No description available.

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

Despite its crucial role in fertility, relatively little has been published on the human endometrial stroma. The first experiment reported in this thesis was designed to provide quantitative baseline data on the human stroma during the mid to late luteal phase, when it plays a major part in normal pregnancy. Subsequent chapters investigate the effects of an antioestrogen (clomiphene citrate-CC); growth hormone (GH) supplementation of patients on hormone replacement therapy (HRT) due to lack of endogenous ovarian steroids; and patients with unexplained recurrent miscarriage. Finally a novel 3-dimensional in vitro model of human stroma is described along with the effects of steroid supplementation on stromal cells grown in the model. In all cases of in vivo studies, conventional stereological methods were used to obtain quantitative morphological data from at least 6 subjects per group, from both control (fertile) and experimental (infertile) subjects using light and electron microscopy. In addition, several staining techniques were also used to demonstrate qualitative changes that occur in human endometrial stroma. During the mid to late luteal phase, endometrial stroma and blood vessels underwent substantial changes, and thus quantitative and qualitative baseline data have been established to fill a gap in this important area of reproductive biology. CC caused no substantial changes in stromal structure in women of proven fertility and therefore its advantageous effects on ovulation are not negated at the level of the endometrium. GH supplementation had no effects on infertile human endometrial stroma from subjects on HRT, (in either premature ovarian failure or Turner's syndrome groups). However, both infertile groups had endometrial stroma which significantly differed from matched fertile endometrium, suggesting an impaired endometrial development in infertile subjects which was not reversed by HRT, either with or without Gil treatment. Stromal morphology did not differ between the recurrent miscarriage groups, however data suggested the existence of several subgroups which made firm conclusions difficult. The tissue culture model provided preliminary data suggesting it to be a potentially very useful technique for the study of stromal cell biology.

No description available.

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

Introduction: Gastrointestinal stromal tumours (GIST) are the commonest tumour of mesenchymal origin; favour the stomach, and account for a very small percentage of gastrointestinal tract tumours. Methods: In this retrospective audit of GISTs presenting to the Groote Schuur Hospital surgical and oncological multidisciplinary team (MDT) between 2004 – 2019, gastric GISTs were evaluated as regards presentation, gastric anatomical position, histological subtype with risk stratification, management and outcomes. Results: Of 126 GIST tumours presenting to this MDT, 82 originated in the stomach. Complete histopathological records could be obtained for 64. With an average of 59 years (50 male: 32 female), 18 (28%) presented with a herald bleed. Other common presentations included anaemia, epigastric mass and pain. The tumours were predominantly found in the body and fundus (64%), with a spindle cell subtype predominance (41%). The association between cancer cell subtype and gastric position was not significantly different (p=0.728). Cystic degeneration was found on 11 (17%) analyzed and cell necrosis on 12 (18%). These findings were not related to larger tumor size or prognosis. Five required downstaging with Imatinib prior to surgery. Thirty-seven patients underwent a surgical procedure: 24 wedge resections and 12 anatomical resections. Risk stratification was performed with the modified National Institutes of Health (NIH/Fletcher) score. Twenty-eight cases had inaccurate mitotic counts and couldn't be scored, 17 scored high risk, 9 intermediate risk, 9 low risk and 1 very low risk. Ten patients died of metastatic disease, 34 were discharged with no disease progression after 3 years, 1 patient with disease progression currently remains on Imatinib, and 19 were lost to follow up. Conclusion: Gastric GISTs appear to have a predilection for the proximal stomach; it is unsure whether this is purely due the greater surface area. The spindle cell subtype dominated in the proximal gastric GISTs. Cystic degeneration and cell necrosis did not seem to be related to larger tumours or outcomes.

No description available.

