"Recent data show that 7.6 million Americans have survived a myocardial infarction (MI), and 5.1 million Americans suffer from severe heart failure. Stem cell therapy has the potential to improve cardiac function after MI. Two promising cells for cardiovascular regeneration therapies include human mesenchymal stem cells (hMSCs) and pluripotent stem cell derived cardiomyocytes (hPS-CM) each with their own unique method for improving cardiac function post-infarct. However, a limiting factor to cell therapies is that the methods currently used to deliver cells to the myocardium, including intramyocardial injection (considered the gold standard), suffer from low retention rates. To promote localization of delivered cells to the infarct and increase retention rates, our lab has developed a fibrin biological suture that can deliver human mesenchymal stem cells (hMSCs) with an efficiency of 64% compared to just 11% with intramyocardial injection in the normal rat heart. In this dissertation we sought to examine the functionality of hMSC and hPS-CM seeded sutures and their impact on cardiovascular regeneration applications. We began by delivering hMSC seeded fibrin sutures to an infarcted rat heart and found that the sutures are an effective method to deliver cells to the infarcted myocardium and demonstrated a trend towards improved regional mechanical function in the infarct region over infarct alone. Next, we transitioned to using hPS-CM and developed methods to seed the sutures, as well as a method to measure hPS-CM contractility with high spatial and temporal resolution, while concurrently capturing calcium transients. This technique allowed us to examine the contractile behavior in terms of contractile strain and conduction velocity of hPS-CM seeded on fibrin microthreads over 21 days in culture. We found that the fibrin microthread is a suitable scaffold for hPS-CM attachment and contraction and that extended culture promotes cell alignment along the length of the suture as well as improvements in contractile function in terms of increases in contractile strain and conduction velocity. Finally, we delivered the hPS-CM seeded microthreads to an uninjured rat heart and found a delivery efficiency of 67%. Overall, we further demonstrated the technology of the fibrin suture to deliver cells to an infarct as well as the ability to support the attachment, contraction and delivery of hPS-CM to cardiac tissue. "

"Recent evidence suggests that delivering human mesenchymal stem cells (hMSCs) to the infarcted heart reduces infarct size and improves ventricular performance. However, cell delivery systems have critical limitations such as inefficient cell retention and poor survival, and lack targeted localization. Our laboratories have recently developed a method to produce discrete fibrin microthreads that can be attached to a needle and delivered to a precise location within the heart wall. We hypothesize that fibrin microthreads will support hMSC proliferation, survival and retention of multipotency, and may therefore facilitate targeted hMSC delivery to injured tissues such as infarcted myocardium. To test this hypothesis, we bundled 100 μm diameter microthreads to provide grooves to encourage initial cell attachment. We seeded hMSCs onto the microthread bundles by applying 50,000 cells in 100 μL of media. The number of cells adhered to the microthreads was determined up to 5 days in culture. Cell density on the fibrin microthreads increased over time in culture, achieving an average density of 730 ± 101 cells/mm2. A LIVE/DEAD assay confirmed that the cells were viable and Ki-67 staining verified the increase in cell number over time was due to proliferation. Additionally, functional differentiation assays proved that the hMSCs cultured on microthreads retained their ability to differentiate into adipocytes and osteocytes. The results of this study demonstrate that delivering 1 to 4 cell seeded microthread bundles to the infarcted rat myocardium has the potential to produce a positive improvement in mechanical function and these microthreads support hMSC proliferation and survival. Additionally these findings suggest that cell-seeded microthreads may serve a platform technology to improve localized delivery of viable cells to infarcted myocardium to promote functional tissue regeneration. "

O diabetes mellitus tipo 1 é uma doença autoimune causada pela destruição das células β produtoras de insulina, presentes nas ilhotas pancreáticas, por células autorreativas do sistema imune. A opção de tratamento mais utilizada são injeções diárias de insulina exógena, o que configura um tratamento não curativo. Para alcançar a independência de insulina, alternativas como o transplante de ilhotas vêm sendo estudadas. Entretanto, a disponibilidade de pâncreas de doadores cadavéricos para o isolamento destas ilhotas é pequena e os métodos de isolamento, pouco eficazes, sendo necessários de 2 a 4 doadores para atingir o número adequado de ilhotas. Além disso, o transplante apresenta problemas relacionados à enxertia, devidos principalmente à baixa vascularização, o que leva à morte de células β nos primeiros dias pós-transplante. Desta forma, estudos explorando alternativas que aumentem a sobrevivência e a funcionalidade dos transplantes e diminuam