Background: Transplantation of marrow stromal cells (MSC) has been shown to improve heart perfusion and cardiac function after ischemia. Erythropoietin (EPO) is capable of inducing angiogenesis and inhibiting cell apoptosis. The aim of this study was to investigate the effect of EPO on the therapeutic potency of MSC transplantation in a rat model of myocardial infarction. Methods: MSC viability was detected by MTT andflow cytometry following culture in serum-free medium for 24 h with or without EPO. Release of vascular endothelial growth factor (VEGF) by MSC incubated with different doses of EPO was assayed using ELISA. Immediately after coronary ligation, autologous MSC (3 × 10 6 cells) were injected into the ischemic myocardium (MSC and MSC-EPO groups). EPO (3,000 U/kg body weight) was injected daily for 3 consecutive days starting 1 day prior to ligation. The same EPO dose was also injected for consecutive 3 days starting 15 days after surgery (EPO and MSC-EPO groups). Control animals were injected saline solution for the same time period. Cardiac function was assessed by echocardiography 2 and 21 days after surgery, respectively. Western blot and immunohistological assessments were performed to examine the effects of treatments. Results: In vitro, EPO inhibited MSC apoptosis induced by serum-free medium and increased vascular endothelial growth factor (VEGF) release by MSC. In vivo, cardiac infarct size was significantly smaller, cardiac function significantly improved, and capillary density obviously higher in the MSC and EPO groups than in the control group. Combined treatment with EPO infusion and MSC transplantation demonstrated a further decrease in infarct size, a further improvement in cardiac function, and a further increase in capillary density compared with MSC or EPO alone. Furthermore, a higher ratio of phosphorylated Akt to total Akt was measured by Western blot; Bcl-2 was upregulated and Bax was downregulated by immunohistochemistry in the MSC-EPO group compared to the other three groups. Conclusion: Transplantation of MSC combined with EPO infusion is superior to MSC monotherapy for angiogenesis and cardiac function recovery.

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Ovarian cancer is considered to be one of the deadliest gynecological diseases. Over 21,000 women are expected to be diagnosed with this fatal disease in 2020 alone. Obesity, but more specifically a high waist-to-hip ratio, is indicative of abdominal obesity and has been correlated with increased risk of ovarian cancer. How abdominal obesity contributes to this increased risk has not been clearly delineated but much of the current research has been focused on the role of adipocytes. However, in addition to the adipocytes, abdominal white adipose tissue contains the stromal vascular fraction (SVF) which includes stem and progenitor cell populations, immune cells, and fibroblasts. Since the SVF can also be recruited by the cancer cells, we investigated how obesity affects the survival and metastatic potential of cancer cells by investigating changes in the expression of genes that contribute to survival, proliferation, migration, adherence, and invasion. We used culture conditions that mimic the non-permissive peritoneal environment. Cancer related genes, such as Dkc1, Ccnd2, Lig4, and Snai2, were upregulated when adipose derived stem cells (ADSC) were added into MOSE-LTICv spheroids. It was found that peritoneal serous fluid (PSF) from obese mice significantly increased migration of MOSE-LTICv (Serum vs PSF, 517.8 vs 1158.6). These studies brought new knowledge into the field of obesity and ovarian cancer risk and provided direction for future studies involving potential cellular and molecular targets for ovarian cancer diagnosis and treatment. / Master of Science / Ovarian cancer affects many women in the United States. Obesity or more specifically, carrying more weight around the waist, can affect a woman's risk of developing ovarian cancer. Abdominal fat needs to be researched to see if abdominal obesity can affect ovarian cancer on the cellular level. Researchers have looked into how fat cells, known as adipocytes, can affect the progression of ovarian cancer, but more research needs to be done on the contributions of other cells found within adipose tissue. Other cells in abdominal fat include cells such as immune cells, stem and progenitor cells and fibroblasts. We have explored how adipose stem cells from obese mice affect the DNA or "the blueprints" of the cells, survival, and progression of mouse ovarian cancer cells. We found that when adipose stem cells are combined with ovarian cancer cells the expression of certain genes or particular "blueprints" increased. The genes whose expression increased included Dkc1, Ccnd2, Lig4, and Snai2 and when deregulated can cause ovarian cancer cells to become more aggressive. The abdominal fluid from obese mice was found to increase migration of ovarian cancer cells which simulates an increase in metastatic potential. This information has given new insight into the obesity and ovarian cancer relationship.

