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STEM CELL BIOLOGY AND STRATEGIES FOR THERAPEUTIC DEVELOPMENT IN DEGENERATIVE DISEASES AND CANCER
Title
STEM CELL BIOLOGY AND STRATEGIES FOR THERAPEUTIC DEVELOPMENT IN DEGENERATIVE DISEASES AND CANCER.
Creator
Alvarez, Angel, Sugaya, Kiminobu, University of Central Florida
Abstract / Description
Stem cell biology is an exciting field that will lead to significant advancements in science and medicine. We hypothesize that inducing the expression of stem cell genes, using the embryonic stem cell gene nanog, will reprogram cells and dedifferentiate human mesenchymal stem cells into pluripotent stem cells capable of neural differentiation. The aims of initial studies are as follows: Aim 1: Demonstrate that forced expression of the embryonic stem cell gene nanog induces changes in human...
Show moreStem cell biology is an exciting field that will lead to significant advancements in science and medicine. We hypothesize that inducing the expression of stem cell genes, using the embryonic stem cell gene nanog, will reprogram cells and dedifferentiate human mesenchymal stem cells into pluripotent stem cells capable of neural differentiation. The aims of initial studies are as follows: Aim 1: Demonstrate that forced expression of the embryonic stem cell gene nanog induces changes in human mesenchymal stem cells to an embryonic stem cell-like phenotype. Aim 2: Demonstrate that induced expression of nanog up-regulates the expression of multiple embryonic stem cell markers and expands the differentiation potential of the stem cells. Aim 3: Demonstrate that these nanog-expressing stem cells have the ability to differentiate along neural lineages in vitro and in vivo, while mock-transfected cells have an extremely limited capacity for transdifferentiation. Alternatively, we hypothesize that embryonic stem cell genes can become activated in malignant gliomas and differentially regulate the subpopulation of cancer stem cells. This study examines the role of embryonic stem cell genes in transformed cells, particularly cancer stem cells. These studies explore has the following objectives: Aim 1: Isolate different sub-populations of cells from tumors and characterize cells with stem cell-like properties. Aim 2: Characterize the expression of embryonic stem cell markers in the sub-population of cancer stem cells. Aim 3: Examine the effects of histone deacetylase inhibitors at inhibiting the growth and reducing the expression of stem cell markers. Our research has demonstrated the potential of the embryonic transcription factor, nanog, at inducing dedifferentiation of human bone marrow mesenchymal stem cells and allowing their recommitment to a neural lineage. Specifically, we used viral and non-viral vectors to induce expression of NANOG, which produced an embryonic stem cell-like morphology in transduced cells. We characterized these cells using real-time PCR and immunohistochemical staining and find an up-regulation of genes responsible for pluripotency and self-renewal. Embryonic stem cell markers including Sox2, Oct4 and TERT were up-regulated following delivery of nanog. The role of nanog in the expression of these markers was further demonstrated in our induced-differentiation method where we transfected embryonic stem cell-like cells, that have been transduced with nanog flanked by two loxP sites, with a vector containing Cre-recominase. We tested the ability of these nanog-transfected cells to undergo neural differentiation in vitro using a neural co-culture system or in vivo following intracranial transplantation. Our next study characterized patient-derived glioblastoma cancer stem cells. We found that cells isolated from serum-free stem cell cultures were enriched for stem cell markers and were more proliferative than the bulk population of cells grown in convention serum-supplemented media. These cancer stem cells expressed embryonic stem cell markers NANOG and OCT4 whereas non-tumor-derived neural stem cells do not. Moreover, the expression of stem cell markers was correlated with enhanced proliferation and could serve as a measure of drug effectiveness. We tested two different histone deacetylase inhibitors, trichostatin A and valproic acid, and found that both inhibited proliferation and significantly reduced expression of stem cell markers in our cancer stem cell lines. These data demonstrate the potential use of stem cell genes as therapeutic markers and supports the hypothesis that cancer stem cells are a major contributor to brain tumor malignancy.
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Date Issued
2011
Identifier
CFE0003641, ucf:48845
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003641
Embryonic Stem Cell-Derived Exosomes Inhibit Doxorubicin-Induced Pyroptosis in Cell Culture Models
Title
Embryonic Stem Cell-Derived Exosomes Inhibit Doxorubicin-Induced Pyroptosis in Cell Culture Models.
Creator
Tavakoli Dargani, Zahra, Singla, Dinender, Masternak, Michal, Siddiqi, Shadab, Steward, Robert, University of Central Florida
Abstract / Description
Doxorubicin (Dox) is a potent chemotherapeutic drug used for the treatment of various cancers. Unfortunately, its use is limited as Dox induces adverse cardiotoxicity (DIC) and muscle toxicity (DIMT), which are mediated through oxidative stress, ER stress, and inflammation. However, it remains unknown whether Dox induces an inflammation mediated cell death, called (")pyroptosis("). The current study is designed to determine whether Dox induces pyroptosis in cardiac and muscle cell culture...
