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Title: Establishment of Methods for Isolation of Pnmt+ Cardiac Progenitor Cells.
Creator: Varudkar, Namita, Ebert, Steven, Parthasarathy, Sampath, Muller, Mark, University of Central Florida
Abstract / Description: Cardiovascular disease is the leading cause of death in the United States. Millions of patients suffer each year from endothelial dysfunction and/or debilitating myocardial damage resulting in decreased quality of life and increased risk of death or disablement. Current pharmacological approaches are only partly effective at treating cardiovascular disease, and hence, better strategies are needed to provide significant improvements in treatment options. Cardiac stem/progenitorcells have the...
Show moreCardiovascular disease is the leading cause of death in the United States. Millions of patients suffer each year from endothelial dysfunction and/or debilitating myocardial damage resulting in decreased quality of life and increased risk of death or disablement. Current pharmacological approaches are only partly effective at treating cardiovascular disease, and hence, better strategies are needed to provide significant improvements in treatment options. Cardiac stem/progenitorcells have the potential to regenerate myocardial tissue and repair damaged heart muscle. There are many different types of cardiac progenitor cells, and each may have certain unique properties and characteristics that would likely be useful for particular clinical applications. A current challengein the field is to identify, isolate, and test specific cardiac stem/progenitor cell populations for their ability to repair/regenerate myocardial tissue. Our laboratory has discovered a new type of cardiac progenitor cell that expresses the enzyme, Phenylethanolamine-n-methyltransferase (Pnmt). My initial studies focused on identification of Pnmt+ cells based on knock-in of a nuclear-localized Enhanced Green Fluorescent Protein (nEGFP) reporter gene into exon 1 of the Pnmt gene in a stable recombinant Pnmt-nEGFP mouse embryonic stem cell (mESC) line. These cells were differentiated into cardiomyocytes, and I identified nEGFP+ cells using fluorescence, immunofluorescence, and phase-contrast microscopy techniques. Our results showed that only about 0.025% ( 1 per 4000) of the cardiac-differentiating stem cells expressed the nEGFP+ marker. Because of the relative rarity of these cells, optimization of isolation methods proved initially challenging. To overcome this technical barrier, I used a surrogate cell culture system to establish the methodsof isolation based on expression of either a fluorescent cell marker (EGFP), or a unique cell surface receptor represented by an inactivated (truncated) version of the human low-affinity nerve growth factor receptor (LNGFR). Plasmid DNA containing these reporter genes was transiently transfected into a permissive cell line (RS1), and reporter gene expression was used to identify and isolate transfected from non-transfected cells using either Fluorescence-Activated Cell Sorting(FACS) or Magnetic-Activated Cell Sorting (MACS) methods. The main objective of the study was to establish the isolation techniques based on the expression of reporter genes (EGFP and LNGFR) in RS1 cells. Following transfection, EGFP+ cells were successfully isolated via FACS as verified by flow cytometric and microscopic analyses, which showed that approximately 96% of the isolated cells were indeed EGFP+. Despite the relative purity of the isolated cell population, however, their viability in culture following FACS was substantially compromised ( 50% attrition). In contrast, MACS enabled efficient isolation of LNGFR+ cells, and the vast majority of these ( 90%) retained viability in culture following MACS. The LNGFR expression was verified using RT-PCR. Further, MACS methods enabled isolation of marked cells in about 5-7 mins, whereas it took 2-4 hours to using FACS to perform similar isolations from the same amount of starting material (10^6 cells). In addition, MACS is a more economical method in that it does not require the use of an expensive laser-based instrument to perform the sorting. These results suggest that MACS was a more efficient, gentle, and feasible technique than FACS for isolation of reporter-tagged mammalian cells. Consequently, future studies aimed at isolation of Pnmt+ cardiac progenitor cells will thus primarily focus on MACS methods.
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Date Issued: 2014
Identifier: CFE0005558, ucf:50287
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005558

