Human mesenchymal stromal/stem cells (MSC) isolated from aborted first trimester fetal bone marrow (BM) hold promise for use in tissue engineering applications and cell-based therapies due to their advantageous characteristics compared to their adult BM-MSC counterparts; faster growth kinetics, active telomerase, smaller size and higher differentiation potency. However, their isolation is restricted ethically and technically and therefore there is a need to identify a cell source with high therapeutic potential that is easily accessible in the clinic without ethical restrictions. The placenta is a potential source of readily-obtainable chorionic stem cells (CSC) throughout pregnancy. The aim of my thesis was to study the evolution of the CSC phenotype (i.e. change of stem cell characteristics) during gestation and to assess their capacity for bone repair in a mouse model of osteogenesis imperfecta (oim). I hypothesised that early fetal placental chorionic stem cells (e-CSC) were physiologically superior to their term counterparts, late chorionic stem cells (l-CSC), with advantages for use in fetal stem cell therapy of osteogenesis imperfecta (OI). In the first chapter I showed that e-CSC and l-CSC shared a common phenotype, which was intermediate between adult BM-MSC and human embryonic stem cells, with characteristics of both. I also showed the phenotype of CSC evolves during gestation with e-CSC displaying characteristics of an earlier state of stemness compared to l-CSC, such as smaller size, faster kinetics, unique expression of OCT4A variant 1 and higher levels of Nanog, Sox2, c-Myc and Klf4 expression, as well as the capacity to differentiate into lineages of the three germ layers through embryoid body formation. In the second chapter I showed the more primitive in vitro characteristics of e-CSC translated to higher tissue repair in vivo compared to l-CSC; accelerating healing when applied to a skin wound and increasing bone quality and plasticity following neonatal transplantation into the oim model. I subsequently used the oim model to assess the therapeutic potential of e-CSC for use in fetal cell therapy for OI. I showed compared to non-transplanted mice, oim transplanted with e-CSC had a two third reduction in fracture incidence, more ductile bones and increased trabecular bone volume. Prevention of fractures was attributed to the differentiation of exogenous cells to osteoblasts that expressed mature osteoblast genes and synthesised human type 1 collagen (COL1A2). However, this beneficial effect may also have resulted from an indirect effect of the transplanted cells on the endogenous cells of the host mouse, since transplanted mice had upregulation of endogenous genes involved in endochondral ossification and osteoblast differentiation. Altogether, my thesis characterises early and late human fetal chorionic stem cells, providing insight into the ontogenesis of stemness phenotype during fetal development and shows the first trimester placenta is a practical source of stem cells that can be used to treat OI during bone development.

Background: Chest pain makes up a quarter of medical admissions in the United Kingdom. A diagnostic strategy that prevents unnecessary hospital admission in a large proportion of this patient group would have significant benefits for healthcare services by reducing hospital admission rates, ED overcrowding, duplication of staff time and resource use. A clinically applicable protocol that allows the discharge of a significant proportion of patients after a single blood draw at presentation to the emergency department remains an attractive yet elusive goal. Objective: To establish whether a novel accelerated diagnostic protocol (ADP) for suspected acute coronary syndrome (ACS) could successfully identify low-risk patients suitable for discharge after a single high-sensitivity troponin T (hs-cTnT) taken at presentation to the Emergency Department (ED). Comparison of the diagnostic accuracy of this ADP with strategies utilising initial undetectable hs-cTnT was made. Methods: This prospective observational study evaluated the ability of the Triage Rule-out Using high-Sensitivity Troponin (TRUST) ADP to identify low-risk patients with suspected ACS. The ADP incorporated a single presentation hs-cTnT of < 14ng/L, a non-ischaemic electrocardiogram and a modified Goldman risk score. Diagnostic performance of the PhD Thesis. Dr Edward W. Carlton ADP was compared with the detection limit cut-offs of hs-cTnT (< 5ng/L and < 3ng/L). The primary endpoint was major adverse cardiac events (MACE) occurring within 30 days. Results: 960 participants were recruited, mean age 58.0 years, 97 (10.1%) had MACE. The TRUST ADP classified 382 (39.8%) as low-risk with a sensitivity for identifying MACE of 99.0% (95%CI 93.7-99.9). Hs-cTnT detection limits (< 5ng/L and < 3ng/L) had a sensitivity of 96.8% (90.6-99.2) and 98.9% (93.8-99.9) respectively. The TRUST ADP identified more patients suitable for early discharge at 39.8% vs 29.3% (<5ng/L) and 7.9% (<3ng/L) (P<0.001) with a lower false-positive rate for MACE detection; specificity 44.1% (95%CI 43.6-44.3) vs 32.3% (95%CI 31.6-32.6) and 8.7% (95%CI 8.1-8.8) respectively. Conclusion: The TRUST ADP, which incorporates structured risk-assessment and a single presentation hs-cTnT blood draw, has potential to allow early discharge in 40% of patients with suspected ACS and has greater clinical utility than undetectable hs-cTnT strategies.

