Browsing by Subject "Stem cells"
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Item Chemically modified hyaluronic acid biomaterials for cell culture and tissue engineering(2016-12) Joaquin, Alysa Marie; Zoldan, Janeta; Suggs, LauraThe fate and behavior of cells is strongly dependent on the cell microenvironment, and this knowledge has been applied to the design of biomaterials to influence cell growth, morphology, and differentiation. However, a dearth of research specifically focused on the effects of material hydrophobicity on cell behavior indicates that this easily controllable material property is being overlooked. The field of tissue engineering has a need for cost-efficient, scalable methods to both increase stem cell stocks and control cell behavior, and hydrophobic biomaterials may be a robust solution to these needs. To evaluate the utility of hydrophobicity in controlling cell behavior, hyaluronic acid was modified with amines representing a wide range of hydrophobicity, resulting in twelve new materials. Both mouse embryonic stem cells (mESCs) and fibroblasts were cultured on these materials to evaluate the differences in pluripotent and differentiated cell behavior in response to hydrophobic materials. The viability of cells cultured on these materials was tested as an indicator of biocompatibility, and cell morphology and spreading area was evaluated to relate cell behavior to biomaterial hydrophobicity. Eight of the twelve materials proved to be biocompatible, and hydrophobic materials inhibited cell spreading; fibroblasts cultured on modified HA hydrogels grew in populations of both compact cell clusters and elongated, multi-polar morphologies, and cell spreading area increased as hydrophobicity decreased. Similarly, mESC spreading area increased with decreasing hydrophobicity; mESCs grown on the least hydrophobic HA hydrogels also multi-polar spreading, while mESCs cultured on the more hydrophobic materials grew exclusively in compact cell clusters. As the morphology of cells is often indicative of cell fate, and as hydrophobic materials tended to inhibit cell spreading, we expected that mESCs cultured on hydrophobic materials would maintain pluripotency. To this end, hybrid scaffolds composed of modified HA and gelatin were developed as a platform for stem cell pluripotency maintenance. The mESCs seeded into these scaffolds had a higher expression of the pluripotency marker SSEA-1 compared to control mESCs grown in complete medium after 24 hours, indicating that hydrophobicity is an important material property to consider in stem cell culture.Item Microfluidic separation of multisized particles using acoustic standing waves for stem cell sorting(2006-12) Moon, Hongdae; Zhang, John X.J.The sorting and isolation of target cells and suspended particles from a medium is of great importance to cell biology, drug delivery, and related fields in biomedicine. Furthermore, the ability to separate and sort stem cells is of profound interest to the biomedical community to obviate current clinical limitations. Stem cells are considered as a repair system for the body and adipose tissue (fat) is a rich source of stem cells. The regenerative medicine discipline is attempting to harness the innate ability of stem cells to form tissues de novo, and innovative strategies are required to repair the tissue defects. The focus of this work is to develop an acoustically driven, microfluidic cell sorter that will separate particles according to size-based differential migration with high throughput and accuracy, tunable spatial-temporal resolution, and low power consumption. We fabricated three types of cell sorters based on polydimethylsiloxane (PDMS), SU-8 and silicon channels with rectangular cross-section through soft lithography, surface micromachining and bulk micromachining technologies. Acoustic standing wave is launched into the microfluidic channels through an array of integrated piezoelectric plate transducers. Acoustic radiation pressure is simulated and experimentally verified. The acoustic sorting technique provides unique features and complements previous sorting methods. The device design is simple, and it is easy to fabricate. While sorting occurs, there is no direct physical contact which prevents cells or particles from being severely defected. The acoustic sorting can potentially be applied to handle many cell types or particles.Item Resistance training as a modality to enhance muscle regeneration in a rat skeletal muscle defect(2009-12) Taylor, Daniel Ryan; Farrar, Roger P.; Suggs, LauraTraumatic skeletal muscle injuries that include loss of large amounts of muscle mass are becoming more common in today’s warfare. Traditional treatments often do not prevent long term functional impairments. Using a decellularized extracellular matrix (ECM) as scaffolding to replace lost muscle tissue allows for transmission of force through the injury site, and provides a suitable microenvironment receptive to myofiber growth. Seeding the ECM with progenitor cells improves cellular content in the defect area. Exercise exposes the muscle to improved blood flow as well as higher than normal loading. This results in increased blood vessel density as well as higher levels of cellular content, and near complete restoration of function.Item Targeting breast cancer with natural forms of vitamin E and simvastatin(2012-05) Gopalan, Archana; Kline, Kimberly; Sanders, Bob G.; Hursting, Stephen; Tucker, Philip; deGraffenried, LindaBreast cancer is the second leading cause of death due to cancer in women. A number of effective therapeutic strategies have been implemented in clinics to cope with the disease yet recurrent disease and toxicity reduce their effectiveness. Hence, there is a need to identify and develop more effective therapies with reduced toxic side effects to improve overall survival rates. This dissertation investigates the mechanisms of action of two natural forms of vitamin E and a cholesterol lowering drug, simvastatin, as a therapeutic strategy in human breast cancer cells. Vitamin E in nature consists of eight distinct forms which are fat soluble small lipids. Until recently, vitamin E was known as a potent antioxidant but emerging work suggests they may be resourceful agents in managing a number of chronic diseases including cancer. Anticancer properties of