Master of Science / Department of Clinical Sciences / Walter Renberg / Objective: To evaluate the safety and effect of a single simultaneous intra-articular and intravenous injection of autologous adipose-derived stromal vascular fraction (SVF) and platelet rich plasma (PRP) on coxofemoral osteoarthritis (OA) in dogs. Methods: This was a randomized, double-blind, placebo-controlled prospective pilot trial of simultaneous intra-articular and intravenous SVF and PRP for coxofemoral OA. Dogs with coxofemoral OA causing signs of lameness or discomfort were evaluated by orthopedic exam, visual lameness score, Canine Brief Pain Inventory (CBPI), goniometry, visual analogue scale (VAS), and pressure-sensitive walkway (PSW) at week 0 (baseline), and at 4, 8, 12 and 24 weeks after injection. Joint radiographs were scored at 0 and 24 weeks. Results: Twenty two client-owned dogs with naturally occurring OA of the coxofemoral joints were enrolled (12 placebo-control, 10 SVF-treated). CBPI pain severity scores were lower in the treatment group at 24 weeks compared to the placebo group (p=0.042). The VAS score for the treatment group was significantly greater at 0 weeks than at 4, 8, or 24 weeks (p<0.05). When dogs with low quartile baseline PVF (25th percentile) were compared, the treatment group had statistically higher PVF at all post-injection time points when compared to the placebo group. After SVF injection, fewer dogs in the treated group were lame compared to the control group. Clinical Significance: This study is the first to utilize objective data from PSW as an outcome measure for dogs treated with SVF and PRP for coxofemoral OA. No adverse events were noted. Improvements in some measured parameters in the treated dogs compared to those in the placebo group.

Orientador: Rogério Martins Amorim / Coorientador: Ana Liz Garcia Alves / Banca: Rui Seabra Ferreira Junior / Banca: Fernanda da Cruz Landim / Resumo: Estudos demonstram o grande potencial do uso das células estromais mesenquimais multipotentes (MSCs) como terapia celular. Seu uso em lesões neurológicas, que usualmente apresentam difícil regeneração, tem sido estudado pelo fato das MSCs apresentarem baixa imunogenicidade efeitos imunomoduladores e neuroregenerativos. Nesse contexto, o presente estudo avaliou a viabilidade e a segurança do transplante de MSCs alogênicas provenientes do tecido adiposo, medula óssea e cordão umbilical de equinos, pela via intratecal através do espaço intervertebral C1-C2, por meio de exame neurológico seriado, análises hematológicas e determinação dos níveis séricos de proteínas de fase aguda. Foram utilizados 16 equinos saudáveis, divididos em quatro grupos: grupo SHAM (SHAM) que recebeu o transplante de salina tamponada com fosfato (PBS); grupo tecido adiposo (GTA), recebeu MSCs de origem do tecido adiposo; grupo medula óssea (GMO), recebeu MSCs de origem da medula óssea; e grupo cordão umbilical (GCU), recebeu células de origem da matriz do cordão umbilical. Foram realizadas três aplicações com intervalo de 30 dias em cada grupo, e coletou-se amostras de sangue, nos momentos que antecederam os transplantes, M0, M30 e M60 e 24 horas, após o transplante, M1, M31, M61. E por último foi realizada uma coleta 30 dias após M60, caracterizando M90. Não foram observadas alterações do exame clinico, hematológico e nas proteínas de fase aguda relacionadas aos sucessivos transplantes intratecais de MSC... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Studies demonstrate the great potential of using multipotent mesenchymal stromal cells (MSCs) as cell therapy. Its use in neurological lesions, which usually present difficult regeneration, has been studied because MSCs have low immunogenicity and immunoregulatory and neuroregenerative effects. In this context, the present study evaluated the viability and safety of transplantation of allogeneic MSCs from the adipose tissue, bone marrow and umbilical cord of horses, through the intrathecal route through the C1-C2 intervertebral space, through serial neurological examination, hematological analyzes and determination of serum levels of acute phase proteins. Sixteen healthy horses were used, divided into four groups: SHAM group (SHAM) that received phosphate buffered saline (PBS) transplantation; adipose tissue group (GTA), received adipose tissue MSCs; bone marrow group (GMO), received MSCs from bone marrow origin; and umbilical cord (UGC) group, received cells from the umbilical cord array. M0, M30 and M60 were collected at the time of the transplantation, M1, M31 and M61 and 24 hours after transplantation. And finally a collection was performed 30 days after M60, characterizing M90. There were no changes in the clinical, hematological and acute phase-related proteins related to successive intrathecal transplantations of MSCs, nor were there alterations that contra indicated the use of any of the cellular sources, demonstrating that the protocol used, as well as any of cellula... (Complete abstract click electronic access below) / Mestre

Introduction. Safe closure of gastric luminal defects remains a big challenge for development of gastric endoscopic surgery. The aims of this thesis are to assess the effect and efficiency of Eagle Claw VIII (endoscopic suturing device)and adipose tissue-derived mesenchymal stem cells (ADMSCs) for closure and enhancing healing of gastric luminal defects. / Methods and Results. 1. Endoscopic suturing is superior to endoclips for closure of gastrotomy after NOTES. A 2cm linear incision on the body of porcine stomach was closed by hand suturing, Eagle Claw VIII or endoclips, respectively (n=17 for each group). The results indicated that all gastrotomies were successfully closed. Closure time was significantly longer in Eagle Claw VIII group. Bursting pressure of gastrotomies for Eagle Claw VIII was significantly higher than endoclips, but lower than hand suturing. Besides, both Eagle Claw VIII and endoclip closure encountered significantly technical challenges. This study suggested that Eagle Claw VIII had potential for endoscopic closure of gastrotomies, but need further refinement. / 2. ADMSCs for Acceleration of Healing of Sutured Gastric Perforation(SGP). ADMSCs were isolated and expanded in vitro, and characterized by stromal differentiations and cell surface markers. A 2cm SGP was produced on gastric body of rats. 5×10⁶ ADMSCs were transplanted into SGP by local injection (LI-ADMSCs) or topical spraying (TS-ADMSCs). Healing of SGP was assessed. LI-ADMCs significantly decreased peritoneal adhesion and wound dehiscence, and increased bursting pressure of SGP, when comparing to other experimental groups. Histologic analysis indicated that SGPs in LI-ADMSCs group had more re-epithelialization and collagen regeneration, and less inflammation. Expression of TGF-β1 was up-regulated, while IL-6 was down-regulated in LI-ADMSCs group, when comparing to fibrin and control groups. This study suggested that local injection of ADMSCs is an effective approach for accelerating the healing of SGP. / 3. Promoting Effect of ADMSCs on Healing of Gastric Ulcer is abrogated by NSAIDs. Gastric ulcer model in rats was successfully produced by using 70% acetic acid. A total of 1×10⁷ ADMSCs was locally injected into ulcer lesion. Ulcer area was measured at different time points. Therapeutic potentialof ADMSCs was assessed when NSAIDs was simultaneously administrated. The results demonstrated that ADMSCs significantly decreased ulcer area. Histologic assessment indicated that ADMSCs increased re-epithelialization, angiogenesis and collagen deposition, and suppressed