Title: INVESTIGATING THE ROLE OF NEURONAL AGING IN FRAGILE X-ASSOCIATED TREMOR/ATAXIA SYNDROME.
Creator: Hencak, Katlin Marie, von Kalm, Laurence, Southwell, Amber, University of Central Florida
Abstract / Description: Fragile X-associated tremor/ataxia syndrome (FXTAS) is an X-linked late-onset neurodegenerative disorder caused by a noncoding trinucleotide repeat expansion in the FMR1 gene. This gene produces fragile x mental retardation protein (FMRP), an RNA binding protein whose targets are involved in brain development and synaptic plasticity. One of the proposed mechanisms of FXTAS pathogenesis is an RNA gain-of-function in which the repeat expansion causes toxic mRNA that sequesters important...
Show moreFragile X-associated tremor/ataxia syndrome (FXTAS) is an X-linked late-onset neurodegenerative disorder caused by a noncoding trinucleotide repeat expansion in the FMR1 gene. This gene produces fragile x mental retardation protein (FMRP), an RNA binding protein whose targets are involved in brain development and synaptic plasticity. One of the proposed mechanisms of FXTAS pathogenesis is an RNA gain-of-function in which the repeat expansion causes toxic mRNA that sequesters important proteins in the cell, interfering with their functions. Another suggested method of pathogenesis is through a mutant protein called FMRpolyG. This protein results from repeat-associated non-AUG (RAN) translation, in which the expanded repeats are translated where they otherwise would not be. This protein co-localizes with intranuclear inclusions and nuclear membrane proteins, causing disorganization of the nuclear lamina in FXTAS patient brain samples and neurons differentiated from FXTAS patient-derived induced pluripotent stem cells (iPSCs). iPSC technology involves reprogramming an adult somatic cell back to an embryonic-like state, allowing it to be differentiated into all cell types. A limit with iPSCs, though, is modeling late-onset disorders because the cells lose all age-related features during reprogramming. Progerin, a truncated form of the lamin A protein, has been used to age neurons differentiated from Parkinson Disease (PD) patient-derived iPSCs. Progerin-mediated aging was found to unmask PD-like phenotypes in those neurons, making it a promising technology for modeling late-onset disorders such as FXTAS. In this study, we investigated the link between the aging process and FXTAS pathogenesis in neurons differentiated from FXTAS patient-derived iPSCs with the use of progerin. Progerin transduction was successful in aging the FXTAS neurons. The presence of FMRpolyG was confirmed and an interaction with Lap2b was observed. In some neurons, there was also an observed interaction between FMRpolyG and progerin. Overall, this data suggests that there is an interaction between the mutant FMRpolyG protein and the nuclear membrane during aging, which may contribute to the cell death that causes neurodegeneration in FXTAS patients.
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Date Issued: 2019
Identifier: CFH2000554, ucf:45678
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH2000554

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

Title: Embryonic Stem Cell Derived Exosomes Enhance Cardiac Stem Cell Differentiation into Heart Cells.
Creator: Hammond, Jamillah, Singla, Dinender, Masternak, Michal, Davidson, Victor, University of Central Florida
Abstract / Description: Transplantation of embryonic stem (ES) cells into the ischemic and infarcted heart has proven to repopulate cardiac cell populations, attenuate structural cardiac remodeling, and rescue cardiac function. Unfortunately, the pluripotency of ES cells increases risk of teratoma formation in vivo. Exosomes, smaller in comparison to ES cells, are cell free carriers of miRNA, proteins, and lipids, and do not suggest risk of teratoma formation. Exosomes have been proposed to mediate and attenuate...
Show moreTransplantation of embryonic stem (ES) cells into the ischemic and infarcted heart has proven to repopulate cardiac cell populations, attenuate structural cardiac remodeling, and rescue cardiac function. Unfortunately, the pluripotency of ES cells increases risk of teratoma formation in vivo. Exosomes, smaller in comparison to ES cells, are cell free carriers of miRNA, proteins, and lipids, and do not suggest risk of teratoma formation. Exosomes have been proposed to mediate and attenuate regeneration following myocardial infarction (MI), however, the role of exosomes derived from ES cells (ES-Exo) in activating resident cardiac stem cells (CSCs) to undergo cardiac differentiation is not established. In the present study, Stem cell antigen 1 positive (Sca-1+ve) CSCs were isolated, incubated with exosomes, and evaluated for differentiation into the major heart cell types in vitro. Observations of in vitro cardiac differentiation were further established in an in vivo model of MI. Ligation of the coronary artery, or a sham surgery was performed in C57BL/6 mice 8-12 weeks of age. Mice were split among four study groups: sham, MI, MI + H9c2-Exo (a cell line control), (&) MI + ES-Exo. ES-Exo were transplanted via intramyocardial (IM) injection immediately following coronary artery ligation. At day 14 (D14), echocardiography was used to evaluate cardiac function. Differentiation into the major heart cells was determined by sarcomeric ?-actin (cardiomyocytes) and smooth muscle ?-actin (vascular smooth muscle cells) immunostaining. Hematoxylin and Eosin and Masson's Trichrome staining assessed cardiomyocyte hypertrophy and fibrosis, respectively. Immunostaining for major heart cellular markers revealed significant activation of resident Sca-1+ve CSCs to undergo cardiac differentiation after ES-Exo treatment. Cardiomyocyte hypertrophy and myocardial fibrosis were significantly increased following coronary artery ligation. Results from histological staining revealed significantly decreased levels of hypertrophy and fibrosis in hearts transplanted with ES-Exo following coronary ligation. In summary, our findings advocate ES-Exo as a viable treatment option to repopulate the myocardium with viable heart cells, attenuate cardiac remodeling, and rescue cardiac function.
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Date Issued: 2018
Identifier: CFE0007188, ucf:52254
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007188

