The potential of embryonic stem (ES) cells to generate any developmental or adult cell type holds much promise for regenerative medicine and in vitro modelling of development and disease. Haematopoietic stem cells (HSCs) regenerate all lineages of the blood throughout adult life and are essential for the treatment of a vast number of haematalogic disorders. Current sources of HSCs for clinical use and research, including adult bone marrow, peripheral blood stem cells and umbilical cord blood, are limited by the number of HSCs they contain and by the availability of a suitable donor. A system that generates a reliable source of HSCs from ES cells would therefore be an ideal alternative. While much progress has been made in the generation of downstream lineages of the haematopoietic system, progress in the derivation of HSCs capable of long-term self-renewal and multilineage reconstitution from ES cells has been limited. Understanding of the developmental steps leading to HSC emergence in the embryo has been advancing in recent years. In particular, precursors of HSCs (preHSCs) have been isolated from the mouse embryo, characterised and matured into HSCs ex vivo using the specialised conditions of aggregate culture systems (Taoudi et al 2008, Rybtsov et al 2011). We hypothesised that application of the aggregate culture system in the differentiation of ES cells could provide a missing link in the in vitro generation of HSCs. Here I have developed a novel ES cell differentiation system that employs the specialised conditions of the aggregate culture system, after an initial stage of mesoderm differentiation. I show that this system creates an environment for efficient haematopoietic and endothelial progenitor formation and generates cells of a preHSC type I (VE-Cadherin+CD45-CD41lo) and preHSC type II (VE-Cadhein+CD45+) surface phenotype. Notably, the system gives rise to cells that achieve low-levels of haematopoietic repopulation in sublethally irradiated NSG mice. The low-level repopulating cells persist for over 4 months in animals and show both myeloid and lymphoid potential. I identify genes that are expressed in cells of a preHSC II surface marker-phenotype from the E11.5 dorsal aorta, but not in cells of this phenotype from the E11.5 Yolk sac or differentiated ES cells. I also show that enforced expression of Notch downstream target Hes1 in Flk1+ mesoderm during ES cell differentiation does not improve levels of ES-derived repopulation.

Les prokinéticines sont des hormones angiogéniques qui exercent leurs fonctions biologiques par l’intermédiaire de deux récepteurs couplés aux protéines G : PKR1 et PKR2. PKR1 a été révélé comme crucial dans l’homéostasie cardiovasculaire. L’objectif de ce projet de thèse était de développer un nouvel agoniste non–peptidique à PKR1 pour la cardioprotection et la régénération cardiaque. Les premiers résultats ont permis de caractériser le premier ligand spécifique à PKR1 : IS20. L’étude in vivo a démontré qu’IS20 est capable de prévenir les lésions après induction d’un infarctus du myocarde chez la souris. Ce composé améliore les fonctions cardiaques en activant la prolifération de cellules progénitrices cardiaques et la néovascularisation (Gasser et al, PlosOne, 2015). Dans une deuxième étude, nous avons évalué le potentiel cardioprotecteur d’IS20 face à la toxicité induite par la doxorubicine (DOX), un anticancéreux de la famille des anthracyclines très efficace mais cardiotoxique. Les résultats montrent qu’IS20 atténue l’apoptose des cardiomyocytes H9c2 et des cellules progénitrices humaines de types EPDC, induite par la doxorubicine, sans affecter la cytotoxicité de la doxorubicine sur les cellules cancéreuses. In vivo, le traitement par IS20 atténue la diminution de la prolifération provoquée par la doxorubicine dans un modèle de cardiotoxicité juvénile. Dans un modèle de cardiotoxicité chronique, IS20 maintient l’intégrité cellulaire et tissulaire des vaisseaux et protège des défaillances produites par DOX. Par ses effets cytoprotecteurs des cardiomyocytes et des cellules progénitrices cardiaques, l’IS20 présente un potentiel thérapeutique prometteur pour protéger les patients cancéreux des effets cardiotoxiques des anthracyclines. / Prokineticins are angiogenic hormones that activate two G protein-coupled receptors (GPCRs): PKR1 and PKR2. PKR1 has emerged as a critical mediator of cardiovascular homeostasis and cardioprotection. The aim of this thesis project was to develop a first non-peptide PKR1 agonist stimulates cardioprotection and cardiac regeneration in mouse model of myocardial infarction (MI) or anti-cancer drug mediated cardiotoxicity. Collaboration with chemist and biomodelization team, we characterized the first selective/specific PKR1 agonist, named IS20. In vivo study demonstrated IS20 prevented cardiac lesion formation and improved cardiac function after myocardial infarction in mice, promoting proliferation of cardiac progenitor cells and neovasculogenesis (Gasser et al., 2015). Since use of a very potent anthracycline chemotherapeutic, Doxorubicin (DOX) is limited by cardiotoxicity, we hypothesized that IS20 could protect heart against DOX-mediated cardiotoxicity. Indeed, IS20 attenuated apoptosis induced by DOX in H9c2 cardiomyocytes and human epicardial progenitors in vitro. However, IS20 did not affect antineoplastic or cytostatic effect of DOX in cancer cell lines. In vivo, in the juvenile model of cardiotoxicity, IS20 significantly attenuated DOX-induced decrease in viability and proliferation cardiac progenitor cells. In the chronic cardiotoxicity model by DOX, IS20 improves heart structure and function by the activation of cardiac progenitor cells, diminishing cardiac cell death, improving vascular stability. IS20 has translational potential for cardioprotection in patients with cancer receiving anthracyclines.

