896 research outputs found
Neuralized Mouse Embryonic Stem Cells Develop Neural Rosette-Like Structures in Response to Retinoic Acid and Produce Teratomas in the Brains of Syngeneic Mice
Several induction protocols can direct differentiation of mouse embryonic stem cells (ESCs) to become neural cells. The B5 and B6 mouse ESC lines display different growth patterns in vitro, and when grown as adherent cultures, the B6 ESCs proliferated at a significantly lower rate than B5 ESCs. Remarkably, after a neural induction protocol that includes removal of LIF and addition of retinoic acid (RA), mature B6 embryoid bodies (EBs) displayed a unique neural rosette-like morphology. On Day 8 of neural induction, B6 EBs revealed mature neuronal markers localized primarily to cells in the center of the EBs and glial markers expressed both in centrally and peripherally located cells. In contrast to B5 cells, when neuralized Day 8 B6 EB cells were dissociated and transplanted into the left striatum of syngeneic C57BL/6 mouse brains, teratomas formed. In addition, teratomas established from undifferentiated B6 cells grew more rapidly and achieved larger volumes when compared to those produced by Day 8, neuralized B6 EBs. The slow growth rate of B6 cells in vitro may have contributed to incomplete neuralization, formation of neural rosette-like structures, and a propensity to form teratomas
Determining an efficient protocol for production of neural stem cells
Abstract only availableMouse embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the developing blastocyst. When cultured in non-adherent dishes, ES cells form free-floating embryoid bodies (EBs). Cells within the EBs can then be induced to form neural stem and progenitor cells. These 'neuralized' mouse ES cells have been used for therapeutic transplantation experiments in mouse models of human neurodegenerative diseases, including neuronal ceroid-lipofuscinoses (NCLs). This study focused on developing a more homogenous population of neural stem cells from ES cells for use in transplantation experiments. A homogenous population of neural stem cells could provide a renewable source of neural stem cells and thus a more consistent fate outcome for transplanted cells. We tested selected protocols for neural induction of mouse ES cells and compared their efficiencies in creating neural stem cells in vitro. Three previously developed protocols were tested in this study. The first induction protocol was specifically used to generate spheres of neural precursor cells, or neurospheres. It used a retinoic acid induction protocol followed by seeding dissociated EBs into neurosphere media. The second protocol involved growing neural stem cell colonies in astrocyte-conditioned media. The third protocol consisted of growing ES cells in flasks in neurosphere media (including FGF) without EGF for four days and then four days in neurosphere media plus EGF. Four variations on the last protocol were also tested. Preliminary results suggest that to produce a larger yield of neurospheres, the first protocol would need to be altered. The second protocol was time consuming and produced a small population of neural stem cells. The third protocol produced promising results with a larger yield of neurospheres than the first two protocols. Future studies will focus on the third protocol and define the optimal conditions whereby it will produce more neural stem cells.NSF-REU Program in Biological Sciences & Biochemistr
High Rate of Obesity-Associated Hypertension among Primary Schoolchildren in Sudan
Cardiovascular disease (CVD) frequently has roots in childhood, including following childhood-onset hypertension. Incidence of CVD has increased in developing countries in East Africa during recent urbanization. Effects of these shifts on childhood hypertension are unclear. Our objectives were to (1) Determine the prevalence of hypertension among primary schoolchildren in Khartoum, Sudan; (2) Determine whether hypertension in this setting is associated with obesity. We performed a cross sectional study of 6-12y children from two schools randomly selected in Khartoum, Sudan. Height, weight, BMI, BP and family history of hypertension were assessed. Age-, height- and gender-specific BP curves were used to determine pre-hypertension (90–95%) and hypertension (>95%). Of 304 children, 45 (14.8%) were overweight; 32 (10.5%) were obese; 15 (4.9%) were pre-hypertensive and 15 (4.9%) were hypertensive. Obesity but not family history of hypertension was associated with current hypertension. In multiple logistic regression, adjusting for family history, children who were obese had a relative-risk of 14.7 (CI 2.45-88.2) for systolic hypertension compared to normal-weight children. We conclude that overweight and obesity are highly prevalent among primary schoolchildren in urban Sudan and are strongly associated with hypertension. That obesity-associated cardiovascular sequelae exist in the developing world at young ages may be a harbinger of future CVD in sub-Saharan Africa