Title: USER-DEFINED PATTERNING OF NEURAL PROGENITOR CELLS ON 3D MICROPILLAR ARRAYS USING ROUND CROSS-SECTIONAL GEOMETRY, SPECIFIC DIMENSIONS AND THIOL-BASED CHEMICAL ADHESION.
Creator: Wesser, Andrea, Cho, Hyoung Jin, University of Central Florida
Abstract / Description: The ability to control stem cell functions, particularly neuronal progenitors, has long since been believed to be the key to successful treatment of neurodegenerative disorders such as Alzheimer's, Parkinson's and accidents involving head trauma. The neurology field calls for many new solutions to address the controlled neural stem cell seeding and placement of cells for neural tissue regeneration. Self-assembled monolayers (SAM) from the alkanethiol group provide a straightforward...
Show moreThe ability to control stem cell functions, particularly neuronal progenitors, has long since been believed to be the key to successful treatment of neurodegenerative disorders such as Alzheimer's, Parkinson's and accidents involving head trauma. The neurology field calls for many new solutions to address the controlled neural stem cell seeding and placement of cells for neural tissue regeneration. Self-assembled monolayers (SAM) from the alkanethiol group provide a straightforward applicable, reliable treatment for cell adhesion. An ODT/gold treatment was used to adhere the cells to patterned areas, due mainly to a high confluence of cells attracted to it, as well as the viable environment it produced for the cells. Arrays of micropillars, made of SU-8 photoresist, then covered with a thin film of gold and treated with the ODT, created scaffolding allowing manipulation of neural stem cells. Based on multiple trials of observing varying cross-sectional geometric parameters, metal layer thicknesses and the ODT/Gold treatment, this study explores seeding density control, base and circumferential cell population dependence on those parameters.
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Date Issued: 2008
Identifier: CFE0002054, ucf:47563
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0002054

Title: STUDIES ON THE NOVEL FUNCTION OF AMYLOID PRECURSOR PROTEIN IN GLIAL DIFFERENTIATION OF NEURAL STEM CELLS.
Creator: Kwak, Young-Don, Sugaya, Kiminobu, University of Central Florida
Abstract / Description: Although amyloid beta (A beta) deposition has been a hallmark of Alzheimer's disease (AD), the physiological function of amyloid precursor protein (APP) is not clear. Our results suggested that high concentration of APP induces glial differentiation while physiological level of APP promotes migration and differentiation of neural stem cell (HNSC). HNSCs were mainly differentiated into astrocytes when they are transplanted into APP transgenic mouse brain or treated with a high...
Show moreAlthough amyloid beta (A beta) deposition has been a hallmark of Alzheimer's disease (AD), the physiological function of amyloid precursor protein (APP) is not clear. Our results suggested that high concentration of APP induces glial differentiation while physiological level of APP promotes migration and differentiation of neural stem cell (HNSC). HNSCs were mainly differentiated into astrocytes when they are transplanted into APP transgenic mouse brain or treated with a high concentration of secreted-type APP (sAPP) in culture. Staurosporine (STS) induced a distinctive astrocytic morphology in NT-2/D1 neural progenitor cells with expressions of APP and astrocyte-specific markers, glial fibrillary acidic protein (GFAP), aspartate transporter, and glutamate transporter-1. Expression of APP is correlated with GFAP expression in both mRNA and protein level in this experiment. Inhibition of APP expression by RNA interference (RNAi) or treatment with MEK1 inhibitor (PD098059), which reduces APP expression by suppressing ERK phosphorylation, abolished GFAP expression. These results indicate that STS induces glial differentiation of neuronal progenitor cells by increasing APP levels through activation of ERK pathway. We also found that APP-induced glial differentiation of neural progenitor NT-2/D1 cells is mediated by activation of IL-6/gp130 and notch signaling pathway. Treatment of APP activated IL-6/gp130 signal pathway via protein-protein interaction between APP and gp130 and it increased the gene expressions of CNTF, gp130 and JAK1, and phosphorylation of STAT3 while gene silencing of CNTF, JAK1 or STAT3 by RNAi, or treatment the cells with antibodies recognizing gp130 suppressed GFAP expression, indicating these molecules are crucial for APP-induced glial differentiation. Thus treatment of sAPP may promote glial differentiation of neural progenitor cells by activation of IL-6/gp130 signaling cascade. Treatment of sAPP increased the generation of notch intracellular domain as well as gene expression of Hes1 but did not change expression levels of notch or its ligands. We also found protein-protein interaction of APP and notch using immunoprecipitation suggesting that glial differentiation of NT-2/D1 cells is mediated by the physical interaction between APP and notch. N-terminal domain of APP (1-205 a.a.) alone can bind to notch and activate these signaling cascade in NT-2/D1 cells. Thus, APP may induce glial differentiation through activation of IL-6/gp130 and notch signal cascade by binding with its N-terminal domain. Taken together, our results suggest that APP regulates neural stem cell (NSC) differentiation through IL-6/gp130 and notch signaling pathway. Furthermore, the activation of both glial differentiation mechanisms may be necessary to potentiate APP-induced glial differentiation of NSC. Altered APP metabolism in Down syndrome and Alzheimer's disease may accelerate premature glial differentiation of NSCs, resulting in gliosis found in these diseases. Although it is not clear that how adult neurogenesis contributes to maintain normal brain function, destruction of neuroreplacement mechanism by NSCs may pose a problem. We may also have to consider effect of APP on the stem cell therapy for these diseases, since HNSCs may not properly differentiate into neurons under these pathological conditions.
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Date Issued: 2006
Identifier: CFE0001375, ucf:46980
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0001375

