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dc.contributor.authorCalhoun, John
dc.description.abstractFew scientific discoveries have generated as much interest and controversy as embryonic stem cells (ESCs). Their discovery has led to a new age in developmental biology and they hold promise as an unlimited supply of material for cell replacement therapy. To date there are no directed differentiation strategies for primate (monkey or human) ESCs that efficiently produce one specific cell type. Further, only recently have efforts been made to characterize the transcriptional profile and mechanism of pluripotency of these cells. For this reason the current study utilizes a technology defined in the mouse to characterize gene expression patterns during early differentiation and create a differentiation protocol for making neural cells from monkey ES cells. By combining microarray analysis and MEDII media, conditioned media from the growth of HepG2 cells, we have characterized gene expression patterns during early differentiation events in hESCs. Treatment of adherent hESCs with 50% MEDII media for 3 days effected differentiation to a cell type with gene expression similar to primitive streak stage cells of the mouse embryo. MEDII treatment up-regulates Cripto, a gene essential for proper anterior-posterior axis and mesoderm formation in mouse embryos. In addition, several genes previously shown to be important for proper development were down-regulated with MEDII treatment even though the pluripotency markers Oct-4, Nanog, and SSEA-4 were unchanged by the treatment. These include but are not limited to Follistatin, Lefty A, HOXA1, Dapper, and EZH2. Genes down-regulated in this manner are thought to play important roles in pluripotency maintenance of primate ESCs. The current study also utilizes MEDII media to develop an efficient neural differentiation protocol of rhesus monkey ESCs. Embryoid bodies (EBs) formed in the presence of 50% MEDII media exhibited high levels of neural differentiation in comparison to untreated controls. Further, a population of neural progenitors (NPs) was isolated from explants of treated EBs that could be maintained in culture in the undifferentiated state. Mitogen withdrawal led to terminal differentiation into neurons that possessed immunostaining and electrophysiological characteristics of functional neurons. The current study demonstrates MEDII’s utility in studying pluripotency and its ability to enrich differentiation to desired lineages.
dc.subjectEmbryonic stem cells
dc.subjectneural differentiation
dc.subjectmesoderm induction
dc.titleGene expression and neural differentiation studies of embryonic stem cells
dc.description.departmentBiochemistry and Molecular Biology
dc.description.majorBiochemistry and Molecular Biology
dc.description.advisorSteven L. Stice
dc.description.committeeSteven L. Stice
dc.description.committeeMichael Pierce
dc.description.committeeMary Bedell
dc.description.committeeAlan Przybyla

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