acase@tulane.edu / Multiple sclerosis (MS) is a common neurodegenerative disease and remains an unmet clinical challenge. In MS, an autoimmune response leads to immune cell infiltration, inflammation, demyelination, and lesions in central nervous system (CNS) tissues resulting in tremors, fatigue, and progressive loss of motor function. These pathologic hallmarks are effectively reproduced in the murine experimental autoimmune encephalomyelitis (EAE) model. Using the EAE mouse, we have defined critical time points during the disease progression that have correlative immunopathology with those that occur in MS. As promising therapeutic alternatives to treat MS, we investigated the fresh, heterogeneous population of cells from adipose called the stromal vascular fraction (SVF), which contains adipose-derived stromal/stem cells (ASCs). With these studies, we evaluated the therapeutic efficacies of fresh SVF cells and culture-expanded ASCs at early and late stage EAE disease after intraperitoneal (i.p.) administration. At early stage EAE disease, autoimmune reactions and inflammation are prevalent in the periphery lead to CNS damage by the infiltration of cells that generate inflammatory and demyelinating lesions. We demonstrated that at this time, treatment with SVF cells and ASCs were incapable of attenuating CNS pathology. However, the potency of SVF cells to suppress the autoimmune reactions in the periphery was strong enough to partially ameliorate motor impairments. Furthermore, we revealed the altered gene expressions of the SVF cells and ASCs when exposed to this pathogenic milieu in vitro. Not only did we show that the majority of the helper T (TH) cells contained within the SVF are of the TH2 phenotype, but the most enhanced cytokines in response to the inflammatory milieu were interleukin-10 (IL-10) and transforming growth factor-β (TGFβ) which promote regulatory T cells (Tregs). The most dominant increase detected in ASCs was interleukin-6 (IL-6) which correlates with the inability of ASCs to suppress the activities of the pathogenic T cells at early stage disease. At late stage disease, we showed the greatest improvements in SVF-treated EAE mice that led to amelioration to pathology in CNS tissues and partial restoration of motor function. The most pronounced changes following SVF treatment were the high levels of IL-10 in the peripheral blood, lymphoid and CNS tissues along with the induction of regulatory T cells in the lymph nodes which indicated potent immunomodulatory effects. These effects were not as robust following ASC treatment. A deeper investigation into the potential mechanisms showed phenotypes of T cells and macrophages skewed towards favorable phenotypes. SVF treatment shifted the TH cell subsets away from the effector TH1 and TH2 and toward the Tregs which promote immune tolerance and anti-inflammatory effects. Furthermore, the Treg-associated effects involve the induction of the alternative activation phenotype of macrophages, or M2, which were evidenced in the spleens and CNS tissues of SVF-treated EAE mice. Moreover, we determined that i.p. injected ASCs, and more so, SVF cells were still present in the spleens of EAE mice after 5 days. Together, we investigated a novel modality for treating an inflammatory, autoimmune disease. By comparison with ASC treatment, we demonstrated potential mechanisms of SVF treatment at early and late stage EAE disease that are translational to the inflammatory and demyelinating phases MS disease, respectively. We determined that the timing of administration is most critical, and once active immune activities subside, SVF treatment provides robust and comprehensive effects for improving CNS damage. Additionally, these mechanisms may translate and help explain the favorable effects with current clinical applications such as cell-assisted liposuction that uses SVF cells for improving fat grafting yet mechanisms are still unclear. / 1 / Annie C. Bowles

archives@tulane.edu / Models that mimic angiogenesis are extremely valuable for elucidating underlining mechanisms and pre-clinical development of therapies. Angiogenesis, defined as the growth of new blood vessels from preexisting vessels, is a multi-cellular process that involves the temporal and spatial coordination between endothelial cells, pericytes, nerves, growth factors, and macrophages. A need exists for biomimetic models that bridge the gap between the mechanistic control of in vitro constructs and the multi-system physiological environment of in vivo models. To meet this need our lab has introduced the rat mesentery culture model as top down approach with intact microvascular networks and a nearly two-dimensional view. Previous development of the model has proven its time-lapse, angiogenic, and lymphangiogenic capabilities. The goal of this work is to advance the model to include the maintenance of peripheral nerves in culture and develop it as a platform for aging and cell therapy studies. The first aim of this study was to expand the rat mesentery culture model to maintain nerves and the spatiotemporal relationship between nerves and blood vessels in culture. We developed a nerve culture media to prevent regression of nerves. Nerve alignment was maintained at the network feeding arteriole and capillary plexus levels. We further demonstrated the presence of neurotransmitter precursors was preserved. We demonstrate for the first time the ability to maintain adult peripheral nerves in an ex vivo model. For the second aim of this study, we developed an aging rat mesentery culture model as a basis for investigating differences in angiogenesis across age groups. We demonstrated that impaired angiogenesis associated with advanced age could be recovered to adult-like levels with serum and individual growth factor stimulation. The discovery of increased vascular island frequency in aged tissues reveals that the method of angiogenesis for older networks can differ. These results establish the rat mesentery culture model as a method for studying aging effect on angiogenesis. The objective of the third aim was to demonstrate the capability of the rat mesentery culture model to study stromal vascular fraction therapy. We developed a protocol to isolate the SVF from adipose tissue and transplant onto the mesentery. We identified unique patterns of vasculogenesis and increased vascular coverage. We confirmed that this increase in vascular area was a combination of the vasculogenesis of SVF, proangiogenic effect on host vessels, and incorporation of SVF into the growing host vessels. We used the aging model in developed in the second aim to show that adult SVF on adult tissue has the greatest therapeutic potential. These results display the investigative potential of the rat mesentery culture model in cell therapy research. This work establishes for the first time, to the best of our knowledge, an ex vivo model capable of maintaining adult peripheral nerves. We demonstrate that angiogenesis can be rescued in aging scenarios. The results, for the first time, reveal the effect that SVF therapy has on preexisting networks as well as how it integrates during microvascular remodeling. / 1 / Nicholas Hodges