Multipotent stromal cells/mesenchymal stem cells (MSCs) can differentiate into multiple lineages including osteogenic and adipogenic cells. Wnt signalling has been implicated in controlling MSC fate, but the mechanism is unclear and apparently conflicting data exists. Here I show that a glycogen synthase kinase 3β inhibitor, AR28, is a potent activator of canonical Wnt signalling using β-catenin translocation studies and TCF-reporter assays. AR28 induced axis duplication and secondary regions of chordin expression in Xenopus laevis embryos, when injected into the ventral marginal zone, indicative of canonical Wnt signalling. When human MSCs were grown under adipogenic conditions, AR28 caused a significant dose-dependent reduction in FABP5/BODIPY double-positive cells with a corresponding rescue of proliferation. In assays to determine the effects of AR28 on MSC osteogenesis using standard differentiation inducers (β-glycerophosphate, L-ascorbic acid and dexamethasone), AR28 caused a significant decrease in alkaline phosphatase (ALP) activity compared to vehicle controls, indicative of a reduced osteogenic response. However, when using mild osteogenic stimulation, excluding dexamethasone, increases in both ALP and Alizarin Red mineral staining were identified following AR28 treatment, with corresponding increases in proliferation and cell number. This AR28-induced osteogenic response was blocked by mitomycin C, identifying cell proliferation as an important step in Wnt-induced osteogenesis under these conditions. Pre-treatment of MSCs with AR28 for 7 days before osteogenic induction also increased ALP activity and mineralisation. BMP2 treatment of MSCs was capable of inducing both osteogenic and chondrogenic differentiation, to which AR28 caused a switch towards the osteogenic lineage, with synergistic increases in ALP. AR28 simultaneously caused a decrease in the chondrogenic differentiation of MSCs treated with BMP2 through the down regulation of Sox9 transcription. Together these results highlight the potential of GSK3β inhibitors as therapeutic modulators of canonical Wnt signalling, and there use to treat a multitude of bone related disorders.

Background The national strategy for palliative care in Vietnam is to integrate palliative care to cancer care and primary care. In Camau province, 70% of rural family doctors are involved in palliative care provision for cancer patients in rural areas. However, 85% have no palliative care training before or after graduation. Objectives • To evaluate the outcomes of an educational intervention in palliative care for rural family doctors in terms of perceived knowledge, confidence and practice behaviour. • To explore and compare views and experiences of hospitalised patients with those receiving palliative care in the community. Methodology A mixed methods approach was conducted using a pre- and post-workshop design. Doctors completed pre- and post-workshop questionnaires, and patients in two settings (hospital and home) participated in semi-structured interviews. Doctor data was analysed using an independent sample t-test to compare scores of knowledge and confidence before and after the workshop. Thematic analysis was used to analyse patient interviews. Findings The overall response rate from doctors was 67%. Pain management, fatigue and communication were the most requested topics for the education workshop (85%, 50%, 45%). As a result of the workshop, one-third indicated an increase in morphine prescribing and caseloads in their workplace. There was a statistically significant increase in scores of knowledge (p = 0.004) and confidence (p <0.001) after workshop. Patients’ choice of place of care was based on their satisfaction with the care, relationship with staff, the severity of symptoms and convenient access. Hospitalised patients claimed that local family doctors lacked expertise and enthusiasm in palliative care provision. The local palliative care service was perceived as acceptable by patients interviewed at home. Conclusions The multi-faceted intervention improved the knowledge, confidence and behaviour of family doctors. Satisfaction with generalist palliative care from the trained doctors was indicated by patients interviewed in the community.

No description available.