Title: THE ROLE OF ACTIVIN A SIGNALING IN GASTRIC REFLUX-RELATED DISEASES AND THE PROGRESSION TO ESOPHAGEAL ADENOCARCINOMA.
Creator: Roudebush, Cedric J., Andl, Claudia, University of Central Florida
Abstract / Description: Gastroesophageal reflux disease (GERD), or acid reflux, affects 6-9 million people in the United States. It is characterized by a reflux of gastric acid and bile salts from the stomach into the esophagus, causing injuries to the esophagus known as Barrett's esophagus (BE). BE is the main risk factor for the development of esophageal adenocarcinoma (EAC), a devastating cancer in the esophagus whose molecular roots remain poorly understood. In recent years, evidence points to the esophageal...
Show moreGastroesophageal reflux disease (GERD), or acid reflux, affects 6-9 million people in the United States. It is characterized by a reflux of gastric acid and bile salts from the stomach into the esophagus, causing injuries to the esophagus known as Barrett's esophagus (BE). BE is the main risk factor for the development of esophageal adenocarcinoma (EAC), a devastating cancer in the esophagus whose molecular roots remain poorly understood. In recent years, evidence points to the esophageal epithelium itself as responsible for causing and promoting inflammation upon injury by gastric reflux, namely via an increase in inflammatory cytokine secretion. This project was focused on a cytokine of interest, Activin A, which is known for its importance during embryogenesis and stem cell differentiation. It has recently been studied for its role in inflammation and tumor formation, but not in the case of esophageal diseases. Here, we demonstrate that Activin A signaling in esophageal epithelial cells is heavily upregulated shortly after exposure to bile salts and acid. We show evidence that this upregulation causes an increase in cell migration upon a reconstituted extracellular matrix. We also provide further evidence that bile and acid injury causes epithelial cells to secrete cytokines, which drive inflammation. We show that the upregulated Activin A secretion and signaling plays an important role in promoting this inflammatory state. Finally, we provide evidence that bile salts and acid exposure, as well as increased Activin A signaling, causes esophageal epithelial cells to upregulate stem cell and transdifferentiation markers, supporting the latest theories on the origin of Barrett' esophagus stem cells as well as paligenosis.
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Date Issued: 2019
Identifier: CFH2000485, ucf:45887
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH2000485

