Stem cell incorporation and differentiation in organotypic rat and mouse brain slice cultures [abstract]

Abstract only availableStem cells can be derived from a variety of sources and have three general properties. They can continuously divide for long periods of time; they are unspecialized and they can differentiate into specialized cell types. Many stem cell types are capable of migrating through brain tissue and can be used to deliver therapeutic agents to regions of injury or disease. Our lab is interested in determining which stem cell type has the highest survival and integration rate when introduced into mammalian tissue. Migration potential and ability to deliver therapeutics are also vital and will be assessed in the future. To study the stem cells in host mammalian tissue, we are using organotypic cultures of neonate rat and mouse brain slices that enable us to track the functional integration of donor cells for up to three weeks. Donor cell types include undifferentiated ES cells, neuralized ES cells and adult peripheral blood stem cells (provided by Dr. Elmer Price) all of which express green fluorescent protein that allows for visual tracking after transplantation. Functional integration is determined by the presence of unique neural morphologies, expression of neural markers and physiological function after integration into the host tissue. Preliminary results indicate that undifferentiated ES cells integrate well and differentiate into cells that display neural morphologies, though their survival rate is much lower than that of adult peripheral blood stem cells. Neuralized ES cells also integrate well and differentiate into cells displaying neural morphologies. These cells also form interesting structures that span injurious gaps in rat brain slices. Unfortunately, neuralized ES cells have extremely low survival rates when compared to the other two cell types. Peripheral blood stem cells show the highest frequency of integration, differentiation and long-term survival within the host tissue.Life Sciences Undergraduate Research Opportunity Progra

The Land Capability Potential Index: a decision support tool for the management of Missouri River floodplain habitat

The Land Capability Potential Index (LCPI) is a hydrogeomorphic model of potential flow-return interval and soil drainage developed as a decision support tool for the restoration and management of floodplain habitat on the lower Missouri River (Chojnacki et al., 2012). The LCPI captures critical abiotic variables known to affect the distribution of plant species, implying that it may be applied at a variety of scales to anticipate the distribution of plant species and communities and to inform management of floodplain vegetation. Relationships between naturally-occurring floodplain vegetation and LCPI classes in a post-agricultural setting were quantified using existing plot data and vegetation community maps. Multivariate analyses indicate that classes in the LCPI often are more strongly correlated to the distribution of species than are other factors known to affect species distribution. Chi-square tests and analyses of variances indicate that the distributions of six exotic species are significantly related to soil drainage, flow-return interval, or both. Five species groups representing herbaceous wetlands (sedges and smartweeds), early-successional woody communities (willows and cottonwood), and late-successional forests also exhibit significant relationships with LCPI classes that are consistent with the known life-history traits of constituent species. Similar analyses of mapped community distributions in relation to LCPI classes confirm the analyses of species abundance data and support the conclusion that the LCPI model is capturing abiotic variables relevant to the distribution and management of plant species and communities on the Missouri River floodplain. Results indicate that the LCPI can be used by resource managers at multiple scales to inform planning, land acquisition, and management of Missouri River floodplain habitat. As the abundance of LCPI classes changes with ecological province, river section, and near tributaries and other hydrologic features, so too do the opportunities for and limitations against management for particular plant species and communities. At broad scales, this information can be used to develop a conservation portfolio that meets broad habitat objectives. At moderate scales, the LCPI can be used to assess the relative values of sites in terms their ability to support target communities, and to plan the spatial arrangement of communities. At the site-level, the LCPI can used to identify areas that are more or less susceptible to invasion by non-native species and more or less likely to support desired species. Though not tested as part of this thesis, results also suggest that the LCPI can also be used in an adaptive management framework to implement and assess the effects of management actions intended to promote target species and communities

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

Rebound weight gain following exercise or moderate caloric restriction in rats

Call number: LD2668 .T4 FN 1989 S77Master of ScienceHuman Nutritio

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

Development of chick embryo explant cultures as an assay system to test mammalian stem cell migration and differentiation after transplantation

Abstract only availableStem cells are undifferentiated cells capable of achieving multiple developmental fates. The embryonic stem (ES) cell can differentiate into all cells of the body. Our stem cell research focuses on the repair and/or renewal of cells in the central nervous system with potential applications for treatment of such diseases as Parkinson's disease, ALS, Alzhiemers disease, Batten's disease, as well as other neurodegenerative disorders and injuries to the CNS. An important problem is to determine the potential of stem cells to achieve various fates after transplantation. In the present study, we developed the chick embryo explant culture as an assay system to test the ability of mouse embryonic stem cells to migrate and differentiate after transplantation. The focus of this study was to monitor the survival, migration, and incorporation of the mouse ES cells after transplantation into chick embryos and to develop an explant culture method. The explant cultures will enable repeated viewing of transplanted stem cells at various time post-transplantation. Initially to establish chick embryo explants, chicken embryos were excised from the yolk at stage 8 and transferred to agar/albumen culture dishes containing antibiotics. The chick embryos explants were incubated at 37oC in a humidified chamber to continue growth and development, and explants were viewed multiple times. To date, chick embryos survived up to 47 hours after explantation. Successful explants were obtained as determined by normal embryonic development and the presence of a normal heartbeat. For stem cell transplant experiments, green fluorescent protein (GFP)-expressing mouse ES cells were transplanted into the head region of stage 10 chick embryos while the embryos were still in the egg (i.e., in ovo). Immediately following transplantation, explant cultures were established and embryonic development was allowed to proceed for up to 28 hours (~stage 15-16). The embryos were screened using a fluorescence microscope to test for the presence and fate of transplanted mouse ES cells. Ten micrometers thick transverse sections of the embryos were examined and GFP-expressing cells were found in the lower head region around the neural tube. The transplanted ES cells appeared to contribute to embryonic mesoderm. Future experiments will test whether transplanted stem cells can incorporate into the neural tube and differentiate into neural cells.NSF-REU Biology & Biochemistr

