of perforator flaps prior to flap harvest. The vascular territories of individual perforator within hemi-DIEP flaps demonstrated variable patterns with unique patterns of perfusion. Concepts including early capture of large calibre direct linking vessels to adjacent perforators or the superficial inferior epigastric artery (SIEA) territories, mostly found in the supra-scarpa’s and subdermal layers of the flap, played a key role in defining overall perfusion area and dynamic perfusion patterns not previously described. In conclusion, this work reported the characterization of the microvasculature within abdominal based perforator flaps to better understand the variation in dynamic perfusion. It also explored the potential role of non-invasive negative pressure treatment to augment flap perfusion that may be translated into the clinical setting.Item Indefinitely restricted Restoring Function, Transporting, Assessing and Transplanting the Donation after Circulatory determined Death (DCD) Heart(2019-10-14) Messer, Simon[Redacted]