Discovery of the ability of mesenchymal stem cells (MSCs) to differentiate into cells of non-mesodermal tissues, particularly neuronal cells, have raised the possibility of utilising MSCs in regenerative/reparative therapies for neurological disorders. However, a number of hurdles remain to be resolved. This thesis aims to address some of these issues by investigating the characteristics of bone marrow-derived human MSCs (hMSCs) during long-term culture, the potential of hMSCs to differentiate in vitro toward the neuronal lineage under the influence of cytokines, and the effects of intracerebral transplantation in the hemiparkinsonian rat model. During expansion culture hMSCs were found to display the expected characteristics of MSC populations, and also constitutively expressed neural and pluripotency markers simultaneously with mesodermal markers. Analysis of hMSC long-term subcultivation revealed an optimal period for commencing neuronal differentiation (first 6-8 passages), and also showed the absence of spontaneous neural differentiation. Application of neural-inducing cytokines and culture conditions resulted in the generation of an immature neuronal-like phenotype by hMSCs. Through live cell microscopy it was demonstrated for the first time that cytokine-based hMSC neuronal differentiation occurs through active and dynamic cellular processes involving outgrowth and motility of cellular extensions. In addition, single- and multiple-stage cytokine-based strategies for inducing dopaminergic neuronal-like cells from hMSCs were investigated. These studies revealed that FGF-2 and EGF exerted the greatest benefits for hMSC neuronal differentiation. Undifferentiated and neuronal-primed hMSCs were transplanted intracerebrally into the striatum and substantia nigra of cyclosporine-treated hemiparkinsonian rats. Grafted hMSCs could be clearly identified at 1-day and 7-days post-transplantation; however, grafts were gradually lost over time, with complete absence by 21-days. Co-transplantation with olfactory ensheathing cells, neuronal-priming prior to grafting, and nigral as well as striatal grafting could not provide engraftment and differentiation advantages. Immunohistological analysis demonstrated the presence of innate inflammatory responses (microglia and astrocyte activation) at graft sites, fibronectin deposition by hMSCs, and lack of endogenous host neurogenesis. The results of my PhD work indicate that cytokine-based culture methods are capable of differentiating hMSCs to an immature neuronal-like phenotype, and host-mediated innate inflammatory responses may be a key contributing factor for the failure of in vivo hMSC engraftment.

Thesis (M.Sc.). / Written for the Division of Experimental Medicine. Title from title page of PDF (viewed 2009/06/18). Includes bibliographical references.

Resumo: Injúrias traqueais são problemas na prática cirúrgica, pois apresentam dificuldades no tratamento. Atualmente, a engenharia tecidual (ET) é a única técnica para substituição traqueal que fornece promessa real. O uso de suportes naturais apresenta vantagens biológicas, mas faz-se necessário a descelularização desses materiais.Assim, o objetivo do presente trabalho é a descelularização de traquéias, com posterior aplicação das células epiteliais respiratórias (CERs) na face interna das traquéias descelularizadas; bem como, a diferenciação das células-tronco mesenquimais (CTMs) em condrócitos. O presente estudo foi efetuado com modelo em coelhos da raça Botucatu, pesando entre 3,0 e 4,0kg, provenientes do Biotério Central da Unesp. A utilização desses animais foi aprovada pelo Comitê de Ética em Experimentação Animal. Foram realizados 54 diferentes protocolos de descelularização, que envolvem métodos físicos (congelamento, agitação, pressão mecânica-PM, irradiação eletromagnética não ionizante-LED 475nm e LED 630nm), associados com métodos químicos (Triton X-100, SDS e DS) e enzimáticos (DNase e RNase). Paralelamente, também foi realizado cultura celular das CTMs, com posterior diferenciação em condrócitos. A expansão ex vivo das CERs foi realizada por diferentes protocolos: fragmentos traqueais, lavado traqueal, raspado traqueal, punch dermatológico de 2mm e digestão por tripsina. As células obtidas foram aplicadas na face interna das traquéias descelularizadas. Com relação a PM, não se observa contribuição nos protocolos de descelularização. A irradiação com LED 475nm provoca remoção das células condrogênicas; já a irradiação com LED 630nm provoca aumento da proliferação celular. Sob o critério custo-benefício, a utilização dos processos enzimáticos não foi validada. Em relação aos detergentes, ocorre... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Tracheal injuries are a problem in surgical practice once they present difficulties in their treatment. Nowadays, tissue engineering (TE) is the only technique for tracheal replacement that provides real promise. There are biological benefits in using natural scaffolds, but the decellularization of such materials is necessary. Thus, the objective of this study is tracheal decellularization with subsequent application of respiratory epithelial cells (RECs) on the inner side of the decellularized cells, as well as the differentiation of mesenchymal stem cells (MSCs) into chondrocytes. This study was carried out with model in Botucatu rabbits weighing between 3.0 to 4.0kg from the Animal Colony at Unesp. The use of such animals was approved by the Ethics Committee of Animal Experiments. Fifty-four different decellularization protocols were used; they involved physical methods (freezing, agitation, mechanical pressure - MP, non-ionizing electromagnetic irradiation - LED 475nm and LED 630nm), associated with chemical methods (Triton X-100, SDS and DS) and enzymatic ones (DNase and RNase). At the same time, a cell culture of the MSCs was done as well, with subsequent differentiation into chondrocytes. The ex vivo expansion of RECs was performed with different protocols: tracheal fragments, tracheal wash, scraped trachea, 2mm dermatological punch, and trypsin digestion. The cells obtained were applied to the inner side of the decellularized tracheae. Regarding MP, no contribution to the decellularization protocols is observed. Irradiation with LED 475nm causes the removal of chondrogenic cells whereas the irradiation with LED 630nm increases cell proliferation. Under the cost-benefit criterion, the use of enzymatic processes was not validated. As for the detergents, there is more destruction of the extracellular matrix of the tracheae treated with SDS when compared to those treated with... (Complete abstract click electronic access below) / Orientador: Elenice Deffune / Coorientador: Raul Lopes Ruiz Júnior / Banca: João Tadeu Ribeiro Paes / Banca: Paulo Francisco Guerreiro Cardoso / Mestre

It has been repeatedly proven that cell therapies can address many current unmet clinical treatment needs and also improve on current treatment options for various diseases, from neurological disorders to bone repair (Rosset et al. 2014; Corey et al. 2017). Though the potential of cell therapies has been demonstrated at a relatively small scale, the realisation of bringing cell based treatments to a larger market is hindered by the complexity of the product along with safety concerned and high production cost. Safety concerns can be informed with more in-depth analytical analysis of the product, however this in turn increase the costs involved in producing a cell therapy (Davie et al. 2012). Consequently the cost of analytical techniques also needs to be reduced, to address this need the area of microfluidic based bioanalytics holds much promise (Titmarsh et al. 2014). The culturing of human mesenchymal stem cells (hMSC) was used as a proof of concept model to demonstrate where improved bioanalytical and bioassay methods could be utilised in the production of cell therapies. Cells from four donors were cultured under three different oxygen environments and the conditioned medium assessed for pro-angiogenic capabilities using a tube formation bioassay and a proportion of the cytokine secretome profile measured using Luminex technology. Thorough secretome analysis it was shown that predicting cytokine levels based solely on the donor was not possible as the handling of the cells also had an influence on the secretome profile. The donor expression profiles did not behave in the same manner across all oxygen environments, for example in some donors IL-8 levels increased per cell at lower oxygen where as other donors showed a decrease per cell. While the tube formation assay showed some differences between donors in pro-angiogenic capabilities it also highlights the challenges with interpreting large data sets. The feasibility of using a microcapillary film (MCF) based enzyme-linked immunosorbent assay (ELISA) to detected two relevant cytokines, IL-8 and hepatocyte growth factor (HGF) was investigated. Following on from this work the development of a combined MCF ELISA assay with hMSC cell culture to produce a fully closed cell screening system was initiated. It was shown that it was feasible to measure IL-8 and HGF using the MCF ELISA platform but further work would need to be done to make the system more compatible with the manufacturing environment. In order to adapt the MCF to also be an hMSC culture platform the first challenge was to functionalise the Fluorinated Ethylene Propylene (FEP) surface of the MCF. It was concluded that a poly (vinyl- alcohol) (PVA) and gelatin mixture produced a homogenous coating to which a consistent level of hMSC would attach. This work was carried out on a flat surface; therefore steps were taken to adapt this knowledge into the MCF, while there was evidence of hMSCs present inside the MCF more work will need to be done to bring this concept to an established platform.