Stem cells transplanted into the brain are immunogenic and are actively rejected by the host [abstract]
Abstract only availableStem cell-based therapies have shown great promise in the treatment of neurodegenerative diseases such as Batten's Disease and Parkinson's Disease. Intracranial stem cell transplantation has the potential to restore function and compensate for neural cells lost due to injury or disease. However, rejection of donor cells by the host immune system may limit the effectiveness of stem cell therapies. Recent research has suggested that some stem cells may be immunoprivileged, able to avoid rejection by the host's immune system in both allogeneic and xenogeneic settings. This can occur regardless of an MHC mismatch. However, several aspects of these studies complicate interpretations of their results. Transplant recipients are often irradiated before the transplant (Drukker et al. 2006) or otherwise immunocompromised and studies often culminate in a time period insufficient for immune rejection to have occurred (under a week) (Li et al. 2004). To evaluate whether stem cells are immunoprivileged when transplanted into the brain, GFP-expressing neural stem cells (NSCs) were transplanted into the brains of immunocompetent, immunologically mismatched mice. Mice were then sacrificed at time points of one, three, five, seven and nine days posttransplantation. The brains were fixed, freeze-embedded with OCT and sectioned. Graft survival was evaluated by observing the amount of GFP-expressing donor cells in the sections. Sections were also immunolabeled for cells expressing CD4, CD8 and CD11b, all of which are markers for infiltrating immune cells. Presence of such cells indicates immune detection and rejection and can be used to quantify the immune response to foreign stem cells. In future studies, the use of Regulatory T Cells may help to alleviate this rejection by suppressing the activity of CD8 (cytotoxic) and CD4 (helper) T Cells.National Institutes of Healt
Glioma cells influence the migration of neuralized mouse embryonic stem cells in vivo [abstract]
Abstract only availableOf the 200,000 brain tumors diagnosed in the United States each year, approximately 23% of them are glioblastomas (Brain Tumor Society 2004). These aggressive tumors spread rapidly and are resistant to standard treatment, and the average survival rate of patients diagnosed with glioblastomas is approximately one year. Current research suggests there is great potential for neural stem cells (NSCs) to be used as a delivery vehicle for therapeutic agents against tumors. Studies have shown NSCs have an innate attraction to tumors and other inflammatory diseases of the brain. This NSC pathotropism is due in part to inflammatory signals, angiogenesis, reactive astrocytosis, and tumor cytokines (Müller, et al. 2006). By harnessing their natural tropism, NSCs engineered with chemotherapeutic properties can be used to track and target tumors for destruction. To demonstrate the therapeutic potential of NSCs as a transplantable, therapeutic delivery system, we are investigating the in vivo migratory behavior and cellular fate of neuralized mouse embryonic stem cells (mESCs) in the presence of glioma cells. In this study, neuralized mESCs and SF767 human glioblastoma cells were injected into opposite hemispheres of the mouse cortex, and frozen sections of the brain tissue were examined to determine the extent of mESC migration and survival. After 3 days in vivo, co-localization of tumor and neuralized mESCs was evident in multiple sections. Previously, we have seen co-localization of neuralized mESCs and tumor cells on organotypic mouse brain slices after approximately one week of migration. NSC migration to tumor cells in vivo lends support to current efforts to use stem cells as a therapeutic deliver system. Furthermore, the neuralized mESCs' proximity to the tumor cells will allow for the specific delivery of chemotherapeutic agents to tumor sites. Expanding our knowledge of fundamental characteristics and behaviors of neural stem cells will facilitate the development of novel and effective stem cell therapies for glioblastomas
Structural Analysis of Test Flight Vehicles with Multifunctional Energy Storage
