Cell based therapy using stem cell derived cardiomyocytes, has emerged as a potential therapeutic approach for cardiac diseases such as myocardial infarction and heart failure. Induced pluripotent stem cells (iPS cells) could be an ideal source of cardiomyocytes (iPS-CM). Challenges facing cell therapy include the high number of viable cells needed to survive in pathological conditions. The Hippo signalling pathway has been described as a key pathway involved in regulating cardiomyocyte proliferation and survival in both embryonic and adult hearts. We hypothesise that modification of the Hippo pathway will enhance the efficiency of iPS-CM generation and will increase iPS-CM survival and viability in pathological conditions. Skin fibroblasts were reprogrammed to iPS cells and then differentiated to cardiomyocytes. The Hippo signalling pathway was modified by genetic ablation of MST1, a major upstream regulator of the Hippo pathway, or by overexpressing YAP, the main downstream effector of the pathway. Cell proliferation was analysed using an EdU incorporation assay and staining for cytokinesis markers Ki67 and phospho-histone H3. Cell death and viability were analysed by measuring caspase 3/7 and MTT activity and by trypan blue staining in both normal and hypoxic conditions (CoCl2 treatment). Analysis of cell proliferation shows that genetic ablation of Mst1 leads to significantly increased proliferation (+12±1.5% P < 0.001), survival and viability (+20±4.3% P < 0.001) of iPS cells in both normal and hypoxic (CoCl2 treatment) conditions compared to controls. In addition, overexpression of YAP, which is normally inhibited by upstream Hippo pathway components, and overexpression of mutated constitutively active form of YAP (S127A) increases cell proliferation in iPS-CM compared to control iPS-CM as shown with EdU assay (46±2.60% P < 0.01) and Ki67 staining (4.9±0.9% P < 0.001). Overexpression of YAP leads to up regulation of genes associated with inhibition of apoptosis and promotion of cell proliferation. Preliminary studies show mouse iPS-CM are retained in the myocardium following intra-cardiac injection and do not cause any adverse effects confirmed with histological, echocardiography and electrocardiogram analysis. In conclusion targeting the Hippo pathway in iPS cells and iPS-CM significantly increases proliferation and survival in both normal and hypoxic conditions. Therefore, modulation of the Hippo pathway could become a new strategy to enhance the therapeutic potential of iPS-CM.

There is a growing literature of clinical studies of bone marrow (BM) cell therapy for liver cirrhosis. At present, the optimum choice of cell type(s) and the mechanism(s) of effect remain undefined. Cells of the monocyte-macrophage lineage have key roles in the development and resolution of liver fibrosis. Therefore, I tested the therapeutic effects of these cells in the context of experimental murine liver fibrosis. The effects of unmanipulated, syngeneic macrophages, their specific BM precursors and unfractionated (whole) BM cells were examined in the iterative carbon tetrachloride model of liver fibrosis. BM-derived macrophage (BMM) delivery resulted in early chemokine upregulation with the hepatic recruitment of endogenous macrophages and neutrophils. These cells delivered matrix metalloproteinases-13 and -9 respectively, into the hepatic scar. The effector cell infiltrate was accompanied by increased levels of the anti-inflammatory cytokine IL-10. A reduction in hepatic myofibroblasts was followed by reduced fibrosis detected 4 weeks after macrophage infusion. Serum albumin levels were elevated at this time. Upregulation of the liver progenitor cell mitogen TWEAK preceded expansion of the progenitor cell compartment. BMM delivery increased hepatic expression of cytokines with reparative effects (including colony stimulating factor-1, insulin-like growth factor-1 and vascular endothelial growth factor). In contrast to the effects of differentiated macrophages, liver fibrosis was not significantly improved by the application of macrophage precursors and was exacerbated by whole BM. BMMs did not affect liver fibrosis or regeneration in the 1% DDC model of biliary disease. These effects were only detected following the intraportal delivery of BM cells. The peripheral (tail) vein administration of BMMs, either singly or repeatedly did not recapitulate the therapeutic phenotype. This was investigated by in vivo tracking of BMMs constitutively expressing green fluorescent protein (GFP). The peripheral administration route resulted in the early (1 hour) accumulation of BMMs within the pulmonary system. This was followed by delayed hepatic engraftment, which was also numerically reduced (< 30%) compared with intraportal administration. Macrophage cell therapy improves clinically relevant parameters in experimental chronic liver injury. Paracrine signalling to endogenous cells amplifies the effect. The benefits from this single, defined cell type suggest clinical potential.