Periosteum and bone marrow (BM) contain cells that, after isolation and culture-expansion, exhibit properties of mesenchymal stromal/stem cells (MSCs). However, these cells have not been identified and characterised in situ due to the lack of specific markers. This study aimed to identify and phenotypically characterise long-term label-retaining cells (LT-LRCs), thought to include stem cells (SCs), in mouse periosteum and BM. Two mouse models were used: nucleoside-analogue labelling, and doxycycline (Dox)-inducible expression of histone 2B–green fluorescent fusion protein (H2B-GFP). LRCs were identified and phenotypically characterised by immunostaining, and microscopy or by flow cytometry (FCM). LRCs were detected throughout the periosteum with no apparent focal concentration, and subsets of cells displayed a phenotype compatible with MSCs but not pericytes. Osteoblasts were also labelled, but osteocalcin-expressing osteoblasts were distinct from Low-affinity nerve growth factor receptor (LNGFR)/P75-expressing MSCs. Similarly, BM contained LRCs expressing MSC markers that were distinct from pericytes. For FCM analyses, two cell isolation methods were compared, which revealed that crushing and collagenase digestion of long bones yielded a higher percentage of LRCs compared with flushing. BM analysed 40 days after the end of nucleoside administration showed that LRCs both within the CD45- and CD45low population were enriched for cells expressing Platelet-derived growth factor receptor α (PDGFRα) together with Stem cell antigen-1 (Sca-1) as well as cells expressing LNGFR/P75+. Furthermore, the CD45-PDGFRα+Sca-1+ population showed an increase in the percentage of LRCs with an increasing washout period, suggesting PDGFRα together with Sca-1 is most suitable to identify stromal LRCs in mouse BM. Comparison of the nucleoside label-retaining model with the H2B-GFP-label-retaining transgenic model showed a good correlation between nucleoside and H2B-GFP-label retention, suggesting the suitability of the H2B-GFP model for identification of stromal LRCs in BM. Future studies characterising the MSC niche in-vivo could reveal novel therapeutic targets for promoting bone regeneration/repair.

Made available in DSpace on 2014-06-11T19:23:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-28Bitstream added on 2014-06-13T18:50:10Z : No. of bitstreams: 1 evaristo_tc_me_botfm.pdf: 1944919 bytes, checksum: f9a5a93860c79f6554bb319a05d333fe (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Injúrias traqueais são problemas na prática cirúrgica, pois apresentam dificuldades no tratamento. Atualmente, a engenharia tecidual (ET) é a única técnica para substituição traqueal que fornece promessa real. O uso de suportes naturais apresenta vantagens biológicas, mas faz-se necessário a descelularização desses materiais.Assim, o objetivo do presente trabalho é a descelularização de traquéias, com posterior aplicação das células epiteliais respiratórias (CERs) na face interna das traquéias descelularizadas; bem como, a diferenciação das células-tronco mesenquimais (CTMs) em condrócitos. O presente estudo foi efetuado com modelo em coelhos da raça Botucatu, pesando entre 3,0 e 4,0kg, provenientes do Biotério Central da Unesp. A utilização desses animais foi aprovada pelo Comitê de Ética em Experimentação Animal. Foram realizados 54 diferentes protocolos de descelularização, que envolvem métodos físicos (congelamento, agitação, pressão mecânica-PM, irradiação eletromagnética não ionizante-LED 475nm e LED 630nm), associados com métodos químicos (Triton X-100, SDS e DS) e enzimáticos (DNase e RNase). Paralelamente, também foi realizado cultura celular das CTMs, com posterior diferenciação em condrócitos. A expansão ex vivo das CERs foi realizada por diferentes protocolos: fragmentos traqueais, lavado traqueal, raspado traqueal, punch dermatológico de 2mm e digestão por tripsina. As células obtidas foram aplicadas na face interna das traquéias descelularizadas. Com relação a PM, não se observa contribuição nos protocolos de descelularização. A irradiação com LED 475nm provoca remoção das células condrogênicas; já a irradiação com LED 630nm provoca aumento da proliferação celular. Sob o critério custo-benefício, a utilização dos processos enzimáticos