Previous studies have transplanted a variety of neural stem cells (NSCs) to the eye in hopes of developing a therapy to replace retinal neurons lost to disease. Successful integration, survival, and differentiation of the cell types has been variably successful. At the moment, little is known about the fundamental biological differences between stem cell or progenitor cell types. Characterization of these differences will not only increase our general understanding of this broadly characterized group of cells, but also lead to development of criteria for sorting cells, evaluating their differentiation, and predicting their suitability for transplantation. We have used two-dimensional gel electrophoresis protein expression profiles to characterize the molecular differences between two populations of murine progenitor cells-retinal progenitor cells (RPCs) and brain progenitor cells (BPCs) isolated from mice of the same age and same genetic background. Our protein expression profiling identified 22 proteins that are differentially expressed in RPCs when compared to BPCs. Four of the differentially expressed proteins correspond to proteins known to be involved in a cellular response to stress, and analysis of potential transcription factor binding sites in the promoter regions of their genes suggests these proteins could be co-regulated at the transcriptional level. On the basis of this discovery, we tested the hypothesis that the addition of the antioxidant vitamin E would decrease the expression of the stress-response proteins and influence differentiation of RPCs. Further investigation of differences between multiple populations of RPCs and BPCs during their maintenance and differentiation will further identify fundamental differences that define 'retinal-like' characteristics and provide tools to assay the success of efforts to influence many populations of stem cells to adapt a retinal cell fate.119
Blinding degenerative retinal diseases including retinitis pigmentosa, macular degeneration and glaucoma are characterized by loss of retinal neurons. At this time there is no way to replace retinal cell loss due to disease or injury since differentiated retinal cells are unable to regenerate. As a potential approach for treating retinal injury, neural progenitor cells have been proposed as a unique source of transplantable cells to replace lost cells in the damaged retina. Previous studies have transplanted a variety of neural stem cells to the eye in hopes of developing a therapy to replace retinal neurons lost to disease. Successful integration, survival and differentiation of the cell types have been variably successful. At the moment little is known about the fundamental biological differences between stem cell or progenitor cell types. Characterization of these differences will not only increase our general understanding of this broadly characterized group of cells, but also lead to development of criteria for sorting cells, evaluating their differentiation and predicting their suitability for transplantation. In this dissertation we used protein expression profiling to characterize the molecular differences between two populations of in vitro expanded progenitor cells, retinal progenitor cells (RPCs) and brain progenitor cells (BPCs) isolated from mice of the same age and same genetic background. From this study we identified 4 stress-response proteins that were increased in expression in RPCs compared to BPCs. To see if these stress-response proteins were expressed during normal development, we used immunohistochemistry to characterize their expression in the developing retina. Finally, we tested the hypothesis that attenuation of oxidative stress would decrease the expression of stress-response proteins. We found that heat shock 60 (Hsp60), heat shock protein 70 (Hsp70), copper-zinc superoxide dismutase viii (Cu-Zn SOD) and catalase (CAT) are dynamically expressed in the developing retina. Further, we report that treatment of cultured progenitors with the antioxidant vitamin E (alpha-tocopherol) decreases expression of these proteins and alters their differentiation. These results are the first to characterize the expression of stress-response proteins during retinal development and demonstrate that reduction of oxidative load on cells can alter their differentiation profile.
Scientists around the world have wondered for many years what distinguishes speciation. Of particular interest is the genetic basis for human/primate (chimpanzee or gorilla) separation. Humans and chimpanzees are 99% identical in their genomic DNA sequence, thus making them very closely related. Despite this high degree of sequence similarity, humans and primates have a number of striking phenotypic differences. We hypothesize that sequence changes that have occurred between humans and primates have altered developmental programs. Because transcription factors alter the expression of numerous genes, we also hypothesize that changes in the expression or activity of transcription factors are responsible for the different phenotypic traits among humans and primates. Using human chromosome 22 as a model for comparison between human and primate DNA, a random selection of noncoding genes approximately 1-2 kilobases (kb) long upstream was sequenced. Focused on promoter regions from the sequence data, significant differences were detected when comparing humans and gorillas (p-value= < 0. 01) and gorillas and chimpanzees (p-value= < 0.01) suggesting that limited similarities existed between the species. When comparing humans and chimpanzees (p-value= > 0. 1), no significant difference was detected. Using this information, transcription factors were analyzed between the human and chimpanzee data to determine if transcription regulation was different between the species. The results indicated no significant difference between humans and chimpanzees at the single-nucleotide level even though the species differ at the genetic and phenotypic levels. The results also indicated that changes in transcription v regulation have played a major role in determining speciation. This research opens new avenues in investigating how many of the differences have functional consequences and the relative contributions of these transcription factors to expression differences. CHAPTER 1: GENERAL INTRODUCTION Thesis Organization Chapter 1, the introduction, covers the evolution of primates, DNA sequence-based comparisons, divergence between humans and primates, and gene expression. Chapter 2 explains how cloning generated large amounts of primate DNA and the conjoining of multiple sequences obtained from the sequencing facility. Chapter 3 gives results of sequence comparison of common genes found between humans and primates, statistical analysis, and the number of transcription factors generated. The final chapter, chapter 4 contains general discussion and conclusion.
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