Made available in DSpace on 2014-06-11T19:23:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-14Bitstream added on 2014-06-13T19:09:55Z : No. of bitstreams: 1 rossetto_vjv_me_botfmvz_parcial.pdf: 44582 bytes, checksum: 3c3725afe05c19146568796a0ee21ac3 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As indicações para a cirurgia reparadora da bexiga abrangem traumas graves com perda tecidual extensa, neoplasias, cistites intersticiais, disfunções neurológicas e anormalidades congênitas. Devido a inúmeras complicações associadas às técnicas de cistoplastia com o emprego de segmentos gastrointestinais, o presente estudo teve como objetivo analisar comparativamente a reparação anatômica da bexiga de cães utilizandose a submucosa intestinal suína (SIS) acelular e semeada com células musculares lisas homólogas (CMLH), bem como avaliar as possíveis complicações e efeitos colaterais inerentes a cada um dos tipos de enxerto empregados. Para tal foram utilizados 10 cães adultos, fêmeas, subdivididos em dois grupos, sendo: Grupo Controle (GC), constituído por cinco animais submetidos à cistoplastia com SIS; e Grupo Tratado (GT), constituído por cinco animais submetidos à cistoplastia com SIS semeada com CMLH. As células foram obtidas a partir de fragmentos de espessura completa, provenientes de bexigas de cães que vieram a óbito por motivos não infecciosos ou neoplásicos, cultivadas e expandidas in vitro. Em todos os grupos foi criado um defeito de 3 cm x 2 cm, preservando-se a região do trígono vesical, o qual foi reparado pelos respectivos implantes. Os cães de ambos os grupos foram submetidos à biópsia cirúrgica com 60 dias de pós-operatório, sendo essas obtidas da área central do tecido neoformado e da adjacência entre o mesmo e a bexiga original. Após a implantação dos enxertos, os animais foram avaliados por meio de exames clínico, ultrassonográfico abdominal e laboratoriais. Todas as amostras foram destinadas à avaliação histológica pelas colorações de H/E e Tricromio de Masson, analisadas com um fotomicroscópio Nikon conectado ao sistema de análise de imagem ImageJ. Não foram... / Not available

Orientador: Cláudia Valéria Seullner Brandão / Coorientador: Lígia Souza Lima Silveira da Mota / Banca: Juliany Gomes Quitzan / Banca: Bruno Watanabe Minto / Resumo: As indicações para a cirurgia reparadora da bexiga abrangem traumas graves com perda tecidual extensa, neoplasias, cistites intersticiais, disfunções neurológicas e anormalidades congênitas. Devido a inúmeras complicações associadas às técnicas de cistoplastia com o emprego de segmentos gastrointestinais, o presente estudo teve como objetivo analisar comparativamente a reparação anatômica da bexiga de cães utilizandose a submucosa intestinal suína (SIS) acelular e semeada com células musculares lisas homólogas (CMLH), bem como avaliar as possíveis complicações e efeitos colaterais inerentes a cada um dos tipos de enxerto empregados. Para tal foram utilizados 10 cães adultos, fêmeas, subdivididos em dois grupos, sendo: Grupo Controle (GC), constituído por cinco animais submetidos à cistoplastia com SIS; e Grupo Tratado (GT), constituído por cinco animais submetidos à cistoplastia com SIS semeada com CMLH. As células foram obtidas a partir de fragmentos de espessura completa, provenientes de bexigas de cães que vieram a óbito por motivos não infecciosos ou neoplásicos, cultivadas e expandidas in vitro. Em todos os grupos foi criado um defeito de 3 cm x 2 cm, preservando-se a região do trígono vesical, o qual foi reparado pelos respectivos implantes. Os cães de ambos os grupos foram submetidos à biópsia cirúrgica com 60 dias de pós-operatório, sendo essas obtidas da área central do tecido neoformado e da adjacência entre o mesmo e a bexiga original. Após a implantação dos enxertos, os animais foram avaliados por meio de exames clínico, ultrassonográfico abdominal e laboratoriais. Todas as amostras foram destinadas à avaliação histológica pelas colorações de H/E e Tricromio de Masson, analisadas com um fotomicroscópio Nikon conectado ao sistema de análise de imagem ImageJ. Não foram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Not available / Mestre

