The term “stem cell” is often broadly applied to a range of cell types that are relatively undifferentiated and have some capacity for proliferation. In this thesis, I employ a strict definition of stem cells as cells that are capable of both self-renewal and multilineage differentiation. These properties are tested in single precursor cells from the forebrain and its derivative, the retina, using clonal assays. Poor survival is a common problem in single cell cultures, and I show that low oxygen dramatically improves viability in neural stem cells clonally derived from mouse embryonic stem cells, as well as in cultured forebrain neural stem cells. Caspase-dependent and apoptosis-inducing factor-dependent cell death pathways were found to be differentially influenced in low oxygen culture of early, primitive and later, definitive neural stem cells. I isolate precursors from 2 separate regions of the adult mouse forebrain, the lateral ventricle and the hippocampus and argue that only cells resident in the lateral ventricle can be classified as stem cells while the hippocampus contains restricted progenitor cells. Unlike neural stem cells, the very existence of retinal precursors in the adult mammal is controversial. I investigate methods to prospectively identify a rare stem cell population in the pigmented ciliary epithelium of the adult mouse eye and show that, although this population intrinsically gives rise to all retinal cell types, cells can be directed specifically towards a photoreceptor fate by the addition of exogenous factors to the culture media. Pigmentation of retinal stem cells is used as a convenient marker to isolate a retinal stem cell from human embryonic stem cells differentiating under conditions known to promote neural differentiation. Retinal stem cells derived from human embryonic stem cells have highly similar properties to those directly isolated from the eye, and their progeny can similarly be driven to differentiate into photoreceptors. The findings presented in this thesis help to define intrinsic properties of adult neural and retinal precursors and provide a basis for manipulating these cells, potentially for future use in clinical applications.

Both cDNA tag-based and DNA chip hybridization assays have revealed widespread transcriptional activity across mammalian genomes, providing a rich source of novel protein-coding and non-coding transcripts. Annotation and functional evaluation of this undefined transcriptome space represents a major step towards the comprehensive definition of biomolecules regulating the properties of living cells, including embryonic stem cells (ESCs) and their derivatives. In this study I analysed 87 rare mRNA transcripts from human ESCs that mapped uniquely to the human genome, in regions lacking evidence for known genes or transcripts. In addition, the transcripts appeared enriched in the hESC transcriptome as enumerated by serial analysis of gene expression (SAGE). Full-length transcripts corresponding to twelve novel LongSAGE tags were recovered and evaluated with respect to gene structure, protein-coding potential, and gene regulatory features. In addition, transcript abundance was compared between RNA isolated from undifferentiated hESCs and differentiated cells. Analysis of full-length transcripts revealed that the novel ORFs did not exceed a size of129 amino acids and no matches were observed to well characterized protein domains. Interesting protein level predictions included small disulfide-bonded proteins, known members of which are important in a variety of biological processes. Transcripts evaluated for differential expression by real-time RT-qPCR (Reverse Transcription followed by real-time quantitative Polymerase Chain Reaction) were found to be variably expressed (0.2- to 4.5-fold) in Day-2 orDay-4 retinoic acid-induced differentiation cultures compared to undifferentiated hESCs. Relative quantitation using a universal reference RNA (derived from pooled adult tissues)showed large differences in novel transcript levels (0.002- to 35-fold) compared to hESCs. Collectively, these results provide a detailed analysis of a set of novel hESC transcripts and their abundance in early and adult differentiated cell types, both of which may advance our understanding of the transcriptional events governing stem cell behavior.