The endometrium undergoes recurrent cycles of dynamic remodelling, involving breakdown and scarless repair, proliferation and differentiation, including decidualisation of the stroma, during the menstrual cycle. Extensive studies have characterised how the steroid hormones oestrogen and progesterone acting via their nuclear receptors coordinate these remarkable changes. Although a few previous studies have postulated a role for androgens the impact of androgens on endometrial function remains understudied. The studies described in this thesis aimed to 1) identify cellular processes, pathways and networks regulated by androgens in human androgen receptor-positive endometrial stromal cells (hESCs), 2) investigate the potential for regulation and determine the regulation of putative dihydrotestosterone (DHT)-regulated gene expression by androgen in hESCs, 3) investigate the expression and regulation of putative androgen-regulated genes in the human endometrium across the menstrual cycle and in early pregnancy and 4) explore the role of androgens in modulating metformin-induced gene expression associated with decidualisation of hESCs. Analysis of data from a whole genome array conducted previously in the laboratory using primary hESCs treated with DHT for 2 or 8 hours identified time dependant putative androgen-regulated mRNAs (34 and 268 genes, respectively). Thereafter, all work was completed by the author. Gene ontology and functional based bioinformatic analyses of the putative androgen-regulated gene sets revealed potential androgen regulation of a variety of cell processes, pathways and networks including those associated with gene transcription, signal transduction pathways (such as phosphatidylinositol, oestrogen receptor alpha (ERα) and Wnt signalling), cancer pathways, metabolism, cell cycle, development, apoptosis/survival. In addition, various transcription factors (e.g. AR, c-Myc, SP1, ERα, p53, E2F1, RUNX2, CREB1 and STAT3) were associated with androgen regulation in hESCs. Consensus androgen receptor binding sites were identified in the promoter sequences of 18 genes by transcription factor binding site sequence analysis. Direct DHT regulation of ten of 15 of these genes was validated in endometrial stromal cells using qRTPCR. Of these genes, RGS2, SIK1, and SNCAIP mRNAs were confirmed as DHT-regulated in hESCs by use of an AR inhibitor (flutamide) and in addition, were not found to be regulated by oestradiol. Discovery bioinformatics predicted these genes may interact in a gene network involving AR and the cAMP transduction pathway. Expression of the 15 putative androgen-regulated genes was confirmed by qRTPCR in intact human endometrial tissue (13 novel) and 9 of these genes were regulated in association with decidualisation i.e. either in the secretory phase, the time at which decidualisation begins and/or in first trimester decidua. Protein expression of RGS2, SIK1 and Synphilin-1 (encoded by SNCAIP) was confirmed by immunohistochemistry in endometrial tissues and protein expression also appeared greater in decidua. Regulation of putative androgen-regulated gene expression by decidualisation was confirmed in 4 out of 8 genes by employing a model of reduced in vivo decidualisation i.e. decidua from ectopic pregnancies. Regulation of 5 out of 7 genes was confirmed in decidualised hESCs (RGS2, SIK1, SLC6A6, SNCAIP and AXIN2) but expression of these genes was not altered by DHT inclusion during decidualisation. Finally, only a high metformin concentration enhanced hESC decidualisation and putative androgen-regulated gene expression (4 genes) in decidualised hESCs. In comparison, in the presence of DHT, a lower clinically relevant metformin concentration (100μM) did enhance decidualisation marker expression but did not alter expression of putative androgen-regulated genes. In summary, these studies have revealed new insights into androgen action in the human endometrium. Studies in hESCs 1) predicted the pathways and interacting transcription factor regulatory networks that may be androgen-dependent in this cell type, these were associated with cell differentiation, apoptosis and proliferation, 2) identified novel putative androgen-regulated genes expressed in hESCs and in endometrial tissues, 3) showed putative androgen-regulated genes are regulated by DHT (possibly via AR) in endometrial stromal cells, some of which are also regulated in association with decidualisation and 4) showed that androgens may enhance decidualisation during exposure to the commonly used drug metformin. Collectively, these new findings support a physiological role for androgens in endometrial function and provide a series of new avenues for further studies of the regulation of differentiation and proliferation.