vitamin E have been identified to be limited to the γ- and δ- forms of both tocopherols and tocotrienols. Gamma-tocopherol ([gamma]T) and gamma-tocotrienol ([gamma]T3) have both already been identified to induce death receptor 5 (DR5) mediated apoptosis in breast cancer cells. Studies here show that similar to [gamma]T3, [gamma]T induced DR5 activation is mediated by c-Jun N-terminal kinase/C/EBP homologous protein (JNK/CHOP) proapoptotic axis which in part contributed to [gamma]T mediated dowregulation of c-FLIP, Bcl-2 and Survivin. Also, both agents activate de novo ceramide synthesis pathway which induces JNK/CHOP/DR5 proapoptotic axis and downregulates antiapoptotic factors FLICE inhibitory protein (c-FLIP), B-cell lymphoma 2 (Bcl-2) and Survivin leading to apoptosis. Simvastatin (SVA) has been identified to display pleiotropic effects including anticancer effects but mechanisms responsible for these actions have yet to be fully understood. In this dissertation, it was observed that simvastatin induced apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 proapoptotic axis and down regulation of antiapoptotic factors c-FLIP and Survivin which are in part dependent on JNK/CHOP/DR5 axis. The anticancer effects mediated by simvastatin can be reversed by exogenously added mevalonate and geranylgeranyl pyrophosphate (GGPP), implicating the blockage of mevalonate as a key event. Furthermore, work has been done to understand the factors responsible for drug resistance and identify therapeutic strategies to counteract the same. It was observed that development of drug resistance was associated with an increase in the percentage of tumor initiating cells (TICs) in both tamoxifen and Adriamycin resistant cells compared to their parental counterparts which was accompanied by an increase in phosphorylated form of Signal transducer and activator of transcription 3 (Stat3) proteins as well as its downstream mediators c-Myc, cyclin D1, Bcl-xL and Survivin. Inhibition of Stat3 demonstrated that Stat3 and its downstream mediators play an important role in regulation of TICs in drug resistant breast cancer. Moreover, SVA, [gamma]T3 and combination of SVA+[gamma]T3 has been observed to target TICs in drug resistant human breast cancer cells and downregulate Stat3 as well as its downstream mediators making it an attractive agent to overcome drug resistance. From the data presented here, the mechanisms responsible for the anticancer actions of [gamma]T, [gamma]T3 and SVA have been better understood, providing the necessary rationale to test these agents by themselves or in combination in pre-clinical models.Item Targeting pancreatic cancer stem cells via Anti-RON Immunoliposomes(2012-05) Padhye, Snehal; Wang, Ming-Hai; Ahsan, Fakhrul; Srivastava, Sanjay; Weidanz, JonCancer stem cells (CSC) contribute to pancreatic cancer tumorigenesis through tumor initiation, drug resistance, and metastasis. Currently, therapeutics targeting pancreatic CSC are under intensive investigation. This study tested a novel strategy that utilizes RON receptor as a drug delivery moiety for increased therapeutic activity against pancreatic CSC. CD24+CD44+ESA+ triple-positive pancreatic CSC (CSC+24/44/ESA) were obtained from spheroids of pancreatic L3.6pl cancer cells by sequential magnetic cell sorting method. These cells displayed typical spheroid growth pattern, expressed unique self-renewal marker Bmi-1, re-differentiated into an epithelial phenotype, acquired an epithelial to mesenchymal phenotype, and caused tumor formation in animal model. Among several receptor tyrosine kinases examined, RON was highly expressed and sustained by CSC+24/44/ESA. This feature provided the cellular basis for validating the therapeutic effectiveness of anti-RON antibody Zt/c9-directing doxorubicin-immunoliposomes (Zt/c9-Dox-IL). Zt/c9-Dox-IL specifically interacted with CSC+24/44/ESA and rapidly caused RON internalization, which led to uptake of liposomal doxorubicin. Moreover, Zt/c9-Dox-IL was effective in reducing viability of L3.6pl cells and CSC+24/44/ESA. The IC50 values between free Dox (62.0±3.1 μM) and Zt/c9-Dox-IL (95.0 ±6.1 μM) treated CSC+24/44/ESA were at relatively comparable levels. In addition, Zt/c9-Dox-IL in combination with small molecule inhibitors lapatinib, sunitinib, or dasatinib further reduced viability of CSC+24/44/ESA. In conclusion, RON expression by CSC+24/44/ESA is a suitable molecule for targeted delivery of chemoagents. Thus anti-RON antibody-directed delivery of chemotherapeutics is effective in reducing viability of pancreatic CSCItem Understanding mechanisms of stem cell tubulogenesis in PEGylated fibrin for improving neovascularization therapies(2013-12) Rytlewski, Julie Ann; Suggs, Laura J.Stem cell-based therapies are an important developing technology for treating cardiovascular ischemic disease, including subsequent co-morbidities such as ulcerative wounds. Mesenchymal stem cells (MSCs) have a proven ability to augment wound healing and neovascularization processes and have been more recently investigated for their endothelial-like behavior. This doctoral work aims to understand mechanisms underlying matrix-driven MSC tubulogenesis within PEGylated fibrin gels, specifically (1) why this behavior occurs and (2) if this behavior has clinical utility. Briefly, a three-dimensional morphological quantification pipeline was first developed for analyzing the maturity of vascular networks (Chapter 2). This method was applied in later studies that examined the full spectrum of MSC behavior in PEGylated fibrin gels, linking biomaterial properties with network development (Chapter 3). Mechanisms underlying the cell-matrix relationship were more fully clarified through gain-of-function cell studies. These studies indicated that PEGylated fibrin promotes endothelial-like MSC behavior through a combination of hypoxic stress and bioactive fibrin cues (Chapter 4). Notably, this endothelial-like MSC behavior closely mirrored vasculogenic mimicry, a process whereby tumors establish non-endothelialized vasculature in response to hypoxic stress. The functionality of these tumor vessels suggests that mature endothelial differentiation of MSCs may not be necessary to achieve therapeutically beneficial tissue perfusion. This hypothesis opens up new mechanisms for exploitation in vascular tissue engineering strategies.