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

A complex network of transcription factors, which are regulated by signalling molecules, is responsible in coordinating the formation of differentiated skeletal and cardiac myocytes from undifferentiated stem cells. The present study aims to understand and compare the transcriptional regulation of skeletal and/or cardiac muscle development in the absence of Sox7 or in the presence of a collagen-based matrix in P19 embyonal carcinoma (EC) and mouse embryonic stem (ES) cells. First, knock-down of Sox7 , by shRNA, in muscle inducing conditions (+DMSO) and in the absence of RA (-RA), decreased muscle progenitor transcription factor and myogenic regulatory factor (MRF) levels, suggesting that Sox7 is necessary for myogenesis. However, knock-down of Sox7 in the presence of RA (+RA) and DMSO increased expression of muscle progenitor markers and MRFs, suggesting that Sox7 is inhibitory for myogenesis +RA. Furthermore, Sox7 overexpression enhanced myogenesis -RA, but inhibited myogenesis and enhanced neurogenesis +RA. These results suggest an important interplay between RA signalling and Sox7 function during P19 differentiation. Second, Q-PCR analysis showed that compared to the mouse ES cells differentiated on the regular TC plates, differentiation on the collagen matrices had a higher expression of skeletal and cardiac precursors, MRFs and terminal differentiation markers. Collagen alone enhanced myotube formation. The enhanced collagen matrix, containing the oligosaccharide sialyl LewisX (sLeX), specifically enhanced cardiomyogenesis. These studies have added to our understanding of the transcriptional regulation of premyogenic mesoderm factors and the role of Sox7 in this process. In addition these studies provide a vision for possible use of biomaterials in directed differentiation of stem cells for the purpose of cell therapy.

Stem cell research, especially the one dealing with human embryonic stem cells, is a major topic nowadays. In the last few years studies about human embryonic stem cell derived cardiomyocytes highlighted the importance of those, as their characteristics are almost identical as of the cardiomyocytes in the heart (i.e. the contraction of those cells). The studies concentrate on the ability of using cardiomyocytes in the drug development for cardiac diseases or in regenerative medicine and cell replacement therapies. In contrast some researchers concentrate on microRNAs (miRNAs) as regulators in the development of cardiomyocytes. This study combines both research topics as it deals with stem cells and miRNAs (as well as their target mRNAs). A main objective is to find differentially expressed genes by using Significance Analysis of Microarrays (SAM) as method. Furthermore miRNA target prediction is applied and the identified targets are compared with the ones found by SAM. With an intersection approach we derived 41 targets of up-regulated miRNAs and 25 targets of down-regulated miRNAs, which can be the basis for further studies (i.e. knock-out experiments).

The incidence and long-term effects of traumatic brain injury (TBI) make it a major healthcare and socioeconomic concern. Cell transplantation may be an alternative therapy option to target prolonged neurological deficits; however, safety and efficacy of the cells must be determined. Bone marrow-derived mesenchymal stem cells (MSCs) are an accessible and expandable cell source which circumvent the many of the accessibility and ethical concerns associated with fetal tissues. A major impediment to recent clinical trials for cell therapies in the central nervous system has been the lack of consistency in functional recovery where some patients receive great benefits while others experience little, if any, effect (Watts and Dunnett 2000; Lindvall and Bjorklund 2004). There are many possible explanations for this patient-to-patient variability including genetic and environmental factors, surgical techniques, and donor cell variability. Of these, the most easily addressable is to increase the reproducibility of donor cells by standardizing the isolation and pre-transplantation protocols, which is the central goal of this dissertation. First, we present an animal study in which transplants of MSCs and neural stem cells (NSCs) were given to brain-injured mice, however, the efficacy of the treatment had high variability between individual subjects. Second, we designed a method to produce MSC-spheres and characterize them in vitro. Last, we employed an in vitro 3-D culture testbed as a pre-transplant injury model to assess the effects of the MSC-spheres on neural cells. The electrophysiological function of the uninjured testbed was assessed, and then MSC-spheres were injected into the testbed and apoptosis of the host cells were measured. The results of this study contribute to our understanding of how extracellular context may influence MSC-spheres and develop MSCs as a donor cell source for transplantation.