Acute myocardial infarction (MI) is the end result of many cardiovascular diseases and is one of the leading causes of death in the western world. Cell therapy, using circulating angiogenic cells (CACs) or CD34+ cells from peripheral blood, is one approach under investigation for restoring blood flow and function to the ischemic heart. However, the numbers of CACs and CD34+ circulating cells are inversely proportional to the severity of cardiovascular disease and age; therefore, there is a need to increase their numbers and/or function for therapy. One possibility is to enhance the therapeutic potential of the cells with the use of a biomaterial. In this study, we used a collagen matrix to culture human CD34+ circulating cells, and evaluated the effect of the matrix on CD34+ cell properties and function. The matrix was able to successfully increase proliferation, migration, CD34+ phenotype and branching in an angiogenesis assay. These functional benefits may be associated with the sonic hedgehog (Shh) pathway. The collagen matrix was previously shown to enhance the function of healthy CACs, but its ability to do the same for CACs from coronary artery disease patients is unknown. In this study, the matrix was shown to enhance the viability, proliferation and angiogenic potential of patient CACs. Furthermore, gene expression for integrins and Shh pathway components in the sub-population of CD34+ cells was similar between patient and healthy donors when isolated from CACs. This work provides insight into the mechanisms for the observed matrix-enhanced function of therapeutic CACs and CD34+ cells from both healthy and CAD patient donors.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22347号 / 医博第4588号 / 新制||医||1042(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 生田 宏一, 教授 江藤 浩之, 教授 濵﨑 洋子 / 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・論文博士 / 博士(医学) / 乙第13371号 / 論医博第2208号 / 新制||医||1047(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 川口 義弥, 教授 妹尾 浩, 教授 浅野 雅秀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Calcification is linked to a high prevalence of cardiovascular events and mortality due to arterial stiffness. Stiffening of the arteries in the case of medial calcification is due to hydroxyapatite mineral deposited in the artery thus leading to the loss of elastin. A possibility of removing this rogue mineral along the vessel walls could be the use of osteoclasts. Osteoclasts, a type of osteocyte, have the unique ability to absorb bone in the bone turnover process. It is proposed that in the future, osteoclasts be delivered to the site of mineralization through oxidized alginate-fibrin microbeads. Alginate hydrogels have proven great in drug delivery and could be a revolutionary cell delivery device to provide care for multitudes of people suffering from adjacent cardiovascular health problems such as arterial stiffness.

Peripheral artery disease (PAD) is characterized by the development of atherosclerotic plaques on arterial walls, leading to the narrowing of blood vessels, resulting in ischemia in the downstream tissue. In the United States, 12% of the adult population is affected by PAD and its related symptoms. Current surgical revascularization techniques can be effective in part of the patient population, but there is a need for other options. Alternatively, collateral blood vessels, or natural bypass arteries, enlarge to increase blood flow to the ischemic tissue in a process called arteriogenesis, which has been studied as a therapeutic option. Cell-based therapies, such as BM-MNCs, have been investigated as means to enhance arteriogenesis, but have largely failed in clinical trials. An alternative cell-based therapy candidate are myoblasts, or muscle progenitor cells. Myoblasts increase arteriogenesis in murine models and are known to interact with macrophages, which are immune cells that are primary regulators of arteriogenesis. Macrophages can polarize to inflammatory (M1) and regenerative (M2) phenotypes, with the M2 phenotype promoting enhanced arteriogenesis. This interaction suggests that myoblasts may be signaling macrophage polarization to enhance arteriogenesis. The purpose of this study was to determine if myoblasts in vitro can affect macrophage polarization into inflammatory (M1) or regenerative (M2) phenotypes. Protocols for macrophage culture and polarization were implemented, and then macrophages were co-cultured with myoblasts for 24 hours to assess the effects in vitro. Concentrations of known inflammatory (TNF-a) and regenerative (IL-10) cytokines released by macrophages were measured after co-culture with myoblasts. Surprisingly, macrophages co-cultured with myoblasts showed a decrease in both TNF-a and IL-10 compared to macrophages cultured alone. Morphology changes of macrophages were also measured after co-culture, with, surprisingly, little difference in the groups co-cultured with myoblasts. Pilot experiments suggest there may be an initial lag time greater than 24 hours for myoblasts to affect macrophage phenotype. Future work ideally will include longer time points and optimizing viability and proliferation of myoblasts in co-culture settings.

