Ischaemic stroke occurs as a result of a blockage in one of the brain’s arteries, leading to neuronal injury and death. Although stroke is a major cause of death and disability, there is no widely available treatment. Inflammation occurs in the brain and in the periphery following stroke, and both contribute to the ischaemic damage. Leukocytes such as neutrophils are key mediators of brain damage and inflammation, particularly in the presence of systemic inflammatory challenges such as interleukin-1 (IL-1). Tumor necrosis factor-stimulated gene 6 (TSG-6) is a potent inhibitor of neutrophil migration, and also modulates the immune response by dampening expression of cytokines and stabilising the extra-cellular matrix (ECM). Mesenchymal stem cells (MSCs) have shown immunomodulatory actions in many inflammatory conditions, and their benefit has often been attributed to the production of TSG-6. This work aimed to evaluate the potential of TSG-6 and TSG-6-expressing MSCs as therapies in cerebral ischaemia, and to investigate the expression profile of endogenous TSG-6 in response to stroke. Mice were subjected to middle cerebral artery occlusion (MCAo) followed by reperfusion. We investigated whether IL-1-induced acute brain injury after stroke is reversed by TSG-6, and long-term recovery was evaluated in mice treated with TSG-6 or MSCs. Functional outcomes were assessed, and brains were sectioned and stained for analysis of lesion volume, haemorrhagic transformation, blood-brain barrier (BBB) disruption and neutrophil infiltration. The expression profile of TSG-6 was evaluated in mice allowed to recover for 4h, 24h, 3, 5 or 7 days. TSG-6 expression was determined by quantitative PCR and immunohistochemistry. Treatment with TSG-6 reduced IL-1-induced neutrophil infiltration into the striatum, and led to decreased BBB disruption and haemorrhagic transformation at 24h. Treatment with TSG-6 in the absence of a systemic inflammatory challenge had no significant effect on lesion volume, BBB disruption or haemorrhagic transformation after 7 days reperfusion, however thalamic neutrophil infiltration was significantly reduced. Treatment with human MSCs had no significant effect on behavioural or histological outcomes, however a heightened inflammatory response in MSC-treated mice suggested rejection of the cells by the murine immune system. TSG-6 expression peaked in the ischaemic hemisphere at 5 days post-reperfusion, and was associated with astrocytes in the glial scar surrounding the infarcted tissue. TSG-6 might be a promising therapy for the treatment of stroke in the presence of systemic inflammation. TSG-6-expressing MSCs might provide a broader therapeutic potential, and further work should optimise experimental conditions to prevent rejection of the cells. Expression of TSG-6 within the glial scar suggests a potential role in repair and recovery following ischaemic stroke. Modulating the peripheral immune response remains an attractive and accessible therapeutic target for the treatment of cerebral ischaemia.

The differentiation of mesenchymal stem cells (MSCs) to endothelium is a critical but poorly understood feature of tissue vascularisation and considerable scepticism still remains surrounding this important differentiation event. Defining features of endothelial cells (ECs) are their ability to exist as contact-inhibited polarised monolayers that are stabilised by intercellular junctions, and the expression and activity of endothelial markers. During vasculogenesis, communication between MSCs and differentiated ECs or vascular smooth muscle cells, or between MSCs themselves is likely to influence MSC differentiation. In this study, the possibility that cell density can influence MSC differentiation along the EC lineage was examined. High density plating of human bone marrow-derived MSCs induced prominent endothelial characteristics including cobblestone-like morphology, enhanced endothelial networks, acetylated-low density lipoprotein uptake, vascular growth and stimulated expression of characteristic endothelial markers. Mechanistically, this density-dependent process has been defined. Cell-cell contact-induced Notch signalling was a key initiating step regulating commitment towards an EC lineage, whilst VEGF-A stimulation was required to consolidate the EC fate. Thus, this study not only provides evidence that MSC density is an essential microenvironmental factor stimulating the in vitro differentiation of MSCs to ECs but also demonstrates that MSCs can be differentiated to a functional EC. Taken together, defining how these crucial MSC differentiation events are regulated in vitro, provides an insight into how MSCs differentiate to ECs during postnatal neovascularisation and an opportunity for the therapeutic manipulation of MSCs in vivo, enabling targeted modulation of neovascularisation in ischaemia, wound healing and tumourigenesis.

