Browsing by Subject "differentiation"
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Item Development of hyaluronic acid – poly(ethylene glycol) hydrogels towards hematopoietic differentiation of mouse embryonic stem cells(2009-08) Erickson, Kathryn Marie; Roy, Krishnendu; Suggs, LauraThe fields of tissue engineering, regenerative medicine, and stem cell engineering are rapidly growing. However, these fields must overcome several obstacles before they can make a significant impact on treating cellular disorders. Two major hurdles that must be addressed are: determining how to control the pluripotency of stem cells and developing systems for high-throughput culture of stem cells. The prospect of using a cell source capable of differentiating into cells of any tissue in the body (embryonic stem cells) has received enormous interest in recent years. The pluripotent attribute of embryonic stem cells seems ideal but developing methods to drive embryonic stem cells to specific lineages, including the hematopoietic lineage, is a complex process dependent on multiple intrinsic and extrinsic factors including chemical, cellular, and environmental signaling. With regards to environmental signaling, the use of three-dimensional culture systems such as scaffolds and hydrogels, have been utilized in an attempt to drive lineage-specific differentiation in a synthetic, biomimetic microenvironment. To determine specific environmental factors responsible for hematopoietic differentiation a systematic biological and engineering process must be implemented. A biodegradable hydrogel composed of the hyaluronic acid, a polysaccharide abundant in the bone marrow microenvironment, and the synthetic polymer, poly(ethylene glycol) was formulated to culture mouse embryonic stem cells (mESCs). Photoencapsulation of mESCs did not significantly decrease cellular viability or proliferation. The FACS data was inconclusive however, from gene expression studies, it was determined that the hydrogel culture system promoted differentiation of mESCs as evidenced by a down-regulation of the gene encoding for stem cell maintenance transcription factor, Oct-3/4. Furthermore, embryoid bodies, necessary for in vitro differentiation were observed in the hydrogel systems. Although an increase in the gene encoding for the cell surface marker, c-kit was up-regulated, the surface marker, sca-1 was not up-regulated. Up-regulation of both c-kit and sca-1 is necessary for the development of hematopoietic progenitor cells. Results indicate that the differentiation of mESCs into the hematopoietic lineage was unsuccessful but differentiation in these hydrogel systems did occur. Future cell marker and gene expression studies are necessary to determine which cell lineage the encapsulated mESCs are differentiating into before the effects of incorporating other environmental, cellular, and chemical factors can be investigated.Item Effective Differentiated Instructional Elements for Improving Student Performance as Perceived by Secondary Principals in Exemplary Public High Schools in Texas: A Delphi Study(2011-10-21) Durrett, Teresa AnnThe primary purpose of this Delphi study was to determine which of the research-identified differentiated instructional elements are the most effective for improving student performance as perceived by secondary principals in 2A to 5A 2009 ?Exemplary? public high schools in Texas. A secondary purpose for this study was to determine what additional differentiated instructional elements are perceived by this study?s targeted principals as being critical for student success. The researcher obtained feedback during three Delphi survey rounds from the twenty-four member expert panel regarding which of the research-identified differentiated instructional elements they perceived to be the most effective for improving student performance. The differentiated instructional elements presented in the survey were based upon a sound theoretical framework resulting from a review of existing research on differentiated instruction. After Round Three, consensus was reached, and the data collection period ended. Each of the surveys for the study, as well as the statistical analysis, can be found in the appendices of this dissertation. The findings of this study determined that using a variety of resources, as well as a variety of strategies, were the top-ranked research-identified differentiated instructional elements that the expert panel perceived to be the most effective for improving student performance. In addition, panelists agreed that the differentiated instructional elements already identified in existing research, as presented in this study, are comprehensive and sufficient for improving student performance. Without a doubt, the conclusions and recommendations of this study could extend the current knowledge base by promoting the use of the most effective research-identified differentiated instructional elements to improve student performance. Furthermore, the implications of the study will be invaluable for ongoing professional development, principal and teacher preparation programs, and for those in the field seeking to improve their daily educational practices for student impact.Item Effects of three-dimensional culture conditions on skeletal muscle myoblasts(2007-03-30) Michele Lynn Marquette; Marguerite Sognier; Robert Leonard; Mary Moslen; Jeff Rabek; Brian HashemiEffects of Three-dimensional Culture Conditions on Skeletal Muscle Myoblasts\r\n\r\nPublication No._____________\r\n\r\n\r\nMichele Lynn Marquette, Ph.D.\r\nThe University of Texas Medical Branch, 2007\r\n\r\nSupervisor: Marguerite A. Sognier\r\n\r\nThe objectives of this research were to: 1) develop a three-dimensional in vitro model; and 2) subsequently, utilize this model to investigate mechanisms of myoblast adhesion, fusion, and differentiation. C2C12 cells were examined as pre-aggregated single cells and multicellular aggregates in the Rotary Cell Culture System (RCCS). At the time intervals tested, RCCS cultured cells maintained viability and did not exhibit increased apoptosis markers such as Caspase 3 (activated) and phosphatydylserine. In contrast, increases in cell death and apoptotic markers were noted in suspension culture (SC) control cells. RCCS cultured cells fused to form multinucleated syncitia and expressed sarcomeric myosin heavy chain (MHC) in significantly higher levels than SC aggregates after cultivation for 3 and 6 days. This occurred in the presence of mitogens without exogenous matrix or support structures. Myoblast fusion was inhibited by exposure to soluble anti-Neural-cadherin antibody, but this treatment increased MHC levels assessed using immunohistochemistry.