Browsing by Author "Winger, Quinton, advisor"
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Item Open AccessGenomic and non-genomic androgen signaling in human placenta cells(Colorado State University. Libraries, 2018) McWhorter, Erin Soisson, author; Bouma, Gerrit, advisor; Winger, Quinton, advisor; Kendall, Lonnie, committee member; Engle, Terry, committee memberTo view the abstract, please see the full text of the document. Item EmbargoModeling human trophoblast development during the peri-implantation period using extended embryo culture(Colorado State University. Libraries, 2023) Logsdon, Deirdre Maria, author; Winger, Quinton, advisor; Krisher, Rebecca, advisor; Yuan, Ye, committee member; Tesfaye, Dawit, committee member; DeLuca, Jennifer, committee memberDuring the peri-implantation period, a human embryo must transition from a pre-implantation stage blastocyst to a gastrulating embryonic disc surrounding by the primitive placenta. The primitive placenta at this time establishes contact, proliferates, invades, modulates the maternal immune system, and provides a primitive form of nutrients to the implanting embryo proper. Insights into this period have been largely stunted due to the ethical and technical challenges that accompany human embryo research. Studies using donated human embryos following fertility treatment are complicated by confounding infertility diagnoses and limited sample sizes. The development of the extended culture system has provided an avenue to functionally study the peri-implantation period. Further, by using a variety of models including mouse embryos, human embryos, and stem cell-derived blastoids in the extended culture system, researchers are finally able to begin to piece together the puzzle of the peri- implantation period. Here, our objectives were to demonstrate the utility of mouse models in modeling human trophoblast during peri-implantation extended culture, examine and summarize human development during peri-implantation in the context of confounding fertility diagnoses, compare human trophoblast in extended culture to other widely available regenerative trophoblast models, and determine to what extent blastoids are able to reflect human peri-implantation development and maternal-fetal crosstalk in extended culture. Further, we show that estrogen signaling in trophectoderm may be conserved between mouse and human embryos, aged embryos exhibit hindered growth in extended culture, peri-implantation trophoblast cells have unique transcriptional priorities, and the presence of endometrial stromal cells encourage fusion of syncytiotrophoblasts. Our studies both reinforce the significance of the extended culture system and lay the groundwork for future studies on early trophoblast and embryo development during peri-implantation. Item Open AccessOncofetal proteins regulate proliferation and differentiation in placental cells(Colorado State University. Libraries, 2018) West, Rachel Claire, author; Winger, Quinton, advisor; Bouma, Gerrit, advisor; Anthony, Russ, committee member; Hamilton, Karyn, committee memberThe chromatin associated transcription factor HMGA2 is a downstream target of let-7 miRNAs and binds to chromatin to regulate gene expression, inducing rapid cell proliferation during embryogenesis. Inhibition of let-7 miRNAs by RNA binding proteins LIN28A and LIN28B is necessary during early embryogenesis to ensure stable expression of HMGA2 and proper cell proliferation. In addition to LIN28, HMGA2 is regulated by a BRCA1/ZNF350/CtIP repressor complex. In normal tissues, the BRCA1/ZNF350/CtIP complex binds to the HMGA2 promoter to prevent transcription. However, in many cancers the oncomiR miR-182 targets BRCA1, preventing BRCA1 translation and allowing for increased HMGA2. Little is known about the regulation of HMGA2 during early placental development therefore we hypothesized that both LIN28 and BRCA1 can regulate HMGA2 in placental cells. Using siRNA and CRISPR gene editing techniques, we found that knockdowns of both LIN28A and LIN28B increase HMGA2 levels in ACH-3P cells. These cells also demonstrated deficiencies in cell differentiation towards the syncytiotrophoblast, secreting higher amounts of hCG and displaying upregulated ERVW-1. Additionally, we found that a knockout of both LIN28A and LIN28B caused a significant increase of miR-182 and a decrease in BRCA1 which allows HMGA2 mRNA levels to increase and protein levels to remain the same. Using chromatin immunoprecipitation, we saw binding of the BRCA1 repressor complex to HMGA2. We also saw a decrease in binding to HMGA2's promoter in the LIN28A/B knockout cells. These findings suggest a novel role for BRCA1 during early human placental development. To test this hypothesis, we used CRISPR-Cas9 gene editing to knockout BRCA1 in the Swan71 cell line as the Swan71 cells had significantly higher BRCA1 levels compared to ACH-3P cells. HMGA2 mRNA and protein was significantly increased in the BRCA1 KO cells compared to control cells. Chromatin immunoprecipitation was used with an antibody for ZNF350 and PCR was run using primers for the promoter region for HMGA2. We saw a loss of BRCA1 repressor complex binding to HMGA2 in the knockout cells compared to our control cells, leading us to conclude that increased HMGA2 was due to decreased binding of the BRCA1 repressor complex. Additionally, we tested levels of apoptosis in our cells. After serum starving cells for 16 hours, we found that Caspase 3 and 7 levels were significantly higher in our BRCA1 KO cells compared to controls. This data suggests that BRCA1 is an important factor in the regulation of the oncofetal protein HMGA2 and promotes cell survival in human placental cells.