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

Title: Regulation of Extra-Pituitary Prolactin in Monocytes and Macrophages.
Creator: Barrett, Richard, Parthasarathy, Sampath, McKinstry, Karl, Masternak, Michal, Zhao, Jihe, University of Central Florida
Abstract / Description: Recently it has been shown that leukocytes are capable of producing prolactin (PRL). Evidence of extra-pituitary PRL (ePRL) production is so far been limited to primates and is not shared across other mammal species such as mice and rats. While ePRL is characterized as an identical protein to traditional pituitary PRL, it is controlled under an alternative promoter and is thus regulated differently from pituitary PRL. Little is known about what regulates ePRL or its direct role in human...
Show moreRecently it has been shown that leukocytes are capable of producing prolactin (PRL). Evidence of extra-pituitary PRL (ePRL) production is so far been limited to primates and is not shared across other mammal species such as mice and rats. While ePRL is characterized as an identical protein to traditional pituitary PRL, it is controlled under an alternative promoter and is thus regulated differently from pituitary PRL. Little is known about what regulates ePRL or its direct role in human physiology, but given that PRL has well over 300 described functions, it is likely that the autocrine and paracrine effects of this hormone could have far reaching implications in overall physiology. This work takes some of the first steps in understanding how leukocyte ePRL is regulated. Our results show that, adrenergic hormones are one key stimulus in ePRL expression in monocytes/macrophages. This is particularly intriguing considering the opposing role of these two signals in settings such as adipose tissue where adipose tissue macrophages are constantly exposed to pro-lipolytic adrenergic hormones that would in turn stimulate production of an anti-lipolytic hormone, PRL. Further, our work shows that the inflammatory phenotype of the leukocytes influences basal expression of PRL and overall ePRL expression increases significantly as monocytes differentiate into macrophages, as is a common occurrence in adipose tissue. The final portion of our work shows how monocytes/macrophages also respond to preadipocytes directly. These stem cell precursors to mature adipose cells release an unknown factor that stimulates ePRL production in monocytes/macrophages. Analysis of our gene array shows many of the genes stimulated by adipose stem cells alongside PRL are important genes in tissue regeneration and remodeling, a possible role that fits well with known effects of PRL. Understanding such primate specific interactions between the immune system and major metabolic tissues such as adipose fills vital gaps in knowledge that may explain why so many treatments fail when transitioning from mouse models to humans.
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Date Issued: 2018
Identifier: CFE0007309, ucf:52164
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007309

Title: TRANSPLANTATION OF IPS CELLS REDUCES APOPTOSIS AND FIBROSIS AND IMPROVES CARDIAC FUNCTION IN STREPTOZOTOCIN-INDUCED DIABETIC RATS.
Creator: Neel, Sarah, Singla, Dinender, University of Central Florida
Abstract / Description: Background: Streptozotocin (STZ) induced diabetes leads to various complications including cardiomyopathy. Recent data suggests transplanted bone marrow stem cells improve cardiac function in diabetic cardiomyopathy. However, whether modified ES, iPS cells, or factors released from these cells can inhibit apoptosis and fibrosis remains completely unknown. The present study was designed to determine the effects of transplanted ES cells overexpressing pancreatic transcription factor 1 a (Ptf1a)...
Show moreBackground: Streptozotocin (STZ) induced diabetes leads to various complications including cardiomyopathy. Recent data suggests transplanted bone marrow stem cells improve cardiac function in diabetic cardiomyopathy. However, whether modified ES, iPS cells, or factors released from these cells can inhibit apoptosis and fibrosis remains completely unknown. The present study was designed to determine the effects of transplanted ES cells overexpressing pancreatic transcription factor 1 a (Ptf1a), a pro-pancreatic endodermal transcription factor, iPS cells, or their respective conditioned media (CM) on diabetic cardiomyopathy. Methods: Experimental diabetes was induced in male Sprague Dawley rats (8-10 weeks old) by intraperitoneal STZ injections (65 mg/kg body weight for 2 consecutive days). Animals were divided into six experimental groups including control, treated with sodium citrate buffer IP, STZ, STZ + ES-Ptf1a cells, STZ + iPS cells, STZ + ES-Ptf1a CM and STZ + iPS CM. Following STZ injections, appropriate cells (1 X 106/mL/injection/day) or CM (2 mL injection/day) were given intravenously for 3 consecutive days. Animals were sacrificed and hearts were harvested at day 28. Histology, TUNEL staining, and Caspase-3 activity were used to assess apoptosis and fibrosis. ERK1/2 phosphorylation was quantified using ELISAs. M-mode echocardiography fractional shortening was used to assess cardiac function. Results: Animals transplanted with ES cells, iPS cells, or both CMs showed a significant (p<0.05) reduction in interstitial fibrosis, and apoptosis compared with STZ group. ERK expression was not significantly different compared with STZ. Echocardiography showed a significant (p<0.05) improvement in fractional shortening in cell and media transplanted groups compared with STZ. Conclusions: Our data suggest that ES cells, iPS cells, and/or CMs inhibit apoptosis, reduce fibrosis, and improve cardiac function in STZ-treated diabetic rats.
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Date Issued: 2010
Identifier: CFE0003512, ucf:48964
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0003512