o número de ilhotas exigido por receptor fazem-se muito necessários. As células-tronco mesenquimais apresentam propriedades interessantes para aplicação em terapia celular. Entre elas, destaca-se o efeito parácrino, que exerce diversas funções benéficas, como o aumento da vascularização, nos locais onde estas células estão presentes. Sendo assim, este trabalho explorou o co-transplante de ilhotas pancreáticas com células-tronco mesenquimais derivadas de tecido adiposo, para o tratamento do diabetes mellitus em modelo murino. Os resultados mostraram que a presença destas células no grupo que recebeu o co-transplante não aumentou a taxa de cura, em relação ao grupo que recebeu somente ilhotas. No entanto, o fenômeno de reversão do diabetes foi antecipado no grupo co-transplantado, o que sugere um possível efeito angiogênico das células-tronco adiposo-derivadas presentes neste grupo. Desta forma, conclui-se que estas células podem exercer atividades benéficas, quando co-transplantadas com ilhotas pancreáticas, para o tratamento do diabetes. / Type 1 diabetes mellitus is an autoimmune disease caused by destruction of insulin-producing β cells, present in pancreatic islets, by auto-reactive cells of the immune system. The most widely used treatment option are daily injections of insulin, which configures a non-curative treatment. To achieve insulin independence, alternatives such as islet transplantation have been studied. However, the availability of pancreas from cadaveric donors for the isolation of these islets is poor and the methods for isolation, ineffective, requiring 2 to 4 donors to achieve the appropriate number of islets. In addition, transplantation presents problems related to engraftment, mainly due to poor vascularization, which leads to β cell death in the first days after transplantation. Thus, studies exploring alternatives that increase the survival and function of transplants and reduce the number of islets required by the recipient are very necessary. Mesenchymal stem cells have interesting properties for application in cell therapy. Among them is the paracrine effect, which has several beneficial functions, such as promoting vascularization in the tissues where these cells are present. Thus, the present study explored the co-transplantation of pancreatic islets with mesenchymal stem cells derived from adipose tissue for the treatment of diabetes mellitus in mice. The results showed that the presence of these cells in the group that received co-transplantation did not increase the cure rate, compared to the group that received islets alone. However, the phenomenon of diabetes reversion was anticipated in co-transplanted animals, which suggests a possible angiogenic effect of adipose-derived stem cells present in this group. Thus, we conclude that these cells may exert beneficial functions when co-transplanted with pancreatic islets for the treatment of diabetes.

Le cancer épithélial de l’ovaire (EOC) a la particularité d’être diagnostiqué à un stade avancé chez 75% des patientes et de récidiver dans un grand nombre de cas malgré une bonne réponse initiale à la chimiothérapie, expliquant ainsi son pronostic sombre. Le rôle du microenvironnement tumoral semble être de premier plan dans le développement et la survie des cellules cancéreuses mais il existe encore peu de données concernant les cellules mésenchymateuses souches (MSC). Dans ce travail nous avons donc cherché à déterminer les mécanismes moléculaires entre les MSC et les cellules tumorales ovariennes. Dans la première partie de ce travail, nous avons mis en évidence l’émergence d’un profile pro-métastatique des cellules tumorales ovariennes après contact avec les MSC. Nous avons ensuite développé un modèle d’infiltration tumorale 3D révélant que les MSC augmentaient la dissémination tumorale ovarienne par la sécrétion d’IL6. Enfin nous avons démontré que les MSC étaient capables d’induire chez les cellules tumorales ovariennes un phénotype thermotolérant lié à la sécrétion CXCL12. Ces données vont donc toutes dans le même sens en démontrant les propriétés pro-tumorales des MSC et ouvrent de nouvelles perspectives de thérapies ciblant les interactions entre le stroma et la tumeur. / Patients with epithelial ovarian cancer (EOC) are diagnosed with advanced stage in 75% of cases and most of them will relapse despite a good primary response to chemotherapy, thus explaining the bad prognosis of EOC. While tumor microenvironment seems to play an important role for the development and survival of cancer cells, there is only few data regarding the mesenchymal stem cells (MSC) in EOC. In this work we therefore aimed at identifying the molecular determinant between MSC and ovarian cancer cells. In the first part of this work, we demonstrated that ovarian cancer cells acquired pro-metastatic profile upon contact with MSC. We then showed that MSC could enhance ovarian cancer cells infiltration through IL6 secretion in an amniochorionic membrane based 3D model. Finally we showed that MSC could protect ovarian cancer cells from hyperthermia through CXCL12 secretion. Taken together, our data are concordant to reveal the pro-tumoral properties of MSC. Cytokine inhibitors interrupting the cross-talk between OCC and MSC should now be tested as new therapies for EOC.