não foi validada. Em relação aos detergentes, ocorre... / Tracheal injuries are a problem in surgical practice once they present difficulties in their treatment. Nowadays, tissue engineering (TE) is the only technique for tracheal replacement that provides real promise. There are biological benefits in using natural scaffolds, but the decellularization of such materials is necessary. Thus, the objective of this study is tracheal decellularization with subsequent application of respiratory epithelial cells (RECs) on the inner side of the decellularized cells, as well as the differentiation of mesenchymal stem cells (MSCs) into chondrocytes. This study was carried out with model in Botucatu rabbits weighing between 3.0 to 4.0kg from the Animal Colony at Unesp. The use of such animals was approved by the Ethics Committee of Animal Experiments. Fifty-four different decellularization protocols were used; they involved physical methods (freezing, agitation, mechanical pressure - MP, non-ionizing electromagnetic irradiation - LED 475nm and LED 630nm), associated with chemical methods (Triton X-100, SDS and DS) and enzymatic ones (DNase and RNase). At the same time, a cell culture of the MSCs was done as well, with subsequent differentiation into chondrocytes. The ex vivo expansion of RECs was performed with different protocols: tracheal fragments, tracheal wash, scraped trachea, 2mm dermatological punch, and trypsin digestion. The cells obtained were applied to the inner side of the decellularized tracheae. Regarding MP, no contribution to the decellularization protocols is observed. Irradiation with LED 475nm causes the removal of chondrogenic cells whereas the irradiation with LED 630nm increases cell proliferation. Under the cost-benefit criterion, the use of enzymatic processes was not validated. As for the detergents, there is more destruction of the extracellular matrix of the tracheae treated with SDS when compared to those treated with... (Complete abstract click electronic access below)

Orientadores: Ângela Cristina Malheiros Luzo, Joaquim Murray Bustorff Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T07:41:35Z (GMT). No. of bitstreams: 1 Volpe_BrunoBosch_M.pdf: 3151194 bytes, checksum: afd54e9f5fdcdd55a24ddfa4b90d02a7 (MD5) Previous issue date: 2013 / Resumo: As fístulas enterocutâneas (FE) são de difícil cicatrização e seu tratamento cirúrgico frequentemente falha, fazendo com que a fistula volte a abrir. Estudos recentes têm demonstrado que a terapia celular pode ser uma nova forma de tratamento nesta área. As células tronco mesenquimais (MSCs) são capazes de se auto- renovar tem alta capacidade proliferativa, podendo se diferenciar em várias linhagens celulares. Ainda apresentam capacidade imunomodulatória. A medula óssea, o sangue de cordão umbilical e o tecido adiposo são as principais fontes de MSCs. O tecido adiposo (TA) é de fácil acesso, sendo que o procedimento de lipoaspiração é um procedimento comum. O tratamento de fístulas enterocutâneas com AT-MSCs já foi testado algumas vezes, porém a fístula em sua grande maioria não se fecha totalmente. O objetivo deste estudo foi analisar o potencial terapêutico das MSCs aderidas a fios de sutura no intuito de melhorar a cicatrização e recuperação no tratamento de FE. O TA foi obtido através do procedimento de lipoaspiração. O TA foi submetido ao processo de digestão com colagenase. As células ficaram em meio de cultura DMEM com baixa glicose e com SFB durante 3 dias. Quando atingiram 80% de confluência, as células aderentes foram tratadas com tripsina e ressuspendidas com o meio citado acima. Na 4ª passagem essas células foram caracterizadas com citometria de fluxo, microscopia confocal e diferenciadas nas três linhagens mesodérmicas para confirmação que realmente são MSCs. Após, a confirmação de que as células eram realmente células tronco mesenquimais, elas foram aderidas em fios de sutura de poliglactina (4-0 Poly Vicryl / Poliglactina 910). Foram gotejadas 106 MSCs em cima de cada fio de sutura e logo em seguida foi adicionada a cola de fibrina (20uL Fibrinogênio, 30uL Trombina e 10uL Cloreto de Cálcio) para ajudar na fixação das células. Os fios de sutura com MSCs aderidas ficaram em meio de cultura durante 24 horas para proliferação celular. As