Under the NASA Aeronautics Research Mission Directorate (ARMD) Convergent Aeronautical Solutions (CAS) project, NASA Glenn Research Center has been leading Multifunctional Structures for High Energy Lightweight Load-bearing Storage (M-SHELLS) research efforts. The technology of integrating load-carrying structures with electrical energy storage capacity has the potential to reduce the overall weight of future electric aircraft. The proposed project goals were to develop M-SHELLS in the form of honeycomb coupons and subcomponents, integrate them into the structure, and conduct low-risk flight tests onboard a remotely piloted small aircraft. Experimental M-SHELLS energy-storing coupons were fabricated and tested in the laboratory for their electrical and mechanical properties. In this paper, finite element model development and structural analyses of two small test aircraft candidates are presented. The finite element analysis of the initial two-spar wing is described for strain, deflection, and weight estimation. After a test aircraft Tempest was acquired, a load- deflection test of the wing was conducted. A finite element model of the Tempest was then developed based on the test aircraft dimensions and construction detail. The component weight analysis from the finite element model and test measurements were correlated. Structural analysis results with multifunctional energy storage panels in the fuselage of the test vehicle are presented. Although the flight test was cancelled because of programmatic reasons and time constraints, the structural analysis results indicate that the mid-fuselage floor composite panel could provide structural integrity with minimal weight penalty while supplying electrical energy. To explore potential future applications of the multifunctional structure, analyses of the NASA X-57 Maxwell electric aircraft and a NASA N+3 Technology Conventional Configuration (N3CC) fuselage are presented. Secondary aluminum structure in the fuselage sub-floor and cargo area were partially replaced with reinforced five-layer composite panels with M-SHELLS honeycomb core. The N3CC fuselage weight reduction associated with each design without risking structural integrity are described. The structural analysis and weight estimation with the application of composite M-SHELLS panels to the N3CC fuselage indicate a 3.2% reduction in the fuselage structural weight, prior to accounting for the additional weight of core material required to complete the energy storage functionality
Camgaroo-2 as an indicator of function in embryonic and neuralized stem cells
Abstract only availableThe transplantation of stem cells to replace cells that have been lost or damaged due to disease or injury is quickly becoming a conceivable treatment method. Embryonic stem (ES) cells have the capacity to become any cell in the body, so the therapeutic possibilities are vast. The ultimate goal of our research on ES cells is to induce them to differentiate into functioning neurons to replace those that are lost in patients suffering from neurodegenerative disorders. However, it is important that the differentiated cells possess the appropriate phenotype and are able to perform the correct function after transplantation. In the past, it was common to accept a differentiated cell's fate based solely on its morphology and the presence of specific membrane markers. Now, it is becoming increasingly important to determine a donor stem cell's fate based on its function, especially if the cell is to be transplanted into a subject as a means of therapy. This study used the calcium-sensitive protein Camgaroo-2 to test the function of embryonic stem cells and cells directed toward a neural lineage. Camgaroo-2 is a fusion protein that consists of calmodulin in between two halves of yellow fluorescent protein. When calcium is present, it binds to the calmodulin portion of the Camgaroo-2, inducing a conformational change that results in increased fluorescence. After mouse embryonic stem cells were transfected with Camgaroo-2, we used reagents such as potassium chloride and ionomycin, known to elevate intracellular calcium, to confirm that the ES cells were stably transfected with the plasmid, and that Camgaroo-2 was functioning correctly. Potassium chloride causes the cell to depolarize while ionomycin (a calcium ionophore) creates large pores in the cell membrane. Both reagents allow for an influx of calcium into the cell, leading to increased fluorescence. The Camgaroo-2 transfected ES cells showed the appropriate responses to KCl and ionomycin by depolarizing and showing visible increases in fluorescence. This confirms that our Camgaroo-2 construct is functioning in the ES cells. We are in the process of testing the responses of neuralized ES cells using appropriate neurotransmitters, the presence of which should induce unique fluorescent signatures in a cell specific manner. Confirming neuronal function from differentiated Camgaroo-2 ES cells is an important step toward neuron transplantation in a neurodegenerative disease model.Arts & Sciences Undergraduate Research Mentorship Progra