inflammation. Transplanted ADMSCs homed into gastric ulcer lesion and differentiated into endothelial and smooth muscle cells. In addition, ADMSCs treatment increased the gene expressions for wound healing, and activated COX-2-PGE₂ and Erk1/2-MAPK signaling pathways. Repeated administration of Indomethacin reduced cell proliferation and angiogenesis, and eliminated ADMSCs-induced ulcer healing on day 10. The results suggested that ADMSCs promoted the healing of peptic ulcer, which is eliminated by NSAIDs. / Conclusions. Endoscopic suturing by Eagle Claw VIII is feasible for closure of gastrotomy, when comparing to endoclips. ADMSC promotes the healing of gastric luminal defects including SGP and ulcer. The promoting effect of ADMSC is PGE₂-dependent, and attenuated by NSAIDs. These evidences implied that combined use of endoscopic suturing and ADMSCs is a helpful approach for safe closure of gastrotomy and gastric perforation. / 引言：胃傷口癒合是胃消化內鏡手術發展的障礙之壹。本課題之目的是評價和探索Eagle Claw VIII和脂肪幹細胞(ADMSCs)縫合和促進胃內傷口癒合的效果和作用。 / 方法和結果：1. 內鏡縫合器Eagle Claw VIII閉合經胃自然腔道手術後傷口的效果評價體外豬胃體上造2cm的胃傷口模型，使用手工縫合、內鏡下Eagle Claw VIII縫合或內鏡夾閉合胃傷口；每組17個樣本。本研究提示所有胃傷口均成功閉合。Eagle Claw VIII縫合胃傷口時間顯著長於其他兩組的閉合時間。Eagle Claw VIII縫合的胃傷口破裂壓顯著高於內鏡夾閉組，但是明顯低於手工縫合組。此外，內鏡縫合和夾閉都面臨較大的技術難度。本研究提示Eagle Claw VIII有臨床運用的潛在價值，但需要進壹步改進。 / 2. 局部移植脂肪幹細胞促進胃穿孔癒合的實驗性研究：建立大鼠2cm胃體穿孔模型，局部註射或傷口表面塗抹法移植ADMSCs，觀察胃傷口癒合情況。局部註射移植ADMSCs顯著減輕胃傷口粘連和裂開發生率，增加胃傷口破裂壓。組織學分析提示ADMSCs治療促進傷口上皮和肉芽組織再生，抑制炎癥反應。此外，局部註射ADMSCs增加TGF-β1抑制IL-6表達。本研究提示局部註射移植ADMSCs是促進胃穿孔傷口癒合的有效方法。 / 3. 局部移植脂肪幹細胞促進胃饋瘍癒合的實驗性研究：使用70%醋酸建立大鼠胃體饋瘍模型；饋瘍病竈內局部註射移植1×107 ADMSCs。研究提示第10和15天ADMSCs顯著減小饋瘍面積。組織學研究提示ADMSCs增加饋瘍傷口上皮和血管再生，促進膠原蛋白分泌和抑制炎癥反應。移植的ADMSCs能夠在饋瘍病竈內成活，並分化成血管內皮細胞和平滑肌細胞。ADMSCs顯著提高促傷口癒合相關基因表達水準。此外，ADMSCs啟動COX-2-PGE2和Erk1/2-MAPK信號通路。第10天，和對照組相比，引哚美辛/ADMSCs組潰瘍病竈內細胞增殖和血管再生顯著降低、饋瘍癒合延遲。本研究提示脂ADMSCs促進胃饋瘍癒合；非甾體抗炎藥顯著減弱ADMSCs的促胃饋瘍癒合作用。 / 結論：與內鏡夾閉相比，Eagle Claw VIII內鏡縫合胃創口有可行性。ADMSCs促進胃穿孔和饋瘍癒合，且依賴於前列腺素E2；引哚美辛抑制前列腺素E2合成從而抑制ADMSCs促胃組織癒合之效能。本研究提示聯合使用內鏡縫合器和ADMSCs是促進胃傷口癒合的潛在有效方法。 / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 152-162). / Abstracts also in Chinese. / Title from PDF title page (viewed on 11, October, 2016). / Liu, Liu. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

The purpose of this study was to identify conditioning strategies for mesenchymal stromal cells (MSC) which optimize cellular immunosuppressive potency. By identifying new treatment strategies and previously unidentified small molecules capable of stimulating MSC we hope to pave the way tailoring licensed MSC phenotypes to be used in a specific disease state, rather than a one size fits all package. We sought to determine how MSC act in response to a changing immune response or environmental condition. MSC are exquisitely sensitive to changes in their environmental conditions and we show that cellular transcriptome and secretome changes are conditionally responsive to their inflammatory stimulus. One of the main subjects of analysis here is the observations of how these cellular profiles evolve over time in the presence of an inflammatory environment. Similarly, this study observes how MSC behavior changes after an inflammatory event has been resolved to address, in part, the plasticity of MSC licensing and the ability of MSC to rapidly recall a previous immunosuppressive state upon secondary challenge with an inflammatory stimulus. Data was obtained from in vitro experiments with human bone marrow derived MSC and donor human peripheral blood mononuclear cells (PBMC), while in vivo data was obtained using C57BL6/J mice. Overall this research demonstrated that MSC potency can be bolstered by small molecule and drug treatment conditioning, and that certain disease conditions may be more effectively paired with specific MSC conditioning strategies to improve their therapeutic effectiveness.