Title: DEVELOPMENT OF THERMALLY PROCESSED NANOCOMPOSITES WITH CONTROLLED SURFACES.
Creator: Georgieva, Petya, Seal, Sudipta, University of Central Florida
Abstract / Description: The ever increasing need for technology development requires the integration of inexpensive, light weight and high strength materials which are able to meet the high standards and specifications for various engineering applications. The intention of this work is to show that the suitable material selection and the utilization of plasma spray processing can be of potential interest to a large number of industrial, biomedical and everyday life applications. This research demonstrates also that...
Show moreThe ever increasing need for technology development requires the integration of inexpensive, light weight and high strength materials which are able to meet the high standards and specifications for various engineering applications. The intention of this work is to show that the suitable material selection and the utilization of plasma spray processing can be of potential interest to a large number of industrial, biomedical and everyday life applications. This research demonstrates also that plasma processing is a promising engineering tool for multifunctional coatings and near-net-shape manufacturing. Further, the theoretical and experimental results are combined in order to explain the mechanisms behind nanostructure retention and enhanced properties. Proper design of experiments, an appropriate material selection and experimental methodology are discussed herein. The experimental conditions were optimized in order to achieve the best materials properties according to their explicit properties and functions. Specific materials were consolidated according to their prospective performance and applications: 1) Plasma spraying of nano-Ceria-stabilized Zirconia free form part for stem cells scaffolds, 2) Plasma spraying of FeCrAlY on Ti-alloy plate, additionally coated with nano-size Hydroxyapatite for bone tissue engineering, 3) Wire-arc spraying of nano-based steel wires for aerospace and automotive applications. The performance and characteristics of all of the developed coatings and free-form-parts are evaluated using state-of-the art characterization techniques.
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Date Issued: 2006
Identifier: CFE0001153, ucf:46871
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0001153

Title: MELATONIN AND NEUROGENESIS: A COMPARATIVE STUDY OF THE EFFICACY OF MELATONIN, ITS PRECURSORS, AND L-DOPA ON NEURAL STEM CELL METABOLISM IN HUMAN ADULT NEUROSPHERES.
Creator: Heriba, Omar, Sugaya, Kiminobu, University of Central Florida
Abstract / Description: Human neurosphere stem cells offer promising potential for the treatment of neurodegenerative diseases. Their well characterized multi-potency of differentiating into neurons, astrocytes, and oligodendrocytes when exposed to the optimum exogenous growth factors make them an exciting area of study (38). Finding novel endogenous methods of modulating stem cell metabolism will allow for the safer treatment of various brain disorders (34). In this experiment, melatonin, N-acetylserotonin, L...
Show moreHuman neurosphere stem cells offer promising potential for the treatment of neurodegenerative diseases. Their well characterized multi-potency of differentiating into neurons, astrocytes, and oligodendrocytes when exposed to the optimum exogenous growth factors make them an exciting area of study (38). Finding novel endogenous methods of modulating stem cell metabolism will allow for the safer treatment of various brain disorders (34). In this experiment, melatonin, N-acetylserotonin, L-tryptophan, and L-DOPA are added in three different concentrations to neurospheres suspended in HNSC/GBM media with less than optimal concentrations of exogenous epidermal growth factor (EGF) and fibroblast growth factor (FGF). The alamarBlue assay (resazurin) was chosen as the most suitable assay for measuring neurosphere metabolism. Metabolic neural stem cells would cause the greatest reduction of the oxidized alamarBlue reagent (resazurin-resorufin), which was detected by a fluorescent plate reader (39-41). The percent reduction in alamarBlue was calculated for all four molecules at three different concentrations and compared to controls without any molecule. Our results illustrate that there was no statistically significant difference at p<0.05 between the biological molecules and the control group except for two exceptions (labeled with asterisks on figures 3 and 5) L-DOPA at a 40 micromolar concentration after 4 hours of incubation and melatonin at a 40 micromolar concentration after 52 hours of incubation.
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Date Issued: 2014
Identifier: CFH0004688, ucf:45239
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004688