During the course of development, specification of the three embryonic germ layers, ectoderm, mesoderm and definitive endoderm (DE), is a critical process by which pluripotent cells acquire the temporal and spatial information needed to form specialised tissues. Of these initial germ layers, the DE arises during gastrulation, which latterly gives rise to the liver, pancreas, lung and epithelial lining of the digestive tract. Elucidation of the molecular mechanisms that govern DE specification not only facilitates our understanding of developmental biology but also aids in the differentiation of human pluripotent stem cells to specific cell types for disease modelling and regenerative therapies. DE formation is largely driven by the cooperation of Activin/Nodal and Wnt/β-catenin signalling, however recent evidence has additionally implicated PI3K/Akt signalling in modulating this process. Although it has been previously reported that PI3K activation acts to antagonise the in vitro differentiation of DE, the molecular mechanisms responsible for this effect remains unclear. To address this issue, this study utilises pluripotent human embryonic stem cells (hESCs) as an in vitro model to interrogate the molecular underpinnings of DE formation through a fully defined differentiation protocol. Modulation of PI3K activity was found to reciprocally downregulate the activation of Smad2/3, which was mitigated in the presence of the PI3K inhibitor LY294002 (LY). Suppression of PI3K/Akt signalling prolongs the activation of Smad2/3 in response to Activin, promoting their nuclear accumulation and the enhancement of transcriptional activity, resulting in the upregulation of mesendoderm and DE gene expression. Activation of PI3K negatively impacts the activity of Smad2/3 via phosphorylation of the Smad2/3 linker T220/T179 residue, which is fully independent of Erk and CDK activity. Phosphorylation of this residue induces the recruitment of the E3 ubiquitin ligase Nedd4L to activated Smad2/3, which in turn promotes their ubiquitin-mediated degradation and attrition of activity. Inhibition of mTORC2 activity by both inhibitor supplementation and genetic manipulation, rather than modulation of Akt or mTORC1 activity, recapitulates the LY-mediated reduction of T220/T179 phosphorylation and increases the duration of Smad2/3 transcriptional activity, promoting a more robust mesendoderm and endoderm differentiation. These findings reveal a new and novel connection between the PI3K/mTOR and TGFβ/Activin pathways, which will greatly impact our understanding of both cell fate determination and the preservation of normal cellular functions. Notably, identification of mTORC2 as a key player in the regulation of this differentiation provides new avenues through which hESCs differentiation protocols can be improved for both regenerative and biomedical applications.

Adult mesenchymal stem cells (MSC) are multipotential cells which can differentiate into various cell types thus giving them utility in tissue engineering regenerative medicine and cell-based therapies. Use of allogeneic MSC offers the prospect of 'off-the-shelf' tissue engineered products and tailor-made 'designer'-therapies with huge benefits to patients and industry. However, their immunological properties are poorly defined and this has hampered their potential clinical utility. Recent studies have suggested that allogeneic MSC are immunoprivileged in addition to possessing immunosuppressive properties. Interestingly, dermal fibroblasts (DF) have also been suggested to be functionally similar to MSC although their immunological properties are controversial. This study sought to systematically investigate the immunomodulatory properties of allogeneic MSC, namely (i) immunosuppression and (ii) (immunogenicity of allogeneic MSC before and after differentiation using an allogeneic mouse model which employed two genetically distinct strains; Balb/c (H2 d) and C3H (H2 k) which are used as responder (recipient) and stimulator (donor) respectively. Immunosuppressive properties were investigated using adaptations of the one-way mixed lymphocyte reaction (MLR) while the immunogenicity was tested using the lymphocyte transformation assays (LTA). DF were similarly tested for comparison. MSC and DF were successfully isolated from the bone marrow and abdominal skin respectively of Balb/c and C3H mice, expanded and characterised by flow cytometry. MSC expressed MHC I, Sca-1, CD29, CD44, CD90.2 and CD105 but not MHC II, CD11b, CD34, CD45, CD80 and CD86. In contrast to MSC, DF were negative for CD29, CD44 and CD105. Both MSC and DF successfully differentiated into adipocytes, chondrocytes and osteocytes in the tri-lineage test; the benchmark test for stem cells. Various parameters including medium changes, cell viability following mitotic inactivation of stimulator cells, type and concentration of serum for medium supplementation, responder to stimulator cell ratio and total cell numbers were tested to determine appropriate conditions for carrying out the MLR and LTA. With regard to the immunosuppressive properties, both syngeneic and allogeneic MSC significantly suppressed one-way MLR and two-way MLR. DF also exhibited similar suppressive potency. In LTA, allogeneic, but not syngeneic MSC stimulated Balb/c lymphocyte proliferation. Interestingly, both syngeneic and allogeneic DF failed to stimulate lymphocyte proliferation. Following chondrogenic differentiation, both syngeneic and allogeneic MSC and DF suppressed one-way MLR, albeit with reduced potency. With regard to their immunogenicity, allogeneic MSC and DF, but not syngeneic MSC and DF significantly stimulated lymphocyte proliferation. Therefore, it was concluded that both allogeneic MSC and DF possess immunosuppressive properties before and after differentiation. Undifferentiated and differentiated allogeneic MSC and differentiated DF however, may not be immunoprivileged. Thus the clinical utility of allogeneic MSC may be limited by their immunogenicity.