Chronic myelogenous leukemia is a myeloproliferative hematopoietic stem cell disease resulting from a reciprocal translocation that gives rise to BCR-ABL, a constitutively active tyrosine kinase. Imatinib and other tyrosine kinase inhibitors are currently standard therapy; however, point mutations often lead to drug resistance and disease relapse often occurs due to the persistence of quiescent leukemia stem cells that are shielded by stromal factors within the bone marrow microenvironment. In an effort to develop new therapies capable of eradicating these elusive cells, a novel approach has been proposed in which the biochemical properties of cancer cells are targeted. It has been established that one such property is oxidative stress due to the increased production of reactive oxygen species, which makes cancer cells especially dependent on their antioxidant systems to maintain redox homeostasis. Recent studies demonstrate that chaetocin, a mycotoxin produced by Chaetomium species fungi, possesses potent and specific antimyeloma activity due in part to its ability to inhibit thioredoxin reductase-1, a central oxidative stress remediation enzyme. In this study, the effectiveness of chaetocin against leukemia stem cells has been investigated using in vitro and in vivo murine chronic myelogenous leukemia models. Our results indicate that: chaetocin and imatinib function synergistically in decreasing cell viability, inducing apoptosis, and inhibiting the colony formation of chronic myelogenous leukemia cells in vitro; that chaetocin in combination with imatinib reduces leukemia stem cell frequency in vivo; that chaetocin increases intracellular reactive oxygen species levels; and that chaetocin does not disrupt the proliferation and differentiation of normal murine hematopoietic stem cells. Surprisingly, our results also show that while bone marrow stromal factors inhibit the activity of imatinib, they potentiate the activity of chaetocin, indicating that chaetocin could potentially be used to target leukemia stem cells within the bone marrow niche.

Bone tissue engineering strategies are currently being developed as alternative mechanisms to address the clinical demand for bioactive and biomechanical graft material. To date, these efforts have been largely restricted by inadequate supply of committed osteoprogenitor cells and loss of osteoblastic phenotype expression following in vitro culture and expansion. The objective of this thesis research was to address the cell sourcing limitations of tissue-engineered bone grafts through constitutive and sustained overexpression of the osteoblast-specific transcriptional activator Runx2/Cbfa1 in osteogenic marrow-derived stromal cells using retroviral gene delivery. Runx2 overexpression enhanced expression of multiple osteoblastic genes proteins and, more importantly, significantly up-regulated matrix mineralization in both monolayer culture and following cell seeding in 3-D polymeric scaffolds. To evaluate in vivo performance, Runx2-expressing cells were seeded into 3-D constructs and implanted both subcutaneously and in a critical size craniotomy bone defect model. Notably, in vitro pre-culture of Runx2-transduced cell-seeded constructs prior to implantation significantly enhanced their capacity to form mineralized tissue in the subcutaneous space and induce new bone formation in the critical size defect model compared to control cells. The described series of analyses provided a novel combination of tissue and genetic engineering techniques toward the development of a Runx2-modified stromal cell/polymeric scaffold composite tissue-engineered bone graft substitute.