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.

Metastasis remains the largest contributor for ovarian cancer mortality. The five-year survival rate decreases dramatically as the disease advances from the primary tumor site to other organ sites within the peritoneal cavity. Thus, characterizing the mechanisms behind this metastatic potential may better elucidate the molecular mechanisms of ovarian cancer progression and may reveal novel targets for preventative and therapeutic treatments. Sphingosine-1-phosphate (S1P) is a critical secondary messenger responsible for many pro-cancer signals, e.g., proliferation, angiogenesis, inflammation, anti-apoptosis, and others. While S1P's role in the aggressive profile of many other cancers is well defined, its function in ovarian cancer development is less understood. The concentration of S1P is significantly increased in the ascites of women with malignant ovarian cancer, suggesting a role in ovarian cancer progression. This study aims to understand the importance of S1P in ovarian cancer metastasis. Using our well-characterized murine cell model for progressive ovarian cancer, we investigate the impact of S1P on ovarian cells and their interactions with the stromal vascular fraction recruited from the adipose tissue in culture conditions that mimic the physiologic environment of the peritoneal cavity. These studies will provide a mechanistic link of obesity, inflammation, and the increased risk of obese women to develop and die from ovarian cancer and identify signaling events as targets for interventions. / Master of Science / The mortality rate of women diagnosed with ovarian cancer increases significantly as the disease metastasizes to other regions. Understanding the progression of this disease can create better detection and treatment methods, improving the outcome of women diagnosed with ovarian cancer. Sphingosine-1-phosphate (S1P) is a lipid molecule that has been implicated in many pro-tumorigenic properties in cancer cells; however, its role in ovarian cancer is less known. Stromal cells excrete high levels of S1P and are recruited into tumors for support and many other functions. Elucidating the role stromal cell incorporation into tumors and the role of S1P in ovarian cancer aggressiveness may highlight key pathways that can be targeted for screening methods and therapeutic treatments. This paper aims to understand the connections between S1P, stromal cells, and ovarian cancer as it progresses from a primary site to a metastatic, highly aggressive disease.

Yes / Interest in adipose tissue is fast becoming a focus of research after many years of being considered as a simple connective tissue. It is becoming increasingly apparent that adipose tissue contains a number of diverse cell types, including adipose-derived stem cells (ASCs) with the potential to differentiate into a number of cell lineages, and thus has significant potential for developing therapies for regenerative medicine. Currently, there is no gold standard treatment for scars and impaired wound healing continues to be a challenge faced by clinicians worldwide. This review describes the current understanding of the origin, different types, anatomical location, and genetics of adipose tissue before discussing the properties of ASCs and their promising applications for tissue engineering, scarring, and wound healing. / The authors thank the Plastic Surgery and Burns Research Unit at the University of Bradford, Bradford, UK for financial support for LVT