Osteoarthritis (OA) is the leading cause of musculoskeletal disability in people and horses, and is characterized by progressive joint degeneration. There is a critical need for a better understanding of disease processes leading to OA in order to develop more efficient therapies. A shared feature among different arthritic conditions is chronic synovitis. Macrophages are the main drivers of synovitis and can display pro-inflammatory (M1) or pro-resolving responses (M2). Macrophages promote joint health through phagocytic and secretory activities; however, when these functions are overwhelmed, macrophages upregulate inflammation, recruiting more cells to counteract damage. Once cell recruitment is efficiently accomplished, macrophages coordinate tissue repair and further resolution of inflammation. Bone marrow mononuclear cells (BMNC) are a source of macrophages used to treat inflammation and produce essential molecules for cartilage metabolism; however, little information exists regarding their use in joints. The studies presented in this dissertation focus on understanding the dual role of macrophages in driving and resolving synovitis and how to harness their therapeutic potential. In the first study, patterns of macrophage phenotypes (M1:M2) in healthy and osteoarthritic equine synovium were compared and correlated with gross pathology, histology, and synovial fluid cytokines. M1 and M2 markers were co-expressed in normal and osteoarthritic joints, varying in intensity of expression according to degree of inflammation. Concentrations of synovial fluid IL-10, a macrophage-produced cytokine that is vital for chondrocyte recovery from injury, was lower in OA joints. The combined findings of this study suggest homeostatic mechanisms from synovial macrophages in OA may be overwhelmed, preventing inflammation resolution. In the second study we investigated the response of BMNC to normal (SF) and inflamed synovial fluid (ISF). BMNC cultured in autologous SF or ISF developed into macrophage cultures that were more confluent in ISF (~100%) than SF (~25%), and exhibited phenotypes that were ultimately similar to cells native to normal joints. BMNC cultured in SF or ISF were neither M1 nor M2, but exhibited aspects of both phenotypes and a regulatory response, characterized by increasing counts of IL-10+ macrophages, decreasing concentrations of IL-1β, and progressively increasing concentrations of IL-10 and IGF-1, all more marked in ISF. These findings suggest that homeostatic mechanisms were preserved over time, and potentially favored by macrophage proliferation. Our data suggest that BMNC therapy could potentiate the macrophage- and IL-10-associated mechanisms of joint homeostasis lost in OA. Finally, using an equine model of synovitis, the last study investigated the response of normal and inflamed joints to autologous BMNC injection. Inflamed joints treated with BMNC showed gross and analytical improvements in synovial fluid and synovial membrane, with increasing numbers of regulatory macrophages and synovial fluid concentrations of IL-10, not observed in saline-treated controls. Autologous BMNC are readily available, downregulate synovitis through macrophage-associated effects, and can benefit thousands of patients with OA. Combined, the results of these studies support the role of macrophage-driven synovial homeostasis and identified a therapeutic way to recover homeostatic mechanisms of synovial macrophages lost during chronic inflammation. Our findings also uncover new research directions and methods for future studies targeting modulation of joint inflammation. / Doctor of Philosophy / Osteoarthritis (OA) is a common cause of joint deterioration in people and horses. Current treatments provide limited recovery of joint function, creating an urgent need for more efficient therapies; however, development of new treatments requires better understanding of the mechanism causing OA. A shared characteristic among many arthritic conditions is long-standing inflammation. Cells called macrophages are the main drivers of joint inflammation and can exert pro- and anti-inflammatory effects. Macrophages promote joint health by clearing aggressor agents and secreting molecules required for optimal joint function. However, when these housekeeping functions are overwhelmed by damage, macrophages drive inflammation recruiting more cells to cope with increased demands for repair. If this process is efficiently accomplished, macrophages then resolve inflammation, recovering joint health. Macrophages in the bone marrow (BMNC - bone marrow mononuclear cells) are used to treat inflammation in several tissues and are known to produce molecules essential for joint health. Although little information exists regarding their use in joints, studies treating different organs suggest it can provide high rewards. The studies presented in this dissertation focused on understanding the dual function of macrophages in driving and controlling joint inflammation, and harnessed their therapeutic potential. In the first study, macrophages were investigated in normal and OA-affected joints, and curiously exhibited a hybrid pro- and anti-inflammatory identity in both groups. The indicators of this mixed identity were more markedly expressed in arthritic joints showing gross inflammation. Low levels of a macrophage-derived anti-inflammatory protein called IL-10 were detected in OA joints. The results of this study suggest that anti-inflammatory mechanisms from macrophages may be overwhelmed in OA-affected joints, preventing inflammation to be resolved, and that recovering this anti-inflammatory function may aid in the treatment of OA. In the second study we investigated how the incubation of BMNC in fluid from normal and inflamed joints affects the response of macrophages. Similar to what we observed in the first study, BMNC incubated in both normal and inflamed joint fluid induced macrophages to develop a hybrid identity that was ultimately similar to native cells from normal joints. Macrophages proliferated more when incubated in fluid from inflamed joints. Macrophages in both groups produced anti-inflammatory effects with high levels of IL-10 that were highest in ISF cultures. These observations suggest that higher proliferation of macrophages in inflamed joint fluid helped preserve anti-inflammatory mechanisms. Therefore, our study suggests that joint injection with BMNC could maximize macrophage- and IL-10-associated mechanisms required to resolve joint inflammation. The third and final study investigated the response of normal and inflamed joints to BMNC injection using a model of joint inflammation in horses. Inflamed joints treated with BMNC showed visual and laboratorial markers of improvement, with increasing numbers of macrophages and concentrations of IL-10 in the joint fluid, which remained lower in joints treated with placebo. BMNC provide means to recover macrophage-associated effects required to control joint inflammation and can benefit thousands of patients with OA. Together, the results of these studies show that macrophages are biased promoters of joint health, leading to inflammation when their anti-inflammatory mechanisms are overwhelmed. Replenishing inflamed joints with healthy macrophages maximizes their anti-inflammatory effects, favoring the recovery of a healthy articular environment.