iPSC technology has the potential to generate patient specific pluripotent cells for use in stem cell therapies and disease modelling. However, current reprogramming methods utilise retroviral vectors, which integrate into the host cell genome disrupting normal gene function. Transient gene delivery methods have been investigated as safer alternatives but demonstrate poor reprogramming efficiency. Therefore, there is a requirement for iPSC gene-delivery vectors which are capable of providing prolonged transgene delivery without integrating into the host cell genome. Herpesvirus saimiri (HVS) is a prototype member of the gamma-2 Herpesviridae, and is capable of persisting as a non-integrated episome in dividing and differentiating cell populations, providing sustained transgene expression. Therefore, this thesis has explored the potential of HVS-based vectors for iPSC generation. This thesis focuses on the generation of three HVS-based vectors expressing the iPSC reprogramming transgenes, Oct4, Lin28 and Nanog. The potential of these HVS-iPSC vectors to reprogram both primary and cancerous cells has been investigated. Human primary Neural Stem Cells demonstrated cytopathology upon transduction with HVS-based vectors, indicating the need for further improvements to the biosafety of these vectors before they are suitable for the generation of clinical grade iPSCs. However, reprogramming attempts utilising the Ewing‟s sarcoma cell line, A673 cells, successfully generated induced pluripotent cancer stem cell (iPC)-like colonies upon transduction with all three recombinant vectors. Results from detailed analysis of these colonies suggest some form of reprogramming has taken place, albeit incomplete, as indicated by elevated expression of Oct4, Rex1 and Klf4; in addition to positive alkaline phosphatase staining and SSEA4 expression. Furthermore, these iPC-like colonies were capable of differentiation down the ectodermal lineage, as evidenced by upregulation of MSX1, MAP2, and Nestin. In conclusion, this thesis has demonstrated the potential of HVS-based reprogramming vectors through the generation of A673-iPCs.

No description available.

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for numerous haematological malignancies. Graft-versus-host disease (GVHD) is the major complication causing mortality and is classified into acute (aGVHD) and chronic. MicroRNAs play a significant role in inflammation and have reported potential as biomarkers of different diseases. This study has investigated the role of microRNAs in allo-HSCT outcomes and had two main aims; (1) identification of microRNAs specific to the aGVHD target organ, skin and (2) an investigation into the role of immune specific miRNAs (miR-146a and miR-155) in peripheral blood. Initially, pathway mining was performed on a list of 18 genes that were shown previously, to have deregulated expression levels with regards to GVHD. The pathway mining identified specific immunological pathways in relation to the genes and potential microRNA targets. Global microRNA profiling was performed on a discovery cohort that identified a signature microRNA list in skin biopsies obtained from patients at the time of cutaneous histopathological aGVHD onset (grades I-III) and healthy volunteers. Twelve microRNAs were selected for further validation and it was shown that miR-34a-5p, miR-34a-3p, miR-503-5p and let-7c-5p were elevated and significantly involved in allo-HSCT outcomes. There was an interaction between miR-34a-3p and miR-503-5p which was significantly diagnostic of aGVHD and let-7c-5p was significantly predictive of disease relapse. MiR-34a-5p protein targets; p53 and c-Myc were then evaluated in the same cohort. MiR-34a-5p expression levels and cells stained positively for p53 were significantly correlated in the epidermis. Preliminary optimization of miR-34a-5p knockdown study was successfully conducted which showed promising results in the reduction of T cell proliferation. The whole blood study showed that miR-146a-5p and its interaction with miR-155-5p was predictive of aGVHD incidence in pre-disease onset (Day+28) samples. Interestingly, the expression levels of miR-146a-5p and miR-155-5p negatively correlated with SPI1 (PU.1). In conclusion, these investigations showed that (1) the microRNAs studied in this investigation may regulate the expression levels of the selected 18 genes, (2) microRNA expression levels in clinical skin biopsies obtained at the time of aGVHD onset could potentially be used as diagnostic biomarkers for aGVHD and as predictive biomarkers for overall survival as well as relapse and (3) miR-146a-5p and miR-155-5p expression levels in whole blood could be used as predictive biomarkers for aGVHD incidence.