Radiotherapy has major therapeutic benefits for cancer patients, but ionizing radiation causes damage of surrounding healthy tissues with poor wound healing a common side effect. Therefore, further oncoplastic, reconstructive surgery is challenging and often problematic. Current research models use normal human dermal fibroblasts irradiated in vitro to mimic radiation damage, but this is not comparable to ionising radiation and only measures acute changes. Since radiotherapy may induce epigenetic changes leading to alterations in dermal fibroblast phenotype, the first aim of this study was to compare fibroblasts cultured from irradiated skin with non-irradiated skin. As mesenchymal stem cells isolated from adipose tissue may offer beneficial effects in the regenerative capacity of irradiated tissue, the second part of this study was to compare those cultured from non-irradiated and irradiated breast tissue. Histological changes in the structural organisation of breast tissue in situ from donors exposed to radiotherapy was compared to untreated breast. Primary cultures of dermal fibroblasts from irradiated and non-irradiated breast skin were established and comparisons quantitated in proliferation (CyQuant), metabolism (Alamar Blue), migration (scratch-wound assay), collagen production (Sircol), levels of proteases and protease inhibitors (human protease/protease inhibitor array) and gene expression of COL1A1, COL3A1, MMP1, MMP2, TIMP1 and PPAR-γ mRNA (qPCR). Cells from the stromal vascular fraction (SVF) were cultured and characterised by immunocytochemistry and compared to human preadipocytes sourced commercially. The secretion of FGF, adiponectin and VEGF by the preadipocyte and the SVF mesenchymal cells was compared and the ability of their secretome to modulate dermal fibroblast proliferation, metabolism and migration was evaluated. Radiotherapy caused extensive disorganisation of the reticular dermis and flattening of the epidermal-dermal junction. Dermal fibroblasts cultured from irradiated skin had a pronounced spindle shaped morphology with longer thinner projections and took approximately twice as long to explant and grow. They had a lower proliferative and higher basal metabolic rate and did not respond to FGF-2. While they secreted similar amounts of total collagen they demonstrated distinct differences in proteolytic enzyme and protease inhibitor expression. This is the first report to culture cells from the SVF of irradiated breast tissue. The cells expressed the preadipocyte markers CD10, CD73 and CD105 and no CD45 (negative marker). SVF cells cultured displayed a typical ASC fibroblastoid morphology. Analysis of the secretome identified the presence of FGF, adiponectin and VEGF, while functional analysis demonstrated a stimulatory effect on normal dermal fibroblast migration, although irradiated dermal fibroblasts were unresponsive. Radiotherapy induces long term, detrimental changes in breast skin. This is the first quantitative characterisation of dermal fibroblasts and mesenchymal cells from the SVF, subjected to ionising radiation in situ. Changes in their phenotype that alter their function will impact on wound healing. Further characterisation of these cells may explain their dysfunctional behaviour, and lead to therapies to reverse or reduce this deleterious side-effect and significantly improve treatments facilitating wound healing following radiation injury. / Plastic Surgery and Burns Research unit

No description available.

Yes / Beneficial effects have been observed following the transplant of lipoaspirates containing adipose-derived stem cells into chronic wounds caused by oncologic radiotherapy. It is not yet certain whether adipose-derived stem cells are resistant to radiation exposure. Therefore, the aims of this study were to isolate stromal vascular fraction from human breast tissue exposed to radiotherapy and determine the presence of adipose-derived stem cells. Stromal vascular fraction from irradiated donor tissue was compared to commercially sourced pre-adipocytes. Immunocytochemistry was used to determine the presence of adipose-derived stem cell markers. Conditioned media from stromal vascular fraction isolated from irradiated donors was used as a treatment in a scratch wound assay of dermal fibroblasts also isolated from irradiated donors and compared to pre-adipocyte conditioned media and serum free control. This is the first report of human stromal vascular fraction being cultured from previously irradiated breast tissue. Stromal vascular fraction conditioned media from irradiated donors had a similar effect in increasing the migration of dermal fibroblasts from irradiated skin to pre-adipocyte conditioned media from healthy donors. Therefore, the ability of adipose-derived stem cells in the stromal vascular fraction to stimulate dermal fibroblasts in wound healing appears to be preserved following radiotherapy. This study demonstrates that stromal vascular fraction from irradiated patients is viable, functional and may have potential for regenerative medicine techniques following radiotherapy. / This research was funded by a Bradford City FC Supporters Fellowship for L.V.T. administered through the Plastic Surgery and Burns Research Unit, University of Bradford. / Research Development Fund Publication Prize Award winner, Mar 2023.

Background: Adipose-derived stem/stromal cells (ASCs) isolated from the stromal vascular fraction are a source of mesenchymal stem cells that have been shown to be beneficial in many regenerative medicine applications. ASCs are an attractive source of stem cells in particular, due to their lack of immunogenicity. This study examines differences between mitochondrial bioenergetic profiles of ASCs isolated from adipose tissue of five peri-organ regions: pericardial, thymic, knee, shoulder, and abdomen. Results: Flow cytometry showed that the majority of each ASC population isolated from the adipose tissue of 12 donors, with an n = 3 for each tissue type, were positive for MSC markers CD90, CD73, and CD105, and negative for hematopoietic markers CD34, CD11B, CD19, and CD45. Bioenergetic profiles were obtained for ASCs with an n = 4 for each tissue type and graphed together for comparison. Mitochondrial stress tests provided the following measurements: basal respiration rate (measured as oxygen consumption rate [pmol O-2/min], ATP production, proton leak, maximal respiration, respiratory control ratio, coupling efficiency, and non-mitochondrial respiration. Glycolytic stress tests provided the following measurements: basal glycolysis rate (measured as extracellular acidification rate [mpH/min]), glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. Conclusions: The main goal of this manuscript was to provide baseline reference values for future experiments and to compare bioenergetic potentials of ASCs isolated from adipose tissue harvested from different anatomical locations. Through an investigation of mitochondrial respiration and glycolysis, it was demonstrated that bioenergetic profiles do not significantly differ by region due to depot-dependent and donor-dependent variability. Thus, although the physiological function, microenvironment and anatomical harvest site may directly affect the characteristics of ASCs isolated from different organ regions, the ultimate utility of ASCs remains independent of the anatomical harvest site.