amostras foram analisadas por microscopia confocal de imnunofluorescência e eletrônica de varredura. O experimento animal utilizou ratos Wistar com 10 semanas de vida que foram distribuídos em três grupos: Grupo Controle (GC) que incluiu 5 animais onde a formação da fístula foi através de cirurgia sem nenhum tipo de suporte. Grupo Injeção (GI) que incluiu 8 animais que receberam uma injeção de 106 AT-MSCs ao redor do local de formação da fístula. Grupo Sutura (GS) que incluiu 9 animais que receberam sutura de 4-0 Vicryl (poliglactina) com 106 AT-MSCs aderidas com a ajuda de cola de fibrina. Após a exposição do ceco foi realizada enterotomia de 5mm, sendo então realizada a sutura da abertura a abertura da parede abdominal (superfície interna da pele) com 4 pontos separados com 4-0 Vicryl - Poly J-304 Polyglactin 910. No grupo GS, o fio para confecção da fístula, como descrita acima, continha 106 AT-MSCs aderidas. As fístulas foram fotografadas no dia da cirurgia e no 3°, 6°, 9°, 12°, 15°, 17°, 19° e 21° dias, onde foram anestesiados e sacrificados. O tamanho da área da fístula foi mensurado através do software ImageJ. Foram utilizados dois métodos estatísticos para analisar os dados do modelo animal. O primeiro método foi o Two-way Anova, fazendo a análise comparativa da cicatrização entre os três grupos, e o segundo o One-way analysis of variance, fazendo a análise comparativa da cicatrização entre os três grupos nos dias D0, D12 e D21. As microscopias confocal de imunofluorescência e eletrônica de varredura demonstraram a presença de AT-MSCs aderidas aos fios de sutura. No experimento animal mostrou que a média de redução do tamanho da área da fístula no 21º dia foi de 46,54% no GC, 71,80% no GI e 90,34% no GS (p<0,05), demonstrando que as MSCs foram eficazes na cicatrização das fístulas enterocutâneas tanto no grupo que recebeu a injeção de células quanto no grupo que utilizou as células aderidas ao fio de sutura. As MSCs foram capazes de se fixarem nos fios de sutura. Quando as fístulas enterocutâneas receberam a sutura com MSCs aderidas, elas mostraram uma melhor recuperação e cicatrização da fístula. AT-MSCs aderidas a fios de sutura pode ser uma nova e efetiva forma no tratamento de fístulas enterocutâneas / Abstract: Enterocutaneous fistulas (EF) are difficult to resolve and surgical failure is frequent. Cell therapy could be a new approach in this area. Mesenchymal stem cells (MSCs) have high proliferative capacity, can differentiate into several lineages and have immunomodulatory capacity. Adipocyte tissue (AT) is an easy source of them. Enterocutaneous fistula (EF) treatment with MSCs was yet performed but sometimes, the fistula did not close completely. The aim of this study is to analyze if AT-MSCs could attached in the suture filament in order to be used for EF treatment. AT obtained from lipoaspirate procedure was submitted to collagenase digestion. Cells were cultured in DMEM low glucose medium, with FBS during 3 days. At the 4ª passages, cells were characterized by flow cytometry, confocal microscopy, differentiated to mesodermal lineages to confirm MSCs and telomerase enzyme activity and karyotype analysis. The experiments were performed with polyester suture filament. MSCs, 106 cells, were fixed in the suture filament by adding fibrin glue.Filaments was led in the medium described above during 3 days. Samples were analyzed by confocal and scanning electron microscopy. The animal experiments were performed on 10 weeks old male Wistar rats divided into 3 groups. Control Group (CG): 5 animals undergoing fistula formation alone. Injection Group (IG):8 animals receiving 106 AT-MSCs injected around the suture line. Suture Filament Group (SG): 9 animals in which suture were performed using 4-0 Vicryl with 106 MSCs attached in the filament with fibrin glue. The cecum was accessed through a standard 7mm stab incision on the lower left side of the abdomen. Upon exposure, a 5mm enterotomy was performed and sutured to the abdominal wall in order to produce the fistula. To ensure normal closure of the fistula the opening in the cecum wall was fixed to the internal surface of the skin, without maturation, using four separate 4-0 Vicryl stitches (Poly J-304 Polyglactin 910 Ethicon). The fistulas were photographed