Camgaroo-2 as an indicator of function in embryonic and neuralized stem cells

Abstract only availableThe transplantation of stem cells to replace cells that have been lost or damaged due to disease or injury is quickly becoming a conceivable treatment method. Embryonic stem (ES) cells have the capacity to become any cell in the body, so the therapeutic possibilities are vast. The ultimate goal of our research on ES cells is to induce them to differentiate into functioning neurons to replace those that are lost in patients suffering from neurodegenerative disorders. However, it is important that the differentiated cells possess the appropriate phenotype and are able to perform the correct function after transplantation. In the past, it was common to accept a differentiated cell's fate based solely on its morphology and the presence of specific membrane markers. Now, it is becoming increasingly important to determine a donor stem cell's fate based on its function, especially if the cell is to be transplanted into a subject as a means of therapy. This study used the calcium-sensitive protein Camgaroo-2 to test the function of embryonic stem cells and cells directed toward a neural lineage. Camgaroo-2 is a fusion protein that consists of calmodulin in between two halves of yellow fluorescent protein. When calcium is present, it binds to the calmodulin portion of the Camgaroo-2, inducing a conformational change that results in increased fluorescence. After mouse embryonic stem cells were transfected with Camgaroo-2, we used reagents such as potassium chloride and ionomycin, known to elevate intracellular calcium, to confirm that the ES cells were stably transfected with the plasmid, and that Camgaroo-2 was functioning correctly. Potassium chloride causes the cell to depolarize while ionomycin (a calcium ionophore) creates large pores in the cell membrane. Both reagents allow for an influx of calcium into the cell, leading to increased fluorescence. The Camgaroo-2 transfected ES cells showed the appropriate responses to KCl and ionomycin by depolarizing and showing visible increases in fluorescence. This confirms that our Camgaroo-2 construct is functioning in the ES cells. We are in the process of testing the responses of neuralized ES cells using appropriate neurotransmitters, the presence of which should induce unique fluorescent signatures in a cell specific manner. Confirming neuronal function from differentiated Camgaroo-2 ES cells is an important step toward neuron transplantation in a neurodegenerative disease model.Arts & Sciences Undergraduate Research Mentorship Progra

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

Potential Role for Programmed Cell Death in the Formation of an In Vitro Neural Stem Cell Niche [abstract]

Abstract only availableFaculty Mentor: Dr. Mark Kirk, Biological SciencesStem cell therapies have the potential to treat neurodegenerative diseases, such as Batten Disease. Batten disease, a rare inherited disease in children, causes severe neurodegeneratoin, which results in blindness, seizures, and eventual death. In Batten disease, the transplantation of stem cells into a patient may replace lost cells or to prevent cell loss due to the disease. In one form of Batten Disease, transplantation of stem cells into the retinas of mutant model mice have shown signs of neuroprotection; including enhanced survival of photoreceptors (Meyer et al. 2006). One possible method to increase the efficiency of this treatment is the transplantation of a functional unit capable of producing its own neural precursors "on demand". Such a structure, known as a neural stem cell (NSC) niche, can be found in two small areas in the brain of mammals, and is the center for adult neurogenesis throughout the lives of these mammals. In our lab, we have developed a way to produce a NSC niche in vitro from neuralized mouse embryonic stem cells. To test how this structure is formed and maintained, I am investigating cell death within this in vitro NSC niche-like structure. I performed two different tests for apoptosis, or programmed cell death, Trypan Blue exclusion and TUNEL. Trypan Blue shows membrane permeability; if cells turn blue it is indicative of a late stage in the apoptotic process. In the TUNEL assay, nicked ends of DNA are labeled, an indication of early stage apoptosis. I also tested the affects of induced cell death on the advancement of in vitro niche formation. We hope that this information will lead to a better understanding of how the in vitro niche forms.College of Arts and Science Undergraduate Research Mentorship Progra

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

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

Implementation of a Novel Social-Emotional Learning Program to Advance Integration of Wellness in Education Practice

Social-emotional learning (SEL) programs aim to enhance emotional intelligence by teaching problem solving, self-awareness, self-management, social awareness, and relationship building skills. SEL interventions have been shown to improve quality of life and wellbeing, increasingly important outcomes in the wake of the staggering effects of the COVID-19 crisis on mental health. HappiGenius is a novel SEL program with the addition of mindful attention and self-compassion. We hypothesized HappiGenius would improve positive emotions, self-compassion, attention, mindful self-awareness, and social skills in a group of students. This observational cohort study took place at a diverse elementary school in a midsize midwestern city and included 48 students across four 3rd grade classrooms. HappiGenius included 12 lessons, approximately 45 minutes each, delivered twice a week for 6 weeks. The results demonstrated increased frequency of positive emotions (Positive Affect Scale for Children, median increase from 3.57 to 4.04, p=0.04) and improved self-compassion (Self-Compassion Scale for Children, 3.04 to 3.25, p=0.0094). Changes in student reported mindful self-awareness (Mindful Attentive Awareness Scale for Children, 3.27 to 3.47, p=0.56) and teacher ratings of hyperactivity/ inattention (Strengths and Difficulties Questionnaire, 3.0 to 2.5, p=0.26) were not statistically significant. Strong positive changes were observed in other teacher-reported behavioral outcomes on the Strengths and Difficulties Questionnaire, including total difficulties (6.0 to 3.5, p=0.0004) and prosocial skills (9.0 to 10.0,

Predictors of indoor absolute humidity and estimated effects on influenza virus survival in grade schools