Introduction: Mesenchymal stem cells (MSC) offer a novel cell therapy within tissue engineering and regenerative medicine (TERM)-based strategies, and the prospect of MSC therapies are widening since the discovery of MSCs within multiple locations of the body including bone marrow (BM-MSCs) and adipose tissue, (AD-MSCs). It is highly recognised that an organisms reparative and regenerative potential declines with advancing age, therefore aged patients are one of the primary target populations for TERM applications. Although information is available regarding the effects of patient age on the quality of human BM-MSCs, little and conflicting information currently exists for AD-MSCs. In addition, few studies have compared the quality of freshly isolated and expanded donor-matched BM and AD-MSCs to elucidate the more appropriate cell source. This study investigated the effect of donor age and in vitro ageing on functional behaviour (i.e. senescence state, population kinetics and differentiation potential) of donor-matched BM and AD-MSCs. Methods: The influence of donor age and in vitro ageing on mature (28-55 years) and elderly (75-86 years) donor-matched BM and AD-MSCs was assessed upon isolation (early life-span) and during extended (mid and late lifespan) timepoints through culture. During culture MSCs were characterised for cumulative population doublings (CPDs) and the expression of senescence associated marker genes, p16INK4A, p21 and p53, and transcription factor NANOG. At each lifespan telomere length was assessed along with differentiation efficiency along the osteogenic, adipogenic and chondrogenic lineages through lineage specific marker genes and histological staining. Results: Elderly BM and AD-MSCs displayed similar characteristics in terms of initial CPD number, p21, p53 and NANOG expression, telomere length and differentiation along osteogenic and adipogenic lineages. With increasing donor age there was a significant decline in p16INK4A expression within BM-MSCs, whilst expression of all chondrogenic markers significantly decreased within AD-MSCs. BM and AD-MSCs were comparable for the majority of outcome measures with the exception of chondrogenic differentiation which was superior with BM-MSCs in terms of COL2A1 expression and histological staining for proteoglycans. Donor age had a negative effect on BM-MSCs with long-term culture leading to a significantly longer PD time and decreased telomere lengths. Similar population kinetics was displayed between BM and AD-MSCs during long-term culture. Increasing culture time had effects on differentiation potential for both MSC sources with complete loss of osteogenic capacity and decreased adipogenic capacity; however chondrogenic capacity was only decreased within AD-MSCs. Differentiation potential after long-term culture between BM and AD-MSCs showed similar osteogenic and adipogenic ability yet superior chondrogenic ability was apparent within mature BM-MSCs compared to AD-MSCs, in terms of ECM deposition. Conclusions: In conclusion the source of MSCs for TERM will need to be considered depending upon the type of tissue regeneration required. The clinical outcome would be greater using MSCs during early stages of culture, as culture expansion has detrimental effects on functional properties of both BM and AD-MSCs.

Mesenchymal stem cells (MSCs) are the most widely used source for bone tissue engineering due to their capability of multipotent differentiation. The use of nanotechnology in biomedical applications and therapy has increased in recent years provides an elegant alternative in comparison to current tissue engineering methods. Magnetic nanowires have a high potential in the medical field, as they are biocompatible, are simple to fabricate, possess low cytotoxic effects and can be operated wirelessly via magnetic fields. A nanowire substrate (NW) can provide a surface with tunable elastic properties. Therefore, magnetic nanowires have many promising applications such as in cell therapy, cell separation, cancer treatment, and as a scaffold for cell culture. This thesis explores the effects of alternating magnetic field (AMF) as a biophysical stimulator of osteogenic differentiation of MSCs by culturing the stem cells on a magnetic iron (Fe) NW. To this end, Fe nanowires were fabricated through electrodeposition and interactions between the NW and cells were analysed by electron microscopy. An AMF was applied to the NW in order to induce a vibration. MSCs were exposed to different magnetic field intensities, 250 mT and 50 mT, for different application times, 12 hours on followed by 12 hours off for two days and 24 hours on followed by 12 hours off. Differentiation was determined through the assessment of osteogenic markers at the mRNA level by RT-PCR and at the protein level by flow cytometry and fluorescence microscopy. Different effects were observed on MSCs grown on Fe NWs following exposure to different magnetic field intensities and duration applications. MSC differentiation towards the osteogenic lineage increased with increased field intensities. The most enhanced osteogenic differentiation of MSCs was observed at 250 mT AMF for 12 hours, as evidenced by elevated osteogenic markers at mRNA level compared to that of an AMF free control. Based on these results, we proposed that culturing MSCs on magnetic nanomaterials has the potential to control and promote osteogenesis under magnetic field and without the addition of external differentiation factors. These findings provide a new tool for stem cell research as an effective technology for bone tissue engineering and regenerative medicine.

Osteoporosis is a systemic skeletal disease that affects millions of people worldwide. There are many possible etiologies for osteoporosis, including inherent variables like genetics and sex, and lifestyle variables like diet and exercise. Characterized by low bone mass and increased fracture risk, the disease places a burden on both the patients and the healthcare industry. Therefore, it is vital that research determine the mechanisms by which the risk factors affect BMD so that better diagnosis and treatment options may be developed. The purpose of this study was to examine the relationship between various osteoporosis risk factors and biochemical markers of osteogenic cell activity derived from bone-marrow MSCs. It was hypothesized postmenopausal white women, having the greatest risk for osteoporosis, would have elevated hydroxyproline and decreased ALP, indicative of greater bone resorption. Acetabular reamings were collected from 26 patients (15 males and 11 females) undergoing total hip arthroplasty at Boston Medical Center. MSCs from the reamings were plated and underwent osteoinduction into osteoblasts. The cells were then harvested and assayed for various indicators of cell growth and bone cell activity, such as DNA, ALP, and hydroxyproline. Our hypothesis was generally supported in that postmenopausal white women did have less ALP, an indicator of bone deposition, than premenopausal women and postmenopausal African American women. Additional findings and directions for future studies are further discussed in this paper.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22039号 / 医博第4524号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 濵﨑 洋子, 教授 武藤 学, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19891号 / 医博第4140号 / 新制||医||1016(附属図書館) / 32968 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 山田 泰広, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Articular cartilage is a smooth, white connective tissue that covers and protects the ends of long bones to allow for a smooth, frictionless surface on which to glide for easy movement. Once the tissue is damaged, articular cartilage lacks a direct blood supply, which results in a limited ability to repair itself. This study explores the effect of the growth factor BMP-13 on the chondroinduction of primary human bone marrow-derived mesenchymal stem cells. The results demonstrate the limited ability of BMP-13 to exert a strong chondroinductive effect on human bone marrow-derived MSCs. However, the results do indicate that BMP-13 has the ability to sustain chondroinduction to a certain extent for up to 18 days following initiation by 3 days of exposure to TGF-β3. Results are encouraging for future work that involves growth factor influence on MSCs in articular cartilage tissue engineering.

No description available.