This dissertation reviews the development and implementation of integrative, systems biology methods designed to parse driver mutations from high- throughput array data derived from human patients. The analysis of vast amounts of genomic and genetic data in the context of complex human genetic diseases such as Glioblastoma is a daunting task. Mutations exist by the hundreds, if not thousands, and only an unknown handful will contribute to the disease in a significant way. The goal of this project was to develop novel computational methods to identify candidate mutations from these data that drive the molecular differentiation of glioblastoma into the mesenchymal subtype, the most aggressive, poorest-prognosis tumors associated with glioblastoma.

The anterior cruciate ligament (ACL) is the most frequently injured ligament of the knee, with upwards of 100,000 ACL reconstructions performed annually. Current grafting techniques are limited by insufficient integration with subchondral bone and donor site morbidity issues related to graft harvest, potentially resulting in revision surgery and long-term joint pain. Therefore, significant demand exists for alternative grafting solutions that do not require additional surgery and can regenerate the native ACL-to-bone interface to promote biological fixation of the implanted ACL graft. To address this need, the ideal system must be able to withstand the functional demands of the native tissue by demonstrating physiologically equivalent mechanical properties, be comprised of compositionally varying phases in order to recapitulate the inherent heterogeneity of the native ligament to bone transition and be biodegradable such that it is gradually replaced by the regenerated tissue following implantation. It is hypothesized that a biomimetic, multi-phased scaffold comprised of optimized bone, interface and ligament regions coupled with controlled chemical and/or mechanical stimulation in vitro will guide phase-specific differentiation of mesenchymal stem cells (MSC) and result in a biologically integrated bone-ligament-bone complex in vivo. Mesenchymal stem cells are particularly attractive for this application as they can be routinely harvested from bone marrow, have been shown to respond to chemical, mechanical and structural cues, and are capable of differentiating towards the primary cell types (fibroblasts, osteoblasts and chondrocytes) found within ligament, bone and the ligament-to-bone interface. To this end, a nanofiber-based synthetic graft was designed with compositionally-varying phases to regenerate ligament, bone and interface tissues. The ligament phase was optimized in terms of nanofiber alignment, composition, mechanical stimulation and chemical stimulation. It was demonstrated that an aligned nanofiber substrate coupled with controlled mechanical stimulation was necessary to differentiate MSCs towards a fibroblastic phenotype. The bone phase was optimized in terms of ceramic content and it was shown that a threshold of mineral incorporation into nanofibers was necessary to differentiate MSCs towards an osteogenic phenotype. Lastly, a mechanoactive nanofiber collar was designed to induce interface formation. It was demonstrated that compressive stimulation applied via nanofiber collar contraction induced chondrogenic differentiation of MSCs. Subsequently, the three phases were incorporated to form a synthetic graft, for which graft architecture and cell seeding density were optimized. The resulting graft was cultured in vitro under the optimized parameters, demonstrating the formation of distinct and structurally continuous regions of bone, interface and ligament tissue. The graft was implanted in vivo where it was shown to be suitable for ACL reconstruction as it maintained knee stability and promoted ligament regeneration. In summary, this thesis focuses on the design of a biomimetic, nanofiber-based, integrated bone-ligament-bone construct, and elucidates chemical, mechanical and scaffold design-related parameters that can guide MSC differentiation towards desired tissue types. The impact of these studies extends beyond ligament reconstruction as they yield valuable scaffold design criteria, establish scaffold and culturing-related parameters to induce stem cell differentiation and can readily be applied to the formation of interfaces between soft-to-hard tissues as well as other complex tissues.