\r\nDuring early RCCS culture, myoblasts exhibited numerous cytoplasmic protrusions (podia). Microscopic examination of cells cultured in RCCS and SC revealed significantly more and slightly longer podia in the RCCS at 3, 6, and 9-hours. Podia were F-actin dependent as shown by exposure to an F-actin depolymerizing agent, Latrunculin A. Podia were inhibited, but recovered upon Latrunculin A removal. \r\nPodia were postulated to play a role in cell-cell adhesion in conjunction with Neural Cadherin (N-cadherin), an adhesion molecule important in myoblast differentiation. To determine if N-cadherin was critical to cell-cell adhesion, RCCS cultured cells were examined for the presence of N-cadherin at both the podia and membrane using confocal microscopy. N-cadherin levels decreased at the podia and membrane of RCCS cultured cells but not in SC cells at 3, 6, and 9-hours. \r\nIn summary, these results revealed: 1) podia formation is F-actin dependent but N-cadherin independent; 2) N-cadherin is critical for myoblast maturation; 3) synctia formation and differentiation can occur with mitogens present, without exogenous substrates in the RCCS; 4) this novel myoblast model test system is suitable for defining muscle development/regeneration processes, identification of molecular targets for development of therapies, and potential regenerative medicine applications.\r\n \r\nItem Regulation and function of the Rho GTPase mediated signaling pathways in metastasis and lenticular differentiation(Texas A&M University, 2007-09-17) Mitchell, Dianne CourtenayModulation of the actin-based cytoskeleton and transcription factor regulation are merely two essential functions in a wide array of cellular activities that the Rho family of small GTPases is responsible for mediating. Aberrations in, or loss of, Rho GTPase signaling has been found to lead to multiple pathologies, including both metastatic progression and lenticular differentiation leading to cataractogenesis. This study has examined the transcriptional regulation of the metastasis suppressor, KiSS-1. Although the mechanism by which KiSS-1 modulates an anti-metastatic effect is not entirely known, it is known that KiSS-1 mediates stress fiber formation, increased adhesion and reduced migratory and invasive properties through modulation of the Rho family of small GTPases. The loss of KiSS-1 that commonly occurs during metastatic progression, leads to a loss of proper Rho GTPase regulation. This study has examined how KiSS-1 is regulated in two tissue types, breast and skin, and how the loss of AP-2(alpha) and DRIP-130, respectively, leads to the progression of breast cancer and melanoma. In addition, this study has also looked at the importance of Rac1 expression and function in the lens epithelium. Activation of Rac1 and its downstream effector, SRF, have been shown to be key regulators in lens cell differentiation, possibly leading to lens opacification via its transcriptional control of the structural crystallins within the lens. The results of this dissertation research have made significant strides in understanding the nature of the anti-metastatic effects registered by the novel KiSS-1 peptide and its cognate GPCR. Additionally, it has shed light on the Rho family regulation of lens epithelial cell differentiation, indicating the elaborate involvement of Rac1 in mediating lens fiber development. In all, this research has determined previously unknown roles of small molecule GTPases in both the progression of metastasis, as well as in normal and abnormal lens cell differentiation.Item Study of Cell Material Interactions for Vascular Tissue Engineering Application(2012-07-16) Qu, XinIn the US alone, more than 500,000 coronary artery bypass procedures are performed annually. Tissue engineering shows the potential to construct functional grafts to overcome the limited availability of autologous saphenous veins, relatively poor elasticity and low compliance of synthetic materials (mainly Dacron and polytetrafluoroethylene). In order to meet the low modulus associate with myocyte differentiation, the high suture retention and an ultimate tensile strength (UTS) sufficient to withstand implantation and peak physiological stresses, we designed and characterized a multi-component scaffold comprised of polyurethane electrospun mesh layers bonded together by a fibrin hydrogel matrix. We have demonstrated this composite construct retains the high tensile strength and suture retention strength but displays a "J-shaped" mechanical response similar to that of native coronary artery. To improve our design, poly(ethylene glycol) diacrylate based hydrogel system was utilized as a blank slate to study the phenotypic regulation by cell material interactions. Fibrinogen, fibronectin, laminin and collagen type IV were incorporated into the hydrogel to mimic the stimuli from extracellular matrix (ECM) proteins. Surprisingly, no significant effect was detected on induction of smooth muscle cell (SMC) differentiation marker expression, activation of mitogen-activated protein (MAP) kinases pathway, or alteration of surface integrin expression profile. However, fibronectin showed repression of undesired phenotypes in SMC differentiation. In contrast to ECM proteins, glycosaminoglycans (GAGs) showed more influence on regulating SMC phenotype. By using a scaffold environment intended to be mimetic of early atherosclerosis, the impact of GAG identity on SMC foam cell formation was explored. We focused on chondroitin sulfate C (CSC), dermatan sulfate (DS), and an intermediate molecular weight hyaluronan (HA_IMW, ~400 kDa), the levels and/or distribution of which are significantly altered in atherosclerosis. CSC and DS hydrogels were associated with greater SMC phagocytosis of apolipoprotein B than HA_IMW gels. However, only SMCs in DS constructs maintained increased expression of adipocyte marker A-FABP relative to HA_IMW gels over 35 days of culture. Combined, our results suggested interesting roles for fibronectin and HA_IMW in repression of undesired phenotypes in SMC differentiation, which could give insights into rational design of novel biomaterials for vascular tissue engineering applications.