Biochemical and mechanical properties of the extracellular matrix (ECM) are known to independently influence cell function. Given the complexity of cellular responses, I hypothesized that the integration of multiple matrix factors as opposed to their individual contribution is key to understanding and controlling cell function. The objective of this thesis was to systematically investigate matrix biochemical and mechanical regulation of multipotent stromal cell (MSC) osteogenesis. First, I demonstrated that substrate stiffness-dependent MSC spreading, proliferation and osteogenic response were differentially regulated by matrix protein type (collagen I vs. fibronectin) and concentration. Second, I developed and characterized a matrix microarray platform that enabled the efficient screening of multiple matrix-derived cues (substrate stiffness, ECM type and density). I implemented the matrix microarray platform together with parametric regression models to elucidate novel matrix interactions in directing mouse MSC osteogenic and adipogenic differentiation. Third, I extended the screening study to examine matrix-dependent human MSC osteogenesis. Non-parametric regression models were used to provide a nuanced description of the multi-factorial matrix regulation in MSC osteogenesis. The response surfaces revealed a biphasic relationship between osteogenesis and substrate stiffness, with the exact location and magnitude of the optimum contingent on matrix composition. Guided by the screening results and perturbation to key cytoskeletal regulators, I identified a novel pathway involving Cdc42 in matrix mechanical and biochemical regulation of MSC osteogenesis. Surprisingly, Cdc42 mediated stiffness-dependent MSC osteogenesis independent of ROCK, suggestive of a contractility-independent mechanism in matrix rigidity signal transduction. In summary, the integration of cell-based arrays and statistical modeling has enabled the systematic investigation of complex cell-matrix interactions. This generalizable approach is readily adaptable to other cellular contexts, complementing hypothesis-driven strategies to facilitate non-intuitive mechanistic discovery. Moreover, the improved understanding of matrix-dependent MSC function also has practical relevance to the development of biomaterials for tissue engineering applications.

Biochemical and mechanical properties of the extracellular matrix (ECM) are known to independently influence cell function. Given the complexity of cellular responses, I hypothesized that the integration of multiple matrix factors as opposed to their individual contribution is key to understanding and controlling cell function. The objective of this thesis was to systematically investigate matrix biochemical and mechanical regulation of multipotent stromal cell (MSC) osteogenesis. First, I demonstrated that substrate stiffness-dependent MSC spreading, proliferation and osteogenic response were differentially regulated by matrix protein type (collagen I vs. fibronectin) and concentration. Second, I developed and characterized a matrix microarray platform that enabled the efficient screening of multiple matrix-derived cues (substrate stiffness, ECM type and density). I implemented the matrix microarray platform together with parametric regression models to elucidate novel matrix interactions in directing mouse MSC osteogenic and adipogenic differentiation. Third, I extended the screening study to examine matrix-dependent human MSC osteogenesis. Non-parametric regression models were used to provide a nuanced description of the multi-factorial matrix regulation in MSC osteogenesis. The response surfaces revealed a biphasic relationship between osteogenesis and substrate stiffness, with the exact location and magnitude of the optimum contingent on matrix composition. Guided by the screening results and perturbation to key cytoskeletal regulators, I identified a novel pathway involving Cdc42 in matrix mechanical and biochemical regulation of MSC osteogenesis. Surprisingly, Cdc42 mediated stiffness-dependent MSC osteogenesis independent of ROCK, suggestive of a contractility-independent mechanism in matrix rigidity signal transduction. In summary, the integration of cell-based arrays and statistical modeling has enabled the systematic investigation of complex cell-matrix interactions. This generalizable approach is readily adaptable to other cellular contexts, complementing hypothesis-driven strategies to facilitate non-intuitive mechanistic discovery. Moreover, the improved understanding of matrix-dependent MSC function also has practical relevance to the development of biomaterials for tissue engineering applications.