The adult Drosophila central nervous system is produced by two phases of neurogenesis: the first phase occurs during embryonic development where the larval brain is formed and the second occurs during larval development to form the adult brain. Neurogenesis in both phases is caused by the activation of neural stem cell division and subsequent progenitor cell division and terminal differentiation. Proper activation of neural stem cell division in the larval brain is essential for proper patterning and functionality of the adult central nervous system. Initiation of neural stem cell proliferation requires signaling from the Fibroblast Growth Factor (FGF) homolog Branchless (Bnl) and by the Hedgehog (Hh) growth factor. I have focused on the interactions between both of these signaling pathways with respect to post-embryonic neural stem cell proliferation using the Drosophila larval brain. Using proliferation assays and quantitative real-time PCR, I have shown that Bnl and Hh signaling is inter-dependent in the 1st instar larval brain and activates neural stem cell proliferation. I have also shown that overexpression of bnl can rescue signaling and neuroblast proliferation in a hh mutant. However, overexpression of hh does not rescue signaling or neuroblast proliferation in a bnl mutant, suggesting that Bnl is the signaling output of the Bnl-Hh feedback loop and that all central brain and optic lobe neural stem cells require Bnl signaling to initiated proliferation.

This dissertation presents three separate studies designed to provide structure and evidence-based insight into the impact of religious beliefs on public perceptions and U.S. government policies regarding embryonic stem cell research. First, a systematic literature review of nine (n=9) empirical studies that examined individuals' religious beliefs and perceptions/utilization of genetic technologies/services will be presented. Based on the finding from the review, there was an equal balance between studies that found that religion was a factor positively affecting intention to submit to genetic testing and those that illustrated a negative association. Secondly, a qualitative examination of college students' from various racial/ethnic and religious backgrounds exploring the definition, interpretation, and conceptualization of the influence of religious beliefs on perceptions regarding embryonic stem cell research will be offered. Employing an emergent design, the data collection process encompassed thirty-seven in-depth interviews. The majority of participants in this study believed that ESCR should be conducted and federally funding in the United States, regardless of their religious beliefs. Lastly, the findings from the analysis of congressional records from the U.S. Congress for areas of convergence and divergence between discussions, voting, and legislation regarding stem cell research with the official stances of the major religious groups in the United States accessing the influence of religious rhetoric on political discourse regarding embryonic stem cell research will be discussed. Findings from this study suggest that religious rhetoric has a substantial influence on political rhetoric regarding ESCR.

Recent studies have discovered that LPS-treated Sprague-Dawley rats induced a reduction (phase I), followed by an augmentation (phase II), and decrease again (phase III) in the power density of the vasomotor component (0-0.8 Hz) in systemic arterial pressure (SAP). It was reported that the vasomotor components were related to the brain stem, even closely related to the rostral ventrolateral medulla (RVLM). But the molecular mechanism involved in the death progression of rat brain stem is mostly unknown. We used suppression subtractive hybridization (SSH) and library construction to find differentially expressed genes between phase I and phase II of LPS-treated RVLM. At present, we have found some genes that are differentially expressed between phase I and phase II of LPS-treated RVLM. Some genes are up-regulation expression and others are down-regulation expression. Thus, these genes may be involved in the molecular mechanism of the death progression in the rat brain stem.

The adult Drosophila central nervous system is produced by two phases of neurogenesis: the first phase occurs during embryonic development where the larval brain is formed and the second occurs during larval development to form the adult brain. Neurogenesis in both phases is caused by the activation of neural stem cell division and subsequent progenitor cell division and terminal differentiation. Proper activation of neural stem cell division in the larval brain is essential for proper patterning and functionality of the adult central nervous system. Initiation of neural stem cell proliferation requires signaling from the Fibroblast Growth Factor (FGF) homolog Branchless (Bnl) and by the Hedgehog (Hh) growth factor. I have focused on the interactions between both of these signaling pathways with respect to post-embryonic neural stem cell proliferation using the Drosophila larval brain. Using proliferation assays and quantitative real-time PCR, I have shown that Bnl and Hh signaling is inter-dependent in the 1st instar larval brain and activates neural stem cell proliferation. I have also shown that overexpression of bnl can rescue signaling and neuroblast proliferation in a hh mutant. However, overexpression of hh does not rescue signaling or neuroblast proliferation in a bnl mutant, suggesting that Bnl is the signaling output of the Bnl-Hh feedback loop and that all central brain and optic lobe neural stem cells require Bnl signaling to initiated proliferation.