Articular cartilage damage associated with joint trauma seldom heals and often leads to osteoarthritis (OA). Current treatment often fails to regenerated functional cartilage close to native tissue. We previously identified a migratory chondrogenic progenitor cell (CPC) population that responded chemotactically to cell death and rapidly repopulated the injured cartilage matrix, which suggested their potential for cartilage repair. To test that potential we filled experimental full thickness chondral defects with an acellular hydrogel containing SDF-1α. We expect that SDF-1α can increase the recruitment of CPCs, and then promote the formation of a functional cartilage matrix with chondrogenic factors. Full-thickness bovine chondral defects were filled with hydrogel comprised of fibrin and hyaluronic acid and containing SDF-1α. Cell migration was monitored, followed by chondrogenic induction. Regenerated tissue was evaluated by histology, immunohistochemistry, and scanning electron microscopy. Push-out tests were performed to assess the strength of integration between regenerated tissue and host cartilage. Significant numbers of progenitor cells were recruited by SDF-1α within 12 days. By 5 weeks chondrogenesis, repair tissue cell morphology, proteoglycan density and surface ultrastructure were similar to native cartilage. SDF-1α treated defects had significantly greater interfacial strength than untreated controls. However, regenerated neocartilage had relatively inferior mechanical properties compared with native cartilage. In addition to that, we developed a 3D bioprinting platform, which can directly print chondrocytes as well as CPCs to fabricated articular cartilage tissue in vitro. We successfully implanted the printed tissue into an osteochondral defect, and observed tissue repair after implantation. The regerated tissue has biochemical and mechanical properties within the physiological range of native articular cartilage. This study showed that, when CPC chemotaxis and chondrogenesis are stimulated sequentially, in situ full thickness cartilage regeneration and bonding of repair tissue to surrounding cartilage could occur without the need for cell transplantation from exogenous sources. This study also demonstrated the potential of using 3D bioprinting to engineer articular cartilage implants for repairing cartilage defect.

The aim of this thesis is to generate original symmetric presentations for finite non-abelian simple groups. We will discuss many permutation progenitors, including but not limited to 2*14 : D28, 2∗9 : 3•(32), 3∗9 : 3•(32), 2∗21 : (7X3) : 2 as well as monomial progenitors, including 7∗5 :m A5, 3∗5 :m S5. We have included their homomorphic images which include the Mathieu group M12, 2•J2, 2XS(4, 5), as well as, many PGL′s, PSL′s and alternating groups. We will give proofs of the isomorphism types of each progenitor, either by hand using double coset enumeration or computer based using MAGMA. We have also constructed Cayley graphs of the following groups, 25 : S5 over 2∗5 : S5, PSL(2, 8) over 2∗7 : D14, M12 over a maximal subgroup, 2XS5. We have developed a lemma using relations to factor permutation progenitors of the form m∗n : N to give an isomorphism of mn : N . Motivated by Robert T. Curtis’ research, we will present a program using MAGMA that, when given a target finite non-abelian simple group, the program will generate possible control groups to write progenitors that will give the given finite non-abelian simple group. Iwasawa’s lemma is also discussed and used to prove PSL(2, 8) and M12 to be simple groups.