The actin-binding protein UNC-115 is an effector of Rac signaling during axon pathfinding in C-elegans

Rac GTPases control cell shape by regulating downstream effectors that influence the actin cytoskeleton. UNC-115, a putative actin-binding protein similar to human abLIM/limatin, has previously been implicated in axon pathfinding. We have discovered the role of UNC-115 as a downstream cytoskeletal effector of Rac signaling in axon pathfinding. We show that unc-115 double mutants with ced-10 Rac, mig-2 Rac or unc-73 GEF but not with rac-2/3 Rac displayed synthetic axon pathfinding defects, and that loss of unc-115 function suppressed the formation of ectopic plasma membrane extensions induced by constitutively-active rac-2 in neurons. Furthermore, we show that UNC-115 can bind to actin filaments. Thus, UNC-115 is an actin-binding protein that acts downstream of Rac signaling in axon pathfinding

Rab11 polarization of the Drosophila oocyte: a novel link between membrane trafficking, microtubule organization, and oskar mRNA localization and translation

The Drosophila embryonic body plan is specified by asymmetries that arise in the oocyte during oogenesis. These asymmetries are apparent in the subcellular distribution of key mRNAs and proteins and in the organization of the microtubule cytoskeleton. We present evidence that the Drosophila oocyte also contains important asymmetries in its membrane trafficking pathways. Specifically, we show that a-adaptin and Rab11, which function critically in the endocytic. pathways of all previously examined animal cells, are localized to neighboring compartments at the posterior pole of stage 810 oocytes. Rab11 and alpha-adaptin localization occurs in the absence of a polarized microtubule cytoskeleton, i.e. in grk null mutants, but is later reinforced and/or refined by Osk, the localization of which is microtubule dependent. Analyses of germline clones of a rab11 partial loss-of-function mutation reveal a requirement for Rab11 in endocytic recycling and in the organization of posterior membrane compartments. Such analyses also reveal a requirement for Rab11 in the organization of microtubule plus ends and osk mRNA localization and translation. We propose that microtubule plus ends and, possibly, translation factors for osk mRNA are anchored to posterior membrane compartments that are defined by Rab11-mediated trafficking and reinforced by Rab11-Osk interactions

Novel mode of ISG15-mediated protection against influenza A virus and Sendai virus in mice

ISG15 is a diubiquitin-like modifier and one of the most rapidly induced genes upon type I interferon stimulation. Hundreds of host proteins and a number of viral proteins have been shown to be ISGylated, and understanding how these modifications affect the interferon response and virus replication has been of considerable interest. ISG15(−/−) mice exhibit increased susceptibility to viral infection, and in the case of influenza B virus and vaccinia virus, ISG15 conjugation has been shown to restrict virus replication in vivo. A number of studies have also found that ISG15 is capable of antagonizing replication of some viruses in tissue culture. However, recent findings have demonstrated that ISG15 can protect mice from Chikungunya virus infection without affecting the virus burden. In order to better understand the function of ISG15 in vivo, we characterized the pathogenesis of influenza A virus and Sendai virus in ISG15(−/−) mice. We found that ISG15 protects mice from virus induced lethality by a conjugation-dependent mechanism in both of these models. However, surprisingly, we found that ISG15 had minimal effect on virus replication and did not have an obvious role in the modulation of the acute immune response to infection. Instead, we observed an increase in the number of diseased small airways in mice lacking ISG15. This ability of ISG15 to protect mice in a conjugation-dependent, but nonantiviral, manner from respiratory virus infection represents a previously undescribed role for ISG15 and demonstrates the importance of further characterization of ISG15 in vivo. IMPORTANCE It has previously been demonstrated that ISG15(−/−) mice are more susceptible to a number of viral infections. Since ISG15 is one of the most strongly induced genes after type I interferon stimulation, analysis of ISG15 function has largely focused on its role as an antiviral molecule during acute infection. Although a number of studies have shown that ISG15 does have a small effect on virus replication in tissue culture, few studies have confirmed this mechanism of protection in vivo. In these studies we have found that while ISG15(−/−) mice are more susceptible to influenza A virus and Sendai virus infections, ISGylation does not appear to mediate this protection through the direct inhibition of virus replication or the modulation of the acute immune response. Thus, in addition to showing a novel mode of ISG15 mediated protection from virus infection, this study demonstrates the importance of studying the role of ISG15 in vivo