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Regulation of trophoblast stem cell maintenance and differentiation by LIN28 and AP-2γ

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dc.contributor.authorFromme, Brittany A., author
dc.contributor.authorWinger, Quinton A., advisor
dc.contributor.authorBouma, Gerrit J., advisor
dc.contributor.authorBailey, Susan M., committee member
dc.contributor.authorAnthony, Russell V., committee member
dc.date.accessioned2022-04-20T14:27:39Z
dc.date.available2022-04-20T14:27:39Z
dc.date.issued2010
dc.descriptionCovers not scanned.
dc.descriptionPrint version deaccessioned 2022.
dc.description.abstractThe placenta is a unique organ essential for survival of the fetus in all eutherian mammals. Failure to develop a normal placenta in humans can lead to diseases, such as pre-eclampsia, with high morbidity and mortality for both the mother and the fetus. These diseases are thought to be caused by abnormal proliferation and differentiation of cells in the placenta. A mouse trophoblast stem (TS) cell culture system is a useful tool in studying TS cell proliferation and differentiation into trophoblast giant cells (TGCs). TS cells cultured in proliferative media (70% conditioned media, 30% TS media, FGF4, and heparin sulfate) will remain proliferative, and TS cells cultured under differentiation media (100% TS media) will differentiate into TGCs. LIN28 is a protein that regulates mRNAs and miRNAs, and is abundantly expressed in many undifferentiated tissues. AP- 2y has been shown to be essential for TS cell maintenance and TGC formation. AP-2y null mutants display embryonic lethality at E7.5 due to a severely disrupted extraembryonic portion of the embryo. In TS cells, AP-2y has been shown to bind to the promoter region of Lin28. This study investigates the hypothesis that Liti28 and Ap-2y are necessary regulators of trophoblast stem cell maintenance and differentiation into TGCs. This study shows the pluripotency genes, Lin28, Sox2, and NrObl, to be differentially expressed in proliferating TS cells and differentiated TGCs. MiRNAs can be used as markers for proliferation or differentiation. 28 significantly different miRNAs were detected between TS cells and TGCs, 18 up-regulated in TGCs and 9 downregulated in TGCs. Expression of the miR-290 family, initially thought to be ES cell specific, was detected in proliferating TS cells suggesting TS cells have similar miRNA mediated regulation of proliferation compared to ES cells. The Let-7 family of miRNAs was found to be up-regulated in differentiated TGCs. The Let-7 family has been shown to be regulated by LIN28, where LIN28 prevents accumulation of mature Let-7 miRNAs. In this study Lin28 was highly expressed in proliferating cells and the Let-7’s are upregulated in differentiated TGCs. Lin28 function in TS cells was assessed by knocking down Lin28 using shRNA lentiviral technology. Lin28 knockdown TS cells were used to observe results of knockdown. We obtained a 78% reduction of Lin28 mRNA, but found that loss of Lin28 in TS cells did not affect morphology, proliferation or differentiation. AP-2y null TS cells grown in culture fail to differentiate morphologically into TGCs. Lin28, Sox2, and NrObl show no difference in expression when grown in conditions to differentiate the cells, indicating a failure of AP-2y null TS cells to differentiate into TGCs. RO3306 is a compound used to block Cyclin-dependent Protein Kinase 1 and force endoreduplication, causing TS cells to differentiate into TGCs. AP-2y null TS cells cannot be forced to differentiate into TGCs, and instead undergo cell death, when cultured with RO3306. Additionally, AP-2y null TS cells express the pluripotency markers Oct4, Stella, and Nanog which only are expressed in ES cells and germ cells. MiRNA profiling of AP-2y null TS cells indicates that cells in proliferative conditions resemble wild type counterparts, but when proliferative conditions are removed we observe an increase in expression of the ES cell specific miR-302 cluster. While there was no effect of proliferation in wild type cells, loss of Lin28 in AP-2y null TS cells via lentiviral knockdown leads to a partial rescue of TGC formation. This suggests that Liii28 must be down-regulated in order for TGC formation, and that AP-2y regulates Lin28 in TS cells. Taken together these data suggest a role for Lin28 in mouse TS cell proliferation and differentiation, where Lin28 must become down regulated in order for differentiation into TGCs. AP-2y has been shown to bind to the Lin28 promoter in TS cells; this regulation enables TS cell differentiation into TGCs. This study also shows the necessity of AP-2y for TS cell differentiation into TGCs; loss of AP-2y leads to a more pluripotent state rather than allowing for differentiation. Loss of AP-2y leads to expression of pluripotency markers Oct4, Nanog, and Stella, and the ES cell specific miR-302 cluster, indicating an increase in pluripotency. We conclude that AP-2y and LIN28 are essential molecular regulators of TS cell proliferation and differentiation.
dc.format.mediummasters theses
dc.identifier.urihttps://hdl.handle.net/10217/234745
dc.languageEnglish
dc.language.isoeng
dc.publisherColorado State University. Libraries
dc.relationCatalog record number (MMS ID): 991014400019703361
dc.relationQH588.S83 .F765 2010
dc.relation.ispartof2000-2019
dc.rightsCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.
dc.subjectTrophoblast
dc.subjectStem cells
dc.titleRegulation of trophoblast stem cell maintenance and differentiation by LIN28 and AP-2γ
dc.typeText
dcterms.rights.dplaThis Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).
thesis.degree.disciplineBiomedical Sciences
thesis.degree.grantorColorado State University
thesis.degree.levelMasters
thesis.degree.nameMaster of Science (M.S.)

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