Browsing by Subject "Oct-4"
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Item Oct-4 expression in equine embryonic cells(Texas A&M University, 2007-04-25) Harding, Heather DarbyThe Oct-4 transcription factor is believed to co-regulate early embryonic development of mammals due to the correlation of its presence with the maintenance of pluripotency. It is commonly used as a marker for the identification of embryonic stem (ES) cells for this reason. Until 1999, Oct-4 studies were limited to in vivo-produced embryos; equine embryos have not been studied for their Oct-4 expression patterns. In addition, equine stem-like cells (defined by marker expression, induced differentiation, passage survival, and morphology) have recently been isolated from in vivo-produced embryos, but no work has been performed in horses to isolate ES cells from in vitroproduced embryos. This study investigated the expression of Oct-4 transcription factor using immunocytochemistry in 42 in vitro-produced embryos aged 1-10 days and in 5 in vivoproduced blastocysts aged 7-10 days. Effective conditions for rapid establishment of a feeder layer of equine fetal fibroblasts were established, and this feeder layer was used to grow isolated equine inner cell mass (ICM) cells from in vitro-produced embryos. The expression of Oct-4 was examined in resultant cell growths. In vitro-produced embryos less than 6 days of age showed variable staining within blastomeres of the same embryo, and the peak of variability correlated with maternal-zygotic transition. After Oct-4 staining of in vitro-produced blastocysts, no cells could be identified as an ICM based on a difference in fluorescent intensity from the other cells of the blasyocysts. However, in vitro-produced blastocysts that were subsequently cultured in vivo contained a presumptive ICM, visible based on greater fluorescent intensity of Oct-4 stain. The trophoblast of all blastocysts also stained positively for Oct-4 protein. Fibroblasts were successfully isolated from equine feti. Treatment with 20 ????g/ml of Mitomycin C arrested cell growth without causing excessive death. Fibroblasts were inactivated and frozen, then thawed as needed to establish a confluent monolayer for ICM isolation overnight. ICMs from in vitro-produced embryos formed outgrowths, but none that could be identified morphologically as ES cells. Outgrowth cells contained about 20% Oct-4 expressing cells in sporadic groupings. Assuming appropriate binding of the Oct-4 antibody, Oct-4 expressing cells (potentially indicating pluripotency) are found throughout the embryo in early development and in the feeder layer after co-culture.Item Signaling pathways regulating self-renewal, differentiation, and multipotency of CD133+ umbilical cord blood stem cells(2008-12-17) Margaret Corbett Howe; Larry Denner; Ronald Tilton; Randall Urban; Ping Wu; Mark Evers; Chunming Liu; Austin CooneyOur goal is to increase the number of immature umbilical cord blood stem cells (UCBSCs) for hematopoietic transplantation. Towards this goal, our lab adapted a culture technique to grow immature CD133+ UCBSCs (CD133+ cells). Although CD133+ cells proliferate rapidly in culture, a minority self-renew and remain CD133+, while a majority differentiate and become CD133-. Therefore, new strategies to identify and grow immature UCBSCs are important. Since little is known about signally mechanisms regulating self-renewal and differentiation of UCBSCs, we sought insight from embryonic stem cell (ESC) literature to guide our studies. \r\nTo identify a population of UCBSCs that grow without differentiating, we focused on Oct-4, a transcription factor essential for self-renewal in ESCs that we previously reported expression in UCBSCs. During our studies, new challenges in the field arose. Two isomers of Oct-4 were discovered, Oct-4A and Oct-4B 3, in which only Oct-4A conferred the ability of ESCs to self-renew 4. We redesigned our experiments to detect Oct-4A and discovered that freshly isolated CD133+ cells expressed Oct-4A mRNA and protein. Since these cells proliferated in culture, they lost expression of adult stem cell markers including CD133, and gained markers of hematopoietic differentiation. However, Oct-4A mRNA and protein were expressed regardless of the differentiation status. Therefore, Oct-4A, despite its essential roles in ESCs, neither defined nor conferred self-renewal of CD133+ cells.\r\nTo discover strategies to grow immature CD133+ cells without differentiation, we focused on the Wnt pathway which is essential for self-renewal in ESCs. Differentiation of CD133+ cells to CD133- cells corresponded to down-regulation of Wnt signaling. Pharmacological activation of the Wnt pathway by (2’Z,3’E)-6-Bromoindirubin-3’-oxime (BIO) inhibition of GSK-3beta resulted in accelerated differentiation, instead of decreased differentiation, of CD133+ cells. BIO-treated CD133- cells that were differentiated maintained multipotency while proliferating at similar rates to vehicle-treated CD133+ cells that self-renewed. Therefore, inhibition of GSK-3beta could be a strategy for differentiating CD133+ cells into hematopoietic progenitor cells while maintaining their proliferation capacity. \r\nIn conclusion, this project demonstrated that pathways regulating UCBSC properties are not similar to pathways regulating ESCs properties. Our findings are the first studies that derive UCBSCs properties of self-renewal, differentiation, and multipotency.\r\n