Glioblastoma is the most common adult malignant primary brain tumor with one of the worst prognosis. With a survival of 10 to 12 months, glioblastoma remains one of the most challenging disease to treat. The standard treatment method involves maximal possible resection of the tumor followed by radiation and chemotherapy. However, the short half-life of most chemotherapeutic drugs, high systemic toxicity and inability to cross the blood brain barrier inhibits effective delivery of the chemotherapeutics to the tumor. An ideal drug delivery system can reach the tumor site with high efficiency and continuously release the drug at the tumor site for an extended period. Adult stem cells including neural stem cells (NSC) and mesenchymal stem cells (MSC) have inherent tumor trophic properties allowing for site-specific delivery of chemotherapeutics. They can also be genetically engineered to secrete the chemotherapeutic drug continuously making them ideal candidates for cell-based delivery system for treating glioblastoma. MSC have been isolated from a wide range of sources including bone marrow, umbilical cord, adipose tissue, liver, multiple dental tissues and induced pluripotent stem cells. MSC are also easily amenable to viral modification allowing for easy manipulation to produce chemotherapeutic drugs. Additionally, more than 350 clinical trials using MSC have successfully established the safety of using MSC for cell-based therapies. Collectively these factors have led to the widespread use of MSC in cancer therapy. MSC have been successfully transduced to produce chemotherapeutic drugs to treat glioma, melanoma, lung cancer, ovarian cancer and breast cancer. Despite the multitudes of advantages that cell therapy provides they are limited in three main domains (1) Low cell retention and survival at the site of the tumor (2) In ability to co-deliver multiple therapeutics and (3) In ability to deliver drugs other than peptide based drugs. This thesis details the work to engineer mesenchymal stem cells to tackle these three issues and develop a system that can increase the efficacy of glioblastoma treatment. To increase the cellular retention and survival we engineered MSC to form multicellular spheroids and cell sheets. To co-delivery multiple therapeutics we engineered MSC to form MSC/DNA-templated nanoparticle hybrid cluster to co-deliver drugs for cancer therapy. The system showed superior performance due to the increased retention of the cells and nanoparticle at the tumor site. Finally, to deliver drugs other peptide based we engineered graphene oxide cellular patches for mesenchymal stem cells. Graphene oxide can carry diverse therapeutics and can kill the cancer cells without affecting the cellular viability of MSC.

<div>Mesenchymal stem cells (MSCs) are responsive to mechanical stimuli that play an essential role in directing their differentiation to the chondrogenic lineage. A better</div><div>understanding of the mechanisms that allow MSCs to respond to mechanical stimuli is important to improving cartilage tissue engineering and regenerative medicine. Hydrostatic pressure (HP) in particular is known to be a primary mechanical force in joints. However, little is known about the underlying mechanisms that facilitate HP</div><div>mechanotransduction. Understanding the signaling pathways in MSCs in transducing HP to a beneficial biologic response and their interrelationship were the focus of this thesis. Studies used porcine marrow-derived MSCs seeded in agarose gel. Calcium ion Ca++ signaling, focal adhesion kinase (FAK) involvement, and sirtuin1 activity were investigated in conjunction with HP application.</div><div><br></div><div><div>Intracellular Ca++ concentration was previously shown to be changed with HP application. In our study a bioreactor was used to apply a single application of HP to the MSC-seeded gel structures and observe Ca++ signaling via live imaging of a fluorescent calcium indicator in cells. However, no fluctuations in Ca++ concentrations were observed with 10 minutes loading of HP. Additionally a problem with the biore actor design was discovered. First the gel was floating around in the bioreactor even without loading. After stabilizing the gel and stopping it from floating, there were still about 16 µm of movement and deformation in the system. The movement and deformation was analyzed for the gel structure and different parts of the bioreactor. </div><div><br></div><div>Furthermore, we investigated the role of FAK in early and late chondrogenesis and also its involvement in HP mechanotransduction. A FAK inhibitor was used on MSCs from day 1 to 21 and showed a dose-dependent suppression of chondrogenesis. However, when low doses of FAK inhibitor added to the MSC culture from day 21 to 42, chondrogenesis was not inhibited. With 4 hour cyclic HP, FAK phosphorylation increased. The beneficial effect of HP was suppressed with overnight addition of the</div></div><div><div>FAK inhibitor to MSC medium, suggesting FAK involvement in HP mechanotransducation by MSCs.