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dc.contributor.authorLadd, Sabine Margareten_US
dc.description.abstractObjectives: The effects of estrogens on immune system formation and function are well documented. Diethylstilbestrol (DES), a synthetic estrogen, has been linked to neoplasia and immune cell dysfunction in humans and animals exposed in-utero. In-vitro effects of DES exposure of murine embryonic stem (ES) cells on the early embryonic immune system development and the expression of cellular surface markers associated with common hemangioblastic and hematopoietic precursors of the endothelial, lymphoid & myeloid lineages were investigated. Hypothesis: Early ES cell