Title: TIMP-1 ACTIVATES A UNIQUE CARDIAC STEM CELL POPULATION, CD63+ve/C-KIT+ve, THEREBY ENHANCING CARDIAC DIFFERENTIATION, AND PROTECTS THE HEART FROM ADVERSE CARDIAC REMODELING FOLLOWING MYOCARDIAL INFARCTION.
Creator: Abdelli, Latifa, Singla, Dinender, Cheng, Zixi, Parthasarathy, Sampath, Jewett, Mollie, University of Central Florida
Abstract / Description: We previously demonstrated that embryonic stem (ES) cells over-expressing tissue inhibitor of metalloproteinase-1 (TIMP-1) have increased potential to engraft and differentiate into cardiac myocytes following transplantation into the infarcted heart. However, the ability of TIMP-1 to activate endogenous stem cells and enhance their differentiation into cardiac regenerative cell types is still unknown. We postulate that TIMP-1 may additionally activate a stem cell population that enhances...
Show moreWe previously demonstrated that embryonic stem (ES) cells over-expressing tissue inhibitor of metalloproteinase-1 (TIMP-1) have increased potential to engraft and differentiate into cardiac myocytes following transplantation into the infarcted heart. However, the ability of TIMP-1 to activate endogenous stem cells and enhance their differentiation into cardiac regenerative cell types is still unknown. We postulate that TIMP-1 may additionally activate a stem cell population that enhances cardiac cell type differentiation in the infarcted myocardium. To prove this hypothesis, we isolated c-kit+ve cells from four weeks old C57BL/6 mice and cultured them in vitro in presence of ES conditioned media (ESCM), ES-TIMP-1-CM or TIMP-1. Our immunostaining data validate the existence of a novel CD63+ve/c-kit+ve cells. When treated with TIMP-1, these cells showed significantly (p(<)0.05) increased proliferation and differentiation into cardiac myocytes, vascular smooth muscle cells, and endothelial cells. Western blot analysis revealed significantly (p(<)0.05) increased expression of CD63, phosphorylated and total ?-catenin proteins. Furthermore, our RT-PCR data showed increased cardiac gene expression (GATA-4, Mef2C, and Nkx-2.5) when compared to ESCM and control cells. Based on the in vitro findings, we investigated the effect of intramyocardial delivery of TIMP-1 on endogenous CD63+ve/c-kit+ve cells following myocardial infarction (MI). C57BL/6 and TIMP-1 KO mice underwent coronary artery ligation followed by intramyocardial delivery of 20(&)#181;l of culture media (CC), ESCM, ES-TIMP-1-CM or TIMP-1. Subsequent immunohistochemistry analysis demonstrated the presence of a CD63+ve/c-kit+ve cell population within the peri-infarct area and confirmed intramyocardial delivery of ES-TIMP-1-CM or TIMP-1 significantly (p(<)0.05) enhanced their proliferation. Percentage of CD63+ve/c-kit+ve cells was significantly (p(<)0.05) lower in TIMP-1 KO mice compared to C57BL/6 animals. RT-PCR analysis revealed TIMP-1 KO animals expressed significantly less CD63 and TIMP-1 mRNAs compared to C57BL/6 mice. Activated CD63+ve/c-kit+ve cells were also able to differentiate into major cardiac cell types as previously shown in vitro. The differentiation potential of these cells was however higher in C57BL/6 mice compared to TIMP-1 KO mice. We also demonstrate that CD63+ve/c-kit+ve cells differentiation is regulated by CD63/?-catenin pathway in vivo. Additionally, we provide evidence that TIMP-1 protects the heart from adverse cardiac remodeling through inhibition of cardiac apoptosis and fibrosis leading to significantly (p(<)0.05) improved contractile function. Collectively, our data show TIMP-1 plays a dual protective role in the MI heart. It activates a unique stem cell population, CD63+ve/c-kit+ve, which proliferates and differentiates into functional myocytes, smooth muscle cells and endothelial cells mediated through CD63/?-catenin pathway. TIMP-1 also protects the heart from adverse cardiac remodeling. Increased cardiac regeneration and inhibition of adverse cardiac remodeling consequently lead to restored cardiac function. ?
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Date Issued: 2015
Identifier: CFE0005750, ucf:50108
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005750