The effect of Camgaroo-2 incorporation on the differentiation potential of embryonic stem cells
Abstract only availableEmbryonic stem (ES) cells are capable of differentiating into any cell type in the body and are a promising therapeutic agent. Our research focuses on the differentiation of ES cells into functional neurons and/or glial that can nurture host cells of the nervous system that are damaged due to disease. Cells must express the appropriate phenotype and perform the proper function after transplantation. Camgaroo-2 is a fluorescence protein that provides a basal fluorescence and responds to a rise in intracellular calcium by producing an increase in fluorescence emission. Our lab transfected a mouse ES cell line (GSI-1) with the Camgaroo-2 gene and is testing this fluorescence indicator to determine the physiological function of cells grown in vitro. There is concern that the incorporation of the Camgaroo-2 gene could alter the cell phenotype, potentially decreasing their differentiation potential. GSI-1 cells were plated on culture slides following a neural induction protocol that uses retinoic acid and allowed to proliferate. Immunohistochemisty of slides was performed to label for neural precursors, immature and mature neurons, astrocytes, and oligodendrocytes (anti-O4). GSI-1 labeling was compared to corresponding immunohistochemistry performed on another ES cell line that had also been 'neuralized' to determine if the differentiation potential of the GSI-1 cells was similar to that of the other ES cell line. Similar labeling was seen for all markers except O4 which did not label for the GSI-1 cells, indicating the GSI-1 cells have the potential to differentiate into all cells of neural lineage except possibly oligodendrocytes. GSI-1 cells retained the ability to differentiate post-transfection with the Camgaroo-2 gene. Because of their unique ability to respond to an influx of intracellular calcium, GSI-1 cells expressing Camgaroo-2 can be transplanted into rodent models for human disease, and can be tested post transplantation for their ability to function as neural cells.Life Sciences Undergraduate Research Opportunity Progra
Potential Role for Programmed Cell Death in the Formation of an In Vitro Neural Stem Cell Niche [abstract]
Abstract only availableFaculty Mentor: Dr. Mark Kirk, Biological SciencesStem cell therapies have the potential to treat neurodegenerative diseases, such as Batten Disease. Batten disease, a rare inherited disease in children, causes severe neurodegeneratoin, which results in blindness, seizures, and eventual death. In Batten disease, the transplantation of stem cells into a patient may replace lost cells or to prevent cell loss due to the disease. In one form of Batten Disease, transplantation of stem cells into the retinas of mutant model mice have shown signs of neuroprotection; including enhanced survival of photoreceptors (Meyer et al. 2006). One possible method to increase the efficiency of this treatment is the transplantation of a functional unit capable of producing its own neural precursors "on demand". Such a structure, known as a neural stem cell (NSC) niche, can be found in two small areas in the brain of mammals, and is the center for adult neurogenesis throughout the lives of these mammals. In our lab, we have developed a way to produce a NSC niche in vitro from neuralized mouse embryonic stem cells. To test how this structure is formed and maintained, I am investigating cell death within this in vitro NSC niche-like structure. I performed two different tests for apoptosis, or programmed cell death, Trypan Blue exclusion and TUNEL. Trypan Blue shows membrane permeability; if cells turn blue it is indicative of a late stage in the apoptotic process. In the TUNEL assay, nicked ends of DNA are labeled, an indication of early stage apoptosis. I also tested the affects of induced cell death on the advancement of in vitro niche formation. We hope that this information will lead to a better understanding of how the in vitro niche forms.College of Arts and Science Undergraduate Research Mentorship Progra
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