on the day of operation and on the 3°, 6°, 9°, 12°, 15°, 17°, 19° and 21° day, in which they were anesthetized and sacrificed. Measure of the size of the fistula was performed using ImageJ software. Statistic comparison between the groups was performed by ANOVA. Confocal and scanning electronic microscopy results demonstrated that the cells were able to attach to the suture filaments. Animal experiments showed that the average size reduction of the fistula area at 21th day was: control group, 46.54%; injected group 71.80% and sutured group 90.34% (p<0,05). MSCs were able to attach to the suture filaments. When the fistulas were sutured with filaments containing MSCs they showed better recovery and healing than the injected and control group. Adipocyte tissue MSCs adhered to suture filament might be a new and effective approach for enterocutaneus fistulas treatment / Mestrado / Fisiopatologia Cirúrgica / Mestre em Ciências

Background: Stem cells are a promising therapy for regeneration following myocardial infarction (MI). Another therapy currently under investigation for MI is glucagon-like peptide-1 (GLP-1), a natural incretin hormone that has cardio-protective properties, although a short half-life in vivo. GLP-1 CellBeads are a novel therapy, combining stem cells and GLP-1. Human mesenchymal stem cells (MSCs) were immortalised, engineered to secrete a fusion protein of GLP-1 and encapsulated in alginate. We have previously demonstrated that GLP-1 CellBeads significantly reduce infarct size and improve ejection fraction post-MI, but the underlying mechanisms are unclear. The therapy was assessed in an in vivo pig MI model and an in vitro cardiomyocyte ischaemia model. Methods: GLP-1 CellBeads were delivered to coronary artery branches in pigs, creating micro-infarcts, as determined by echocardiography. Cell-free beads (Beads) and CellBeads containing hMSCs without GLP-1 (Beads-MSC) were delivered as controls (n=3-5/group). Pigs were sacrificed one and four weeks post-MI. Tissue was analysed for: apoptosis, collagen, cardiomyocyte cross sectional area and myofibroblasts. The localised response around the beads was also measured using immunohistochemistry. Atomic force microscopy (AFM) was used to examine the ultra-structure of the collagen scar. The expression profiles of genes involved in collagen remodelling were measured using qRT-PCR. Viability of MSCs was measured using GFP-tagging and confirmed using qRT-PCR. To examine effects on apoptosis in vitro, human adult cardiomyocytes underwent ischaemia for 1 hour before incubation with: media conditioned with MSCs or MSC+GLP-1, GLP-1, Exendin-4 or media. Apoptosis and viability were measured at 24 and 48 hours respectively. Results: In the in vivo pig model, significant increases in apoptosis were observed in the infarct of all groups one week post-MI, with no differences between treatments. Despite decreased numbers of myofibroblasts, significantly more collagen was observed in MSC treated groups, with increased collagen fibril periodicity and a more organised collagen scar. The altered scar structure was reflected in differences in gene expression between groups, with an accelerated healing response in the MSC groups. However, significantly fewer myofibroblasts were observed in the MSC treated groups. Viability of MSCs was confirmed up to four weeks post-infusion, with GLP-1 secretion confirmed up to one week. In the in vitro ischaemia model, MSC+GLP-1 conditioned media significantly reduced cardiomyocyte apoptosis 24 hours post-ischaemia, compared to media alone. All agonists (GLP-1, MSC media and MSC+GLP-1 media) significantly improved viability compared to media alone 48 hours post-ischaemia. Conclusions GLP-1 CellBeads have a beneficial effect on healing following MI by significantly decreasing infarct size and improving ejection fraction post-MI. these benefits are associated with decreased cardiomyocyte apoptosis and altered collagen scar formation. The CellBeads act as local hubs for regeneration and are viable up to one month post-infusion. The effects observed are due to a combination of the GLP-1 and paracrine factors released from the hMSCs.

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.

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.