Show moreDoxorubicin (Dox) is a potent chemotherapeutic drug used for the treatment of various cancers. Unfortunately, its use is limited as Dox induces adverse cardiotoxicity (DIC) and muscle toxicity (DIMT), which are mediated through oxidative stress, ER stress, and inflammation. However, it remains unknown whether Dox induces an inflammation mediated cell death, called (")pyroptosis("). The current study is designed to determine whether Dox induces pyroptosis in cardiac and muscle cell culture models. Moreover, the protective effects of embryonic stem cell-derived exosomes (ES-Exos) in inhibiting pyroptosis will also be determined. For this purpose, we designed two different cell culture models using H9c2 cadiomyoblasts and Sol 8 cells. For the DIC model, H9c2 were exposed to Dox to induce pyroptosis and then treated with exosomes. Cells were divided into 4 groups: Control, Dox, Dox+ES-Exos, and Dox+MEF-Exos (negative control). Furthermore, to generate the DIMT model, Sol 8 cells were incubated with Dox+THP-1 conditioned medium (TCM) to induce toxicity and inflammation, which was followed by exosomes treatment. We assigned cells into 5 groups: Control, Dox+TCM, Dox+TCM+ES-Exos, Dox+TCM+MEF-Exos (negative control), and Dox+TCM+ES-Exos+GW4869 compound (exosomes inhibitor, negative control). Our data shows that Dox treatment significantly increased pyroptotic marker expression including TLR-4, NLRP3, caspase-1, IL1-?, Caspase-11, and gasdermin-D as well as increased pro-inflammatory TNF-? and IL-6 expression in H9c2 cells. There was also a significant increase in caspase-1, IL1-?, and IL-18 expression in Dox+TCM treated Sol 8 cells. Conversely, increased pyroptosis and inflammation post-Dox treatment were inhibited by ES-Exos in both culture models. No significant changes observed upon MEF-Exos and GW4869 compound treatments. In conclusion, our data shows Dox induces pyroptosis and inflammation within cardiac and skeletal muscle cells, which can be inhibited following treatment with ES-exosomes. This is a novel study with new mechanistic observations on the pathophysiological role of pyroptosis in Dox-induced cardio and muscle toxicities.
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Date Issued
2018
Identifier
CFE0007416, ucf:52700
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007416
Dissertation Title: Development of molecular and cellular imaging tools to evaluate gene and cell based therapeutic strategies in vivo
Title
Dissertation Title: Development of molecular and cellular imaging tools to evaluate gene and cell based therapeutic strategies in vivo.
Creator
Xia, Jixiang, Ebert, Steven, Khaled, Annette, Cheng, Zixi, Daniell, Henry, University of Central Florida
Abstract / Description
Molecular imaging modalities are important tools to evaluate the efficacy of gene delivery systems and cell-based therapies. Development and application of these modalities will advance our understanding of the mechanism of transgene expression and cell fate and functions. Physical gene transfer methods hold many advantages over viral vectors among gene therapeutic strategies. Here, we evaluated the efficacy of biolistic ((")gene gun(")) gene targeting to tissues with non-invasive...