We have investigated several monomial and permutation progenitors, including 2*8 : [8 : 2], 2*18 : [(22 x 3) : (3x2)], 2*16 : [22 : 4], and 2*16 : 24, 5*2 :m [4•22], 5*2 :m [(4x2) :• 2], 103∗2 :m [17 : 2] and 103∗4 :m [17 : 4]. We have discovered original, to the best of our knowledge, symmetric presentations of a number of finite groups, including PSL(2, 7), M12 , A6 : 2, A7 , PSL(2, 25), 25 :• S4, 24 : S3, PSL(2, 271), 12 x PSL(2, 13), and U(3, 7) : 2. We will present our construction of several of these images, including the Mathieu sporadic simple group M12 over the maximal subgroup PSL(2, 11), PSL(2, 17) over D9, PSL(2, 16) : 2 over [24 : 5] and PGL(2, 7) over S3. We will also give our method of finding isomorphism classes of images.

The goal of this presentation is to find original symmetric presentations of finite groups. It is frequently the case, that progenitors factored by appropriate relations produce simple and even sporadic groups as homomorphic images. We have discovered two of the twenty-six sporadic simple groups namely, M12, J1 and the Lie type group Suz(8). In addition several linear and classical groups will also be presented. We will present several progenitors including: 2*12: 22 x (3 : 2), 2*11: PSL2(11), 2*5: (5 : 4) which have produced the homomorphic images: M12 : 2, Suz(8) x 2, and J1 x 2. We will give monomial progenitors whose homomorphic images are: 17*10 :m PGL2(9), 3*4:m Z2 ≀D4 , and 13*2:m (22 x 3) : 2 which produce the homomorphic images:132 : ((2 x 13) : (2 x 3)), 2 x S9, and (22)•PGL4(3). Once we have a presentation of a group we can verify the group's existence through double coset enumeration. We will give proofs of isomorphism types of the presented images: S3 x PGL2(7) x S5, 28:A5, and 2•U4(2):2.

The foundation of the work of this thesis is based around the involutory progenitor and the finite homomorphic images found therein. This process is developed by Robert T. Curtis and he defines it as 2^{*n} :N {pi w | pi in N, w} where 2^{*n} denotes a free product of n copies of the cyclic group of order 2 generated by involutions. We repeat this process with different control groups and a different array of possible relations to discover interesting groups, such as sporadic, linear, or unitary groups, to name a few. Predominantly this work was produced from transitive groups in 6,10,12, and 18 letters. Which led to identify some appealing groups for this project, such as Janko group J1, Symplectic groups S(4,3) and S(6,2), Mathieu group M12 and some linear groups such as PGL2(7) and L2(11) . With this information, we performed double coset enumeration on some of our findings, M12 over L_2(11) and L_2(31) over D15. We will also prove their isomorphism types with the help of the Jordan-Holder theorem, which aids us in defining the make up of the group. Some examples that we will encounter are the extensions of L_2(31)(center) 2 and A5:2^2.