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

Title: Investigating changes in quiescence in oral and esophageal epithelium in response to injury.
Creator: Rothaus, Alexandra, Andl, Claudia, Chakrabarti, Ratna, Singla, Dinender, University of Central Florida
Abstract / Description: More than 570,000 new cases of esophageal cancer are estimated to be diagnosed annually worldwide. Risk factors include gender, age, tobacco use and dietary habits leading to tissue injury and ultimately cancer. While prognoses for other cancers have improved, the 5-year survival for patients with esophageal cancer is only 20%. During the repair process, cell proliferation is increased and is associated with inflammation. Slow-cycling lifetime residential stem cells, called quiescent cells,...
Show moreMore than 570,000 new cases of esophageal cancer are estimated to be diagnosed annually worldwide. Risk factors include gender, age, tobacco use and dietary habits leading to tissue injury and ultimately cancer. While prognoses for other cancers have improved, the 5-year survival for patients with esophageal cancer is only 20%. During the repair process, cell proliferation is increased and is associated with inflammation. Slow-cycling lifetime residential stem cells, called quiescent cells, facilitate repair but are thought to accumulate mutations during DNA replication eventually giving rise to cancer. We hypothesize that esophageal stem cells become activated upon injury and are regulated by Transforming Growth Factor beta 1 (TGF?1), a known regulator of cell proliferation and differentiation. We established an in vitro model of quiescence using normal esophageal epithelial (STR) and oral (OKF6) cells treated with recombinant human TGF?1. Flow cytometry showed increases in cells arrested in G1/G0 phase of the cell cycle in TGF?1 treated cells for both cell lines (STR p(<)0.01, OKF6 p(<)0.05). EdU (5-ethynyl-2'-deoxyuridine) positive recovery cells indicated quiescence in both cell lines (p(<)0.01). Analysis of TGF?1 regulation of putative stem cell markers via western blot and qRT-PCR showed increases in ITGB1, PDPN and K15 as well as XPC, and MeCP2 in treated cells. To apply our in vitro findings, we performed immunohistochemistry staining on tissue microarrays. Proliferation marker Ki67 increased in disease progression from normal to inflammation to hyperplasia (p(<)0.001) while TGF?1 target markers decrease. Our data indicate that the onset of cancer-associated inflammation correlates with the loss of TGF?1 mediated stemness markers and increased basal proliferation suggesting cancer is a stem cell disease.
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Date Issued: 2019
Identifier: CFE0007903, ucf:52754
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007903

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

stem cells (3)	+ -
differentiation (2)	+ -
3D environment (1)	+ -
?-catenin (1)	+ -
Activin A (1)	+ -

Alzheimer's disease (1)	+ -
Amyloid precursor protein (1)	+ -
BE Stem Cells (1)	+ -
Barrett's Esophagus (1)	+ -
CD63 (1)	+ -
Cardiac Progenitor Cells (1)	+ -
Cardiac Stem Cells (1)	+ -
Cardiotoxicity (1)	+ -
DETA (1)	+ -
Doxorubicin (1)	+ -
Embryonic Stem Cell (1)	+ -
Exosomes (1)	+ -
GERD (1)	+ -
Glial differentiation (1)	+ -
Inflammation (1)	+ -
L-DOPA (1)	+ -
Muscle Toxicity (1)	+ -
Myocardial Infarction (1)	+ -
Myocardial Infarction Cardiac Regeneration Stem Cell Exosomes (1)	+ -
Neural stem cells (1)	+ -
Phenylethanoamine-N-methyltransferase (1)	+ -
Pnmt (1)	+ -
Pyroptosis (1)	+ -
Stem Cells (1)	+ -
TIMP-1 (1)	+ -

ETD Collection (13)	+ -
Honors Collection (3)	+ -

University of Central Florida (16)	+ -
Singla, Dinender (5)	+ -
Masternak, Michal (3)	+ -
Parthasarathy, Sampath (3)	+ -
Sugaya, Kiminobu (3)	+ -

Andl, Claudia (2)	+ -
Cheng, Zixi (2)	+ -
Ebert, Steven (2)	+ -
Abdelli, Latifa (1)	+ -
Alvarez, Angel (1)	+ -
Barrett, Richard (1)	+ -
Chakrabarti, Ratna (1)	+ -
Cho, Hyoung Jin (1)	+ -
Daniell, Henry (1)	+ -
Davidson, Victor (1)	+ -
Davis, Hedvika (1)	+ -
Georgieva, Petya (1)	+ -
Hammond, Jamillah (1)	+ -
Hencak, Katlin Marie (1)	+ -
Heriba, Omar (1)	+ -
Hickman, James (1)	+ -
Jewett, Mollie (1)	+ -
Khaled, Annette (1)	+ -
Kwak, Young-Don (1)	+ -
McKinstry, Karl (1)	+ -
Muller, Mark (1)	+ -
Neel, Sarah (1)	+ -
Rothaus, Alexandra (1)	+ -
Roudebush, Cedric J. (1)	+ -
Seal, Sudipta (1)	+ -

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