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The Effect of Gelatin Microgel Stiffness on the Morphology of Encapsulated Mesenchymal Stem Cells
03/11/2015The use of injectable hydrogel scaffolds for encapsulating stem cells has been studied as an effective approach for cell delivery for regenerative medicine. The objective of this study is to examine the effect of hydrogel stiffness on the morphology of encapsulated mesenchymal stem cells (MSCs). Mouse MSCs were encapsulated within gelatin microspheres using a water-in-oil phase bulk emulsification method and the extent of gel stiffness was controlled by changing crosslinking time (5, 30 and 60 minutes) of gelatin matrix. The change in MSC morphology and spreading within three-dimensional microgel space was quantified by measuring cell circularity and spreading. Results shows that MSCs encapsulated within low stiffness gels exhibited high circularity compared with those within high stiffness gels.
Optimizing Liquid Crystalline Elastomers for Three Dimensional Neuronal Cultures
03/11/2015Two dimensional (2D) tissue samples have been successfully grown using usual cell culture techniques. However, in order for cell cultures to be representative of three dimensional (3D) tissues it is necessary that they are grown in a spatial environment. Elastomers can be used for this purpose. Elastomers are a form of polymer that have elastic properties. There are multiple properties of elastomers that can be adjusted in order to provide a suitable environment for growing different cell types. The elastomer used in this project is liquid crystalline based. Its main chain is constructed of ε-caprolactam and side chains of cholesterol which gives the elastomer biocompatible properties. The use of liquid crystalline elastomers (LCE) provides a material that is anisotropic which is important for neuronal cell growth. This LCE is porous in its structure which creates a 3D matrix for cell growth making it a good candidate for supporting spatial growth of cellular structures. Elastomer pore size was measured using scanning electron microscopy (SEM) and adjusted in order to better support the growth of neuroblastoma cells. In this project human neuroblastoma cells (SHSY-5Y) were seeded within the LCE. Cells were allowed to grow for an extended period of time and imaged using confocal microscopy to better understand how the cells are growing within the LCE. The purpose of this project is to create the optimal conditions for the neuroblastoma cells by manipulating the LCE properties to control and enhance neuroblastoma cell growth.
Inhibition of OV2008 Ovarian Cancer Cell Proliferation in the presence of Oleolyl Ethanolamide and Chlorpyrifos-Oxon.
03/11/2015We have previously shown that acylethanolamide oleoylethanolamide (OEA) and its metabolically stable analog AM3102 induce apoptosis in OV2008 ovarian adenocarcinoma cells independent of PPAR-α signaling pathway. This cytotoxicity is reversed in the presence of α-tocopherol, indicative of reactive oxygen species involvement in cell death. We have also shown that palmityl trifluoromethyl ketone (PTK), independent of its inhibitory effect on phospholipase A2, enhances the toxicity of OEA (ASCB 2012, Abstract #888). The enzyme neutral cholesterol ester hydrolase 1 (NCEH1) has also been shown to be a target of trifluoromethylketones (Nat Biotechnol 21:687, 2003). Using Chlorpyrifos-Oxon (CPO), a potent inhibitor of the enzyme NCEH1, we explored the relationship between OEA and NCEH1 in the OV2008 cell line. Cytotoxicity was observed in response to OEA (IC50: 14-21 µM) and CPO (IC50: 48-50 µM). When the compounds were administered together, cytotoxicity was enhanced (OEA IC50: 8-9 µM; CPO IC50: 15-19 µM) with evidence of weak synergism. However, Western blot analysis of OEA and CPO treated cells indicated no change in NCEH1 expression. Live cell labeling with dihydroethidium showed an increase superoxide by about 10% in the presence of OEA. CPO itself did not increase ROS in cells. Live cell fluorescence microscopy using MitoSOX Red also revealed an increase in superoxide in the mitochondria in the presence of OEA. The results of our study suggest that one mechanism by which OEA induces cytotoxicity in OV2008 cells is via superoxide generation. It is possible that OEA-mediated increase in ROS makes the OV2008 cells further susceptible to CPO.
Histological Investigation of the Intestinal Tracts of Lean and Obese-Prone Rats.
03/11/2015Obesity is a growing concern for the nation. Increased interest has focused on the gastrointestinal tract (GI) properties that may impact nutrient absorption. To compare potential differences in GI morphology, we examined the histological characteristics of the digestive tracts of lean and obese rats. High-capacity runners (HCR) are lean, whereas low-capacity runners (LCR) are obese, yet HCR eat more than LCR. Intestines were collected from 12 HCR and 12 LCR adult male rats. After fixation with 10% formalin, a sample from the jejunum was paraffin embedded and sectioned cross-sectionally on a microtome, followed by hematoxylin and eosin (H&E) staining. Using microscopy, we were able to quantify aspects of jejunum such as villi length, diameter, and surface area, and number of crypts per unit area. Preliminary findings showed thinner mucosal layers in the LCR than in the HCR. Longer villi were seen in the HCR than LCR jejunum; however, the number of villi was greater in the LCR. Ongoing analyses will calculate the surface area of villi. The total number of villi and surface area could differentially impact nutrient absorption.
Demyelination in Multiple Sclerosis as an Effect of N-Acetylaspartate Depletion”
03/11/2015Multiple sclerosis (MS) is characterized by the demyelination of the central nervous system, which causes progressive neurological disability. Mitochondrial defects in the MS brain, including decreased levels of the neuronal mitochondrial metabolite N-acetylaspartate (NAA), have previously been identified in our lab. We suspect that demyelination in MS is due in part to depletion of NAA through two possible mechanisms. In one mechanism, oligodendrocytes break down NAA into acetate and aspartate which can enter the tricarboxylic acid (TCA) cycle altering oligodendrocyte metabolism. Changes in levels of TCA intermediates have been shown to regulate the activity of histone H3 demethylase enzymes. Preliminary data suggests that neuronal release of NAA enhances myelination by oligodendrocytes through altering trimethylation of histone H3 on lysine 4 (H3K4me3), which regulates genes associated with myelin lipid synthesis. In mechanism two, additional acetyl-CoA resulting from the catabolism of NAA may serve as a substrate for the synthesis of myelin lipids. Here, we will investigate how defects in myelin can be caused by changes in NAA by studying primary cultures of oligodendrocytes and mice that are deficient for NAA (NAT8L-KO). TCA cycle intermediates will be quantitated by mass spectrometry. Western blotting will be used to determine H3K4me3 levels. Quantitative RT-PCR and immunohistochemistry will be used to measure changes in oligodendrocyte differentiation markers. Mass spectrometry, thin layer chromatography, and magnetic resonance imaging (MRI) will be used to measure changes in myelin lipid levels in vivo in the NAA deficient NAT8L-KO mice.