Articular cartilage underwent serious joint injuries seldom repair spontaneously and might progress to post-traumatic osteoarthritis. This is majorly because articular cartilage’s unique properties that lack blood and nerve supply intrinsically. This peculiar structure, in addition, generates an unfavorable environment for certain phagocytes (macrophages, monocytes, neutrophils, etc) to infiltrate to cartilage to scavenge debris from cartilage matrix and cell caused from joint injuries. Therefore, physiological and functional regeneration of damaged cartilage is urgently needed and several clinical techniques have been developed, including microfracture, autograft transplantation, autologous chondrocytes implantation. We previously identified highly migratory cells emerged and repopulated in cartilage damaged surface after ~10 days of artificial cartilage injury. These cells were later named chondrogenic progenitor cells (CPCs) due to their enhanced potential of chondrogenic differentiation. However, this important finding contrasts the conventional theory that cartilage harbors only one cell type, chondrocytes. Here we hypothesize that CPCs are a distinct cell type in cartilage, and more importantly, one of CPCs’ crucial natures is to phagocytose debris more effectively than chondrocytes. To test these, we first harvested CPCs from cartilage surfaces, chondrocytes, synovial cells (synoviocytes and synovial fluid cells) for microarray assay to evaluate the closeness among these joint cells on whole gene expression level. Quantitative PCR were then conducted to verify gene expression of certain functional interests. Moreover, debris from cell and extracellular matrix were generated and incubated with CPCs and chondrocytes to compare their phagocytic capacity via multiple experimental assessments. In confocal microscopy examination, the emergence of CPCs could be clearly observed after cartilage injury. Aside from their distinguishable morphology compared to chondrocyte, CPCs possess several vital properties including highly migratory, chemotactic, clonogenic. Microarray data revealed that CPCs, from gene expression profile, are distinctively isolated from chondrocytes and are more akin to synovial cells. Additionally, the series of phagocytosis related experiments showed that CPCs are dramatically superior to chondrocytes in engulfing debris, along with enhanced lysosomal activities indicating the following debris degradation. Taken all these data together, CPCs, activated by cartilage injury, emerged and migrated to damaged sites. They are a distinct cell type residing in cartilage apart from chondrocytes. Their enhanced capacity to sustainably phagocytose and clear debris provides a novel insight for cartilage regeneration and prevention of osteoarthritis.

No description available.

Articular cartilage is an avascular, aneural, and alymphatic tissue with a structure consisting of a superficial, a middle and a deep zone, overlie a calcified zone at the cartilage border between. Each zone has biological and mechanical properties. Self-repair of damaged cartilage seldom if ever occurs, and joint injuries that harm cartilage surfaces often result in osteoarthritis. This has prompted researchers to explore diverse approaches to cartilage regeneration. The superficial zone shows the highest cellularity and the lowest matrix density. Cartilage cells (chondrocytes) residing in the superficial zone had been thought to be a subpopulation of chondrocytes. However, our laboratory identified a second population of cells that were distinguishable from chondrocytes based on their clonogenicity, multipotency, migratory activity, higher proliferate rate and substantial morphological differences. These cells later proved to be chondrogenic progenitor cells (CPCs). Our continuing studies have shown that CPCs are less chondrogenic than normal chondrocytes and their function is to protect the cartilage surface rather than to regenerate cartilage matrix as previously supposed. In addition, we found evidence to suggest that CPCs act as pro-inflammatory cells in the context of cartilage injury. For these reasons, we undertook a more comprehensive comparison of the phenotypic differences between CPCs and normal chondrocytes and between CPCs and joint cells (tissue synoviocytes from the joint capsule and cells present in synovial fluid) which have been shown to be play roles in joint inflammation. Gene expression microarray analysis of >25,000 genes revealed that the overall pattern of gene expression in CPCs was distinct from normal chondrocytes, but closely related to synoviocytes and synovial fluid cells. Analysis of specific genes by quantitative PCR (qPCR) showed profound differences between CPCs and normal chondrocytes in terms of cartilage matrix gene expression (Collagen Type ІІ, Aggrecan, Link Protein and COMP) and pro-inflammatory gene expression (IL6, IL8, CCL2 and CXCL12). In contrast, the pattern of CPC gene expression closely resembled. Sulfated glycosaminoglycan assays revealed that cartilage matrix deposition by CPCs, as well as synoviocytes and synovial fluid cells, was significantly inferior to normal chondrocytes. However, chondrogenic and osteogenic differentiation assays, showed no significant differences among the four cell types. In addition to establishing that CPCs are distinct from chondrocytes, this work suggests significant revisions to our understanding of CPC function in cartilage. The weak chondrogenic ability and higher expression of inflammatory cytokines, suggests these cells don't play a regenerative role as previously thought. On the other, we found evidence that CPCs may form a protective layer on the top of the injured cartilage surfaces, preventing further cartilage injury. In vivo studies are needed to fully elucidate the significance of these roles in cartilage health and disease.