Show moreMolecular imaging modalities are important tools to evaluate the efficacy of gene delivery systems and cell-based therapies. Development and application of these modalities will advance our understanding of the mechanism of transgene expression and cell fate and functions. Physical gene transfer methods hold many advantages over viral vectors among gene therapeutic strategies. Here, we evaluated the efficacy of biolistic ((")gene gun(")) gene targeting to tissues with non-invasive bioluminescence imaging (BLI) methods. Plasmids carrying the firefly luciferase reporter gene were transfected into mouse skin and liver using biolistics, and BLI was measured at various time points after transfer. With optimized DNA loading ratio (DLRs), reporter gene expression reached to peak 1day after transfer to mouse skin, and the maximum depth of tissue penetration was between 200-300?m. Similar peak expression of reporter gene was found in mouse liver but the expression was relatively stable 4-8 days post-biolistic gene transfer and remained for up to two weeks afterward. Our results demonstrated BLI was an efficient strategy for evaluation of reporter gene expression in the same animals over a period of up to two weeks in vivo. Different tissues showed different expression kinetics, suggesting that this is an important parameter to consider when developing gene therapy strategies for different target tissues. We also employed BLI to measure differentiation of mouse embryonic stem (ES) cells into beating cardiomyocytes in vitro and in vivo. A subset of these cardiomyocytes appears to be derived from an adrenergic lineage that ultimately contribute to substantial numbers of cardiomyocytes primarily on the left side of the heart. At present, it is unclear what the precise role of these cardiac adrenergic cells is with respect to heart development, though it is known that adrenergic hormones (adrenaline and noradrenaline) are essential for embryonic development since mice lacking them die from apparent heart failure during the prenatal period. To identify and characterize cardiac adrenergic cells, we developed a novel mouse genetic model in which the nuclear-localized enhanced green fluorescent protein (nEGFP) reporter gene was targeted to the first exon of the Phenylethanoamine N-transferase (Pnmt) gene, which encodes for the enzyme that converts noradrenaline to adrenaline, and hence serves as a marker for adrenergic cells. Our results demonstrate this knock-in strategy effectively marked adrenergic cells in both fetal and adult mice. Expression of nEGFP was found in Pnmt-positive cells of the adult adrenal medulla, as expected. Pnmt-nEGFP expression also recapitulated endogenous Pnmt expression in the embryonic mouse heart. In addition, nEGFP and Pnmt expression were induced in parallel during differentiation of pluripotent mouse ES cells into beating cardiomyocytes. This new mouse genetic model provides a useful new tool for studying the properties of adrenergic cells in different tissues. We also identified two limitations of the Pnmt-nEGFP model. One is that the amount of nEGFP expressed within individual adrenergic cells was highly variable. Secondly, expression of nEGFP in the embryonic heart was of low abundance and difficult to distinguish from background autofluorescence. To overcome these limitations, we developed two alternative genetic models to investigate adrenergic cells: (1) Mouse embryonic stem cells, which have been previously targeted with Pnmt-Cre recombinase gene, were additionally targeted with a dual reporter plasmid which covered both a loxP-flanked cDNA of red fluorescence protein (HcRed) and also EGFP. Under the undifferentiated status, cells emit red fluorescence as transcription stops before EGFP coding sequence. After differentiation into beating cardiomyoctyes, some cells switch fluorescence from red to green, indicating that excision of loxP-flanked sequences by Cre since Pnmt had been activated. (2) A surface marker, truncated low-affinity nerve growth factor receptor (?LNGFR) was used as the reporter gene as cells expressing this marker can be enriched by magnetic-activated cell sorting (MACS), a potentially efficient way to yield highly purified positive cells at low input abundance in a population. Through a series of subcloning steps, the targeting construct, Pnmt-?LNGFR-Neo-DTA was created and electroporated into 7AC5EYFP embryonic stem cells. Correctly targeted cells were selected by positive and negative screening. These cells provide a new tool with which to identify, isolate, and characterize the function of adrenergic cells in the developing heart, adrenal gland, and other tissues where adrenergic cells make important contributions.
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Date Issued
2011
Identifier
CFE0004491, ucf:49287
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004491
GLIAL DIFFERENTIATION OF HUMAN UMBILICAL STEM CELLS IN 2D AND 3D ENVIRONMENTS
Title
GLIAL DIFFERENTIATION OF HUMAN UMBILICAL STEM CELLS IN 2D AND 3D ENVIRONMENTS.
Creator
Davis, Hedvika, Hickman, James, University of Central Florida
Abstract / Description
During differentiation stem cells are exposed to a range of microenvironmental chemical and physical cues. In this study, human multipotent progenitor cells (hMLPCs) were differentiated from umbilical cord into oligodendrocytes and astrocytes. Chemical cues were represented by a novel defined differentiation medium containing the neurotransmitter norepinephrine (NE). In traditional 2 dimensional (2D) conditions, the hMLPCs differentiated into oligodendrocyte precursors, but did not progress...
Show moreDuring differentiation stem cells are exposed to a range of microenvironmental chemical and physical cues. In this study, human multipotent progenitor cells (hMLPCs) were differentiated from umbilical cord into oligodendrocytes and astrocytes. Chemical cues were represented by a novel defined differentiation medium containing the neurotransmitter norepinephrine (NE). In traditional 2 dimensional (2D) conditions, the hMLPCs differentiated into oligodendrocyte precursors, but did not progress further. However, in a constructed 3 dimensional (3D) environment, the hMLPCs differentiated into committed oligodendrocytes that expressed MBP. When co-cultured with rat embryonic hippocampal neurons (EHNs), hMLPCs developed in astrocytes or oligodendrocytes, based on presence of growth factors in the differentiation medium. In co-culture, physical cues provided by axons were essential for complete differentiation of both astrocytes and oligodendrocytes. This study presents a novel method of obtaining glia from human MLPCs that could eliminate many of the difficulties associated with their differentiation from embryonic stem cells. In addition, it reveals the complex interplay between physical cues and biomolecules on stem cell differentiation.
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Date Issued
2011
Identifier
CFE0003570, ucf:48894
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003570