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

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

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

Organo-arsenic molecular layers on silicon for high-density doping

This article describes for the first time the controlled monolayer doping (MLD) of bulk and nanostructured crystalline silicon with As at concentrations approaching 2 x 10²⁰ atoms cm⁻³. Characterization of doped structures after the MLD process confirmed that they remained defect- and damage-free, with no indication of increased roughness or a change in morphology. Electrical characterization of the doped substrates and nanowire test structures allowed determination of resistivity, sheet resistance, and active doping levels. Extremely high As-doped Si substrates and nanowire devices could be obtained and controlled using specific capping and annealing steps. Significantly, the As-doped nanowires exhibited resistances several orders of magnitude lower than the predoped materials

A simple and versatile 2-dimensional platform to study plant germination and growth under controlled humidity

We describe a simple, inexpensive, but remarkably versatile and controlled growth environment for the observation of plant germination and seedling root growth on a flat, horizontal surface over periods of weeks. The setup provides to each plant a controlled humidity (between 56% and 91% RH), and contact with both nutrients and atmosphere. The flat and horizontal geometry of the surface supporting the roots eliminates the gravitropic bias on their development and facilitates the imaging of the entire root system. Experiments can be setup under sterile conditions and then transferred to a non-sterile environment. The system can be assembled in 1-2 minutes, costs approximately 8.78$per plant, is almost entirely reusable (0.43$ per experiment in disposables), and is easily scalable to a variety of plants. We demonstrate the performance of the system by germinating, growing, and imaging Wheat (Triticum aestivum), Corn (Zea mays), and Wisconsin Fast Plants (Brassica rapa). Germination rates were close to those expected for optimal conditions

Selective migration of neuralized embryonic stem cells to stem cell factor and media conditioned by glioma cell lines

BACKGROUND: Pluripotent mouse embryonic stem (ES) cells can be induced in vitro to become neural progenitors. Upon transplantation, neural progenitors migrate toward areas of damage and inflammation in the CNS. We tested whether undifferentiated and neuralized mouse ES cells migrate toward media conditioned by glioma cell lines (C6, U87 & N1321) or Stem Cell Factor (SCF). RESULTS: Cell migration assays revealed selective migration by neuralized ES cells to conditioned media as well as to synthetic SCF. Migration of undifferentiated ES cells was extensive, but not significantly different from that of controls (Unconditioned Medium). RT-PCR analysis revealed that all the three tumor cell lines tested synthesized SCF and that both undifferentiated and neuralized ES cells expressed c-kit, the receptor for SCF. CONCLUSION: Our results demonstrate that undifferentiated ES cells are highly mobile and that neural progenitors derived from ES cells are selectively attracted toward factors produced by gliomas. Given that the glioma cell lines synthesize SCF, SCF may be one of several factors that contribute to the selective migration observed

Effect of Airfoil Parametrization on the Optimization of Counter Rotating Open Rotors

The present study compares two optimizations performed on Counter Rotating Open Rotors (CRORs) running at the same operating condition. The main difference between the two optimizations is the airfoil profile used to construct the blades. The first, uses the NACA 16 family of airfoils, whereas the second one, uses a parametrized airfoil type, CST. Two independent multi-objective optimizations are carried out using approximately the same computational resources. All the design variables except those concerning the airfoil profile, are kept with the same design freedom so that a fair comparison can be made. Both sets of configurations are aerodynamically optimized for maximum thrust coefficient and efficiency at top of climb conditions. The optimization is performed using multi-objective Differential Evolution (DE) coupled with 3D RANS simulations and Radial Basis Function (RBF) meta-modeling

A DNA-based method for studying root responses to drought in field-grown wheat genotypes

Root systems are critical for water and nutrient acquisition by crops. Current methods measuring root biomass and length are slow and labour-intensive for studying root responses to environmental stresses in the field. Here, we report the development of a method that measures changes in the root DNA concentration in soil and detects root responses to drought in controlled environment and field trials. To allow comparison of soil DNA concentrations from different wheat genotypes, we also developed a procedure for correcting genotypic differences in the copy number of the target DNA sequence. The new method eliminates the need for separation of roots from soil and permits large-scale phenotyping of root responses to drought or other environmental and disease stresses in the field.Chun Y. Huang, Haydn Kuchel, James Edwards, Sharla Hall, Boris Parent, Paul Eckermann, Herdina, Diana M. Hartley, Peter Langridge & Alan C. McKa