Background: Low absolute humidity (AH) has been associated with increased influenza virus survival and transmissibility and the onset of seasonal influenza outbreaks. Humidification of indoor environments may mitigate viral transmission and may be an important control strategy, particularly in schools where viral transmission is common and contributes to the spread of influenza in communities. However, the variability and predictors of AH in the indoor school environment and the feasibility of classroom humidification to levels that could decrease viral survival have not been studied. Methods: Automated sensors were used to measure temperature, humidity and CO2 levels in two Minnesota grade schools without central humidification during two successive winters. Outdoor AH measurements were derived from the North American Land Data Assimilation System. Variability in indoor AH within classrooms, between classrooms in the same school, and between schools was assessed using concordance correlation coefficients (CCC). Predictors of indoor AH were examined using time-series Auto-Regressive Conditional Heteroskedasticity models. Classroom humidifiers were used when school was not in session to assess the feasibility of increasing indoor AH to levels associated with decreased influenza virus survival, as projected from previously published animal experiments. Results: AH varied little within classrooms (CCC >0.90) but was more variable between classrooms in the same school (CCC 0.81 for School 1, 0.88 for School 2) and between schools (CCC 0.81). Indoor AH varied widely during the winter (range 2.60 to 10.34 millibars [mb]) and was strongly associated with changes in outdoor AH (p < 0.001). Changes in indoor AH on school weekdays were strongly associated with CO2 levels (p < 0.001). Over 4 hours, classroom humidifiers increased indoor AH by 4 mb, an increase sufficient to decrease projected 1-hour virus survival by an absolute value of 30% during winter months. Conclusions: During winter, indoor AH in non-humidified grade schools varies substantially and often to levels that are very low. Indoor results are predicted by outdoor AH over a season and CO2 levels (which likely reflects human activity) during individual school days. Classroom humidification may be a feasible approach to increase indoor AH to levels that may decrease influenza virus survival and transmission

Developmental cues and persistent neurogenic potential within an in vitro neural niche

<p>Abstract</p> <p>Background</p> <p>Neurogenesis, the production of neural cell-types from neural stem cells (NSCs), occurs during development as well as within select regions of the adult brain. NSCs in the adult subependymal zone (SEZ) exist in a well-categorized niche microenvironment established by surrounding cells and their molecular products. The components of this niche maintain the NSCs and their definitive properties, including the ability to self-renew and multipotency (neuronal and glial differentiation).</p> <p>Results</p> <p>We describe a model <it>in vitro </it>NSC niche, derived from embryonic stem cells, that produces many of the cells and products of the developing subventricular zone (SVZ) and adult SEZ NSC niche. We demonstrate a possible role for apoptosis and for components of the extracellular matrix in the maintenance of the NSC population within our niche cultures. We characterize expression of genes relevant to NSC self-renewal and the process of neurogenesis and compare these findings to gene expression produced by an established neural-induction protocol employing retinoic acid.</p> <p>Conclusions</p> <p>The <it>in vitro </it>NSC niche shows an identity that is distinct from the neurally induced embryonic cells that were used to derive it. Molecular and cellular components found in our <it>in vitro </it>NSC niche include NSCs, neural progeny, and ECM components and their receptors. Establishment of the <it>in vitro </it>NSC niche occurs in conjunction with apoptosis. Applications of this culture system range from studies of signaling events fundamental to niche formation and maintenance as well as development of unique NSC transplant platforms to treat disease or injury.</p

Exosomal microRNAs in breast cancer: towards theranostic applications

Breast cancer is one of the top two reproductive cancers responsible for high rates of morbidity and mortality among women globally. Despite the advancements in the treatment of breast cancer, its early diagnosis remains a challenge. Recent evidence indicates that despite the adroit use of numerous strategies to facilitate rapid and precision-oriented screening of breast cancer at the community level through the use of mammograms, Fine-needle aspiration cytology (FNAC) and biomarker tracking, no strategy has been unequivocally accepted as a gold standard for facilitating rapid screening for disease. This necessitates the need to identify novel strategies for the detection and triage of breast cancer lesions at higher rates of specificity, and sensitivity, whilst taking into account the epidemiologic and social-demographic features of the patients. Recent shreds of evidence indicate that exosomes could be a robust source of biomaterial for the rapid screening of breast cancer due to their high stability and their presence in body fluids. Increasing evidence indicates that the Exosomal microRNAs- play a significant role in modifying the tumour microenvironment of breast cancers, thereby potentially aiding in the proliferation, invasion and metastasis of breast cancer. In this review, we summarize the role of ExomiRs in the tumour microenvironment in breast cancer. These ExomiRs can also be used as candidate biomarkers for facilitating rapid screening and triaging of breast cancer patients for clinical intervention

Characterization of an in vitro neural stem cell niche with educational component : stem cells and society

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Title from title screen of research.pdf file (viewed on August 3, 2009)Vita.Thesis (Ph. D.) University of Missouri-Columbia 2008.[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] We describe a novel neural stem cell (NSC) culture system, derived from embryonic stem (ES) cells, that maintains and expands a population of neural stem cells similar to those in the developing and adult brain. The identity of cellular and molecular components within the cultures was documented using quantitative RT-PCR, immunocytochemistry, neurosphere forming assays, and FLOW cytometry. Furthermore we compare the in vitro neural stem cell niche to neurogenic tissue of the adult brain and to other neural induction protocols and explore the use of 3-D physical scaffolding to better mimic the physical growth environment of living tissue. We show that embryonic stem cells can be induced to form structures that exhibit key properties of a developing neural stem cell niche. We believe this system can serve as a usefull model for studies of neurogenesis and stem cell maintenance in the NSC niche as well as for applications in stem cell transplantation.Includes bibliographical reference