</div></div><div><br></div><div>Moreover, sirtuin1 participation in MSC chondrogenesis and mechanotransduction was also explored. The results indicated that overnight sirtuin1 inhibition increased chondrogenic gene expression (Agc, Col2, and Sox9) in MSCs. Additionally, the activity of sirtuin1 was decreased with both 4 hour cyclic hydrostatic pressure and inhibitor application. These two together demonstrated that sirtuin1 inhibition enhances chondrogenesis.</div><div><br></div><div><div>In this research we have investigated the role of Ca++ signaling, FAK involvement, and sirtuin1 activity in the mechanotransduction of HP in MSCs. These understandings about the mechanisms regulating the chondrogenesis with respect to HP could have important implications for cartilage tissue engineering and regenerative studies.</div></div>

Les cellules souches mésenchymateuses (CSM) de la moelle osseuse ont été intensivement étudiées pour leur capacité de régénération et leurs propriétés immunomodulatrices. Beaucoup d’études ont montré que la thérapie qui utilise les CSM améliore les fonctions de tissu ostéo-articulaire particulièrement le cartilage en vue de leur capacité de différenciation en chondrocytes. Les CSM présentent un certain nombre d'avantages pour la médecine régénérative, ces cellules peuvent être facilement isolées et multipliées en culture pour obtenir un nombre approprié pour la thérapie cellulaire. De plus, elles ont une faible immunogénicité, ce que les rende aptes à la transplantation allogénique. Depuis les années 1960, de nombreuses études ont souligné les propriétés médicinales des polysaccharides notamment les β-glucanes qui ont une place particulière du fait de leurs effets immunostimulants. L’objectif de notre travail était de mettre en évidence les capacités d’un β-glucane particulier, la laminarine, sur la prolifération et la différenciation des CSM dans la perspective d’applications dans l’arthrose. Les CSM ont été cultivés dans les milieux de croissance et de différenciation chondrocytaire. La viabilité et l'apoptose des cellules ont été explorées par le comptage, les tests MTT et la coloration à l'annexine V. En outre, l'analyse des protéines spécifiques de la prolifération a été effectuée par le western blott. De plus, l'expression des marqueurs spécifiques des CSM et des chondrocytes a été étudiée à l'aide de la RT-qPCR et de l’immunofluorescence. Nos résultats ont démontré que la stimulation des CSM à la laminarine avec la dose de 1 mg/ml soit en condition de culture de croissance basique ou en chondrogenèse a inhibé la prolifération des cellules sans induire leur apoptose. Encore, dans les conditions de culture chondrogénique, la laminarine à une dose similaire a empêché la différenciation des CSM en chondrocytes. / Mesenchymal stems cells (MSCs) are a population of multipotent cells residing in several readily available adult tissue compartments, thus allowing for their ex vivo expansion. MSCs have a reliable potential for differentiation (plasticity) into cells of the mesodermal lineage (chondrocytes, osteoblasts, adipocytes). Bone marrow-derived MSCs have been a focus of stem cell research in light of their relative ease of isolation and expansion and of their high potential for differentiation. Herein, the aim of the present PhD is to explore the potential of a β-glucan (laminarin) on Mesenchymal stem cell proliferation and differentiation for future benefit for osteoarthritis treatment. MSCs were cultured in MSC growth and chondrogenic differentiation mediums. Cells viability and apoptosis were explored by cell count, MTT assays and Annexin V staining. In addition, Analysis of the specific protein of cell proliferation was performed by western blott. Furthermore, mRNA and protein expression of specifics markers for MSCs and chondrocytes were studied using qPCR and immunofluorescence. Our results demonstrated that stimulation of MSC with laminarin at a dose of 1 mg/ml in either basic growth culture or chondrogenesis inhibited cell proliferation without inducing their apoptosis. Furthermore, under chondrogenic culture conditions, laminarin at a similar dose prevented the differentiation of MSC into chondrocytes.

有證據顯示基質細胞在腫瘤的發生發展中可以發揮重要的作用，基質細胞可以提供適宜腫瘤細胞增殖的腫瘤微環境。腫瘤相關成纖維細胞是一種特殊的與腫瘤生成高度相關的基質細胞。而通过我们的论证，小鼠胚胎成纖維細胞可以作為一種腫瘤相關成纖維細胞的細胞模型。 / 腫瘤誘導因子（TIF）是本實驗室在成瘤實驗中發現的一種新的倉鼠CXC 趨化因子。基于蛋白質序列的分析，TIF 属于Gro CXC 趨化因子家族。這個家族主要通過激活其受體CXCR2 來發揮作用。為了研究TIF 在腫瘤發生中的作用，我們在CHO-K1 細胞中建立了過表達TIF 的穩定細胞株。 / 我們發現共同注射的永生化MEF 與過表達TIF 的D12 細胞導致了腫瘤生長的抑制。為了研究這種現象，重組TIF 蛋白在大腸桿菌中表達，并且用鎳柱進行了提純。純化的蛋白被用于處理CHO-K1 細胞與永生化MEF。我們發現高水平的TIF 可以導致CXCR2 下游的Erk 磷酸化水平下降。其可能的機制為CXCR2 在高水平的TIF 作用下的脫敏作用。同時高水平TIF 可以導致永生化MEF 中CD133 水平的下降。因此，CXCR2 脫敏為TIF 導致腫瘤抑制的可能機制。 / Lines of evidence indicate that stromal cell is one of the determinants in tumor formation by providing a favorable microenvironment for the growth of cancer cells. Cancer associated fibroblast (CAF) is a special form of stromal cells which are shown to be derived from bone marrow. Upon reaching the tumor, the bone marrow-derived mesenchymal stem cells differentiate into CAF, which secrets various growth factors and cytokines to promote cancer growth. Furthermore, genetic study shows that CAF displays p53 mutations and other genetic changes. / Tumor induced factor (TIF) is a CXC chemokine that is originally identified from a xenograft tumor. Sequence analysis suggests TIF is a family member of the Gro CXC chemokines, and exerts its cellular function via activating CXCR2 receptors. In order to investigate the functional roles of TIF, a stable cell line over-expressing TIF in hamster CHO-K1 was established. / To explore the cancer-stromal interactions in xenograft, mouse embryonic fibroblast (MEF) were used as a study model for CAF. MEF was sub-cultured by a conventional protocol that was used for developing the NIH3T3 cells. Based on the growth patterns and expressions of cell markers, growth of MEF can be divided into three stages: the early stage, the senescent stage and the immortalized stage. Our results suggested that MEF might mirror the various developmental stages of CAF. / To examine the contributions of MEF in tumorigenesis, CHO-K1 cells and MEF were co-injected into nude mice. Intriguingly, MEF that in senescent and immortalized stages, rather than in early stage, promoted tumor formation. A possibility arose that the contribution of senescent and immortalized MEF in promoted tumorigenesis may due to CD133 and CXCL1, as the expression of CD133 and CXCL1 in senescent and immortalized MEF were higher than that of MEF in early stage. Moreover, as MEF could gradually develop into a fibroblast promoted tumor formation, MEF could be used as a crucial model to illustrate the origination and development of CAF. / Surprisingly, in nude mice co-injected with immortalized MEF with TIF-overexpressing D12 cells, suppression instead of promotion of tumor growth was found. In order to explore the underlined mechanism of tumor suppression, recombinant TIF protein was purified based on a bacterial expression system. Using purified TIF protein to treat CHO-K1 cells and MEF, it was found that low concentration of TIF promoted Erk phosphorylation but high concentration of TIF suppressed it, which might resulted from desensitization of CXCR2 receptors. Reduction of Erk phosphorylation resulted in decreased proliferation in CHO-K1 cells and alleviated expression of CD133 in MEF, which could be the mechanisms for TIF-induced tumor suppression in nude mice. / Taken together, a CAF model was established to examine the function of TIF in tumor-fibroblast interactions. Mechanistic studies indicated that TIF-induced tumor suppression in nude mice was mediated via desensitization of CXCR2 receptors by high concentration of TIF in the tumor microenvironment. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qi, Wei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 189-206). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Tumorigenesis --- p.4 / Chapter 1.1.1 --- Virus transformation --- p.4 / Chapter 1.1.2 --- Proto-oncogene and oncogene --- p.5 / Chapter 1.1.3 --- Tumor suppressor gene --- p.7 / Chapter 1.1.4 --- Epigenetic alteration --- p.9 / Chapter 1.1.5 --- Cancer stem cell --- p.11 / Chapter 1.1.6 --- Tumor microenvironment --- p.14 / Chapter 1.2 --- Cancer associated fibroblast (CAF) --- p.17 / Chapter 1.2.1 --- Markers for CAF --- p.17 / Chapter 1.2.2 --- CAF and normal fibroblast --- p.20 / Chapter 1.2.3 --- CAF, a important player in tumor growth --- p.22 / Chapter 1.2.4 --- CAF and angiogenesis --- p.23 / Chapter 1.2.5 --- CAF and tumor invasion --- p.25 / Chapter 1.3 --- Chemokine --- p.27 / Chapter 1.3.1 --- Structure of chemokine --- p.27 / Chapter 1.3.2 --- Chemokine and cell Recruitment --- p.30 / Chapter 1.3.3 --- Chemokine and tumor microenvironment --- p.30 / Chapter 1.4 --- Tumor Induced Factor and its induced tumor suppression --- p.38 / Chapter 1.5 --- The aims of the project --- p.47 / Chapter Chapter Two --- Purification of Tumor Induced Factor / Chapter 2.1 --- Introduction --- p.49 / Chapter 2.2 --- Materials --- p.52 / Chapter 2.2.1 --- Chemical --- p.52 / Chapter 2.2.2 --- Enzyme --- p.52 / Chapter 2.2.3 --- Antibody --- p.52 / Chapter 2.3 --- Method --- p.53 / Chapter 2.3.1 --- Overview of protein expression system --- p.53 / Chapter 2.3.2 --- Purification of Trx-His₆-S-TIF protein --- p.54 / Chapter 2.3.3 --- BCA assay --- p.60 / Chapter 2.3.4 --- SDS-PAGE --- p.60 / Chapter 2.3.5 --- Western blotting --- p.61 / Chapter 2.3.6 --- Preparation of pET28/His₆-Sumo-TIF bacterial expression vector --- p.62 / Chapter 2.3.7 --- Optimization of culture condition for BL21 expressed His₆-Sumo-TIF protein --- p.67 / Chapter 2.3.8 --- Purification of His₆-Sumo-TIF protein --- p.68 / Chapter 2.3.9 --- Homology model of TIF --- p.68 / Chapter 2.4 --- Results --- p.69 / Chapter 2.4.1 --- Purification of Trx-His₆-S-TIF --- p.70 / Chapter 2.4.2 --- Optimization of purification protocol of His₆-Sumo-TIF --- p.71 / Chapter 2.4.3 --- Large scale purification of mature TIF --- p.75 / Chapter 2.4.4 --- Homology modeling of TIF --- p.80 / Chapter 2.5 --- Discussion --- p.83 / Chapter Chapter 3 --- Three Stages Hypothesis / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.2 --- Material --- p.93 / Chapter 3.2.1 --- Chemical --- p.93 / Chapter 3.2.2 --- Enzyme --- p.93 / Chapter 3.2.3 --- Animal --- p.93 / Chapter 3.2.4 --- Antibody --- p.94 / Chapter 3.3 --- Methods --- p.95 / Chapter 3.3.1 --- Isolate MEF from 13.5 days mouse embryo --- p.95 / Chapter 3.3.2 --- Culture of MEF following 3T3 protocol --- p.96 / Chapter 3.3.3 --- X gal staining --- p.96 / Chapter 3.3.4 --- Analysis of MEF cell size and complexity by flow cytometry --- p.98 / Chapter 3.3.5 --- MTT assay --- p.98 / Chapter 3.3.6 --- Analysis of CD133 by flow cytometry --- p.99 / Chapter 3.3.7 --- ROS detected by DCFH-DA fluorescent probe --- p.99 / Chapter 3.3.8 --- Double staining of cancer stem cell marker and ROS fluorescent probe --- p.100 / Chapter 3.3.9 --- Reverse transcription --- p.101 / Chapter 3.3.10 --- Analysis CXCL1 mRNA expression level by PCR --- p.102 / Chapter 3.3.11 --- Gelatin zymography --- p.103 / Chapter 3.3.12 --- In-vivo tumorigenicity assay --- p.104 / Chapter 3.4 --- Results --- p.106 / Chapter 3.4.1 --- Three Stages of MEF --- p.106 / Chapter 3.4.2 --- X gal staining --- p.106 / Chapter 3.4.3 --- Flow cytometric analysis of cell diameter and cellular complexity of MEF --- p.109 / Chapter 3.4.4 --- MTT assay --- p.109 / Chapter 3.4.5 --- CD 133 expression of MEF detected by flow cytometry --- p.110 / Chapter 3.4.6 --- Reactive oxygen species of MEF detected by flow cytometry --- p.118 / Chapter 3.4.7 --- The level of ROS and CD133 of MEF detected by flow cytometry stimultaneously --- p.121 / Chapter 3.4.8 --- TIF treatment reduces the small CSC subpopulation in senescent stage MEF --- p.124 / Chapter 3.4.9 --- Increased CXCL1 expression in senescent stage and immortalized stage MEF --- p.125 / Chapter 3.4.10 --- Matrix metalloproteinase 2 activities in different stages of MEF . --- p.129 / Chapter 3.4.11 --- In vivo tumorigenicity assay --- p.130 / Chapter 3.5 --- Discussion --- p.133 / Chapter Chapter Four --- Biphasic Effect of TIF in Cancer-Fibroblasts Interaction / Chapter 4.1 --- Introduction --- p.140 / Chapter 4.2 --- Material --- p.143 / Chapter 4.2.1 --- Chemical --- p.144 / Chapter 4.2.2 --- Kit and Instrument --- p.144 / Chapter 4.2.3 --- Antibody --- p.144 / Chapter 4.3 --- Method --- p.145 / Chapter 4.3.1 --- Purification of TIF-His₆-Flag --- p.145 / Chapter 4.3.2 --- Western blotting to detect purified TIF-His₆-Flag --- p.145 / Chapter 4.3.3. --- Measurement of cell proliferation by cell counting --- p.145 / Chapter 4.3.4 --- MTT assay --- p.146 / Chapter 4.3.5 --- Western blotting to detect pErk and total Erk --- p.146 / Chapter 4.3.6 --- Soft agar assay --- p.148 / Chapter 4.3.7 --- Gelatinase detection --- p.148 / Chapter 4.3.8 --- Wound healing assay --- p.149 / Chapter 4.3.9 --- Colony formation assay --- p.149 / Chapter 4.3.10 --- Detection of CD133 by flow cytometry --- p.150 / Chapter 4.4 --- Results --- p.151 / Chapter 4.4.1 --- Purification of TIF-His₆-Flag --- p.151 / Chapter 4.4.2 --- Reduced cell proliferation of D12 in long time culture --- p.153 / Chapter 4.4.3 --- Reduced metabolic activities of D12 cells in time culture --- p.155 / Chapter 4.4.4. --- TIF-CXCR2-pErk signal axis in CHO cells --- p.155 / Chapter 4.4.5 --- Bigger colonies formed by D12 cells in soft agar assay --- p.161 / Chapter 4.4.6 --- TIF-CXCR2-pErk-MMP9 signal pathway in D12 cells --- p.162 / Chapter 4.4.7 --- Reduced migration of D12 cells --- p.164 / Chapter 4.4.8 --- Reduced cell invasion of D12 cells --- p.165 / Chapter 4.4.9 --- Reduced colony number of D12 cells in colony formation assay --- p.168 / Chapter 4.4.10 --- Bi-phasic “bell shape“ bi-phasic response on Erk activation of TIF in CHO-K1 cells --- p.169 / Chapter 4.4.11 --- Bi-phasic “bell shape“ effect of TIF to pErk in immortalized MEFs --- p.172 / Chapter 4.4.12 --- Reduced CD133 in immortalized MEF by high concentration of TIF --- p.173 / Chapter 4.5 --- Discussion --- p.177 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Project Summary --- p.183 / Chapter 5.2 --- Significances of the project --- p.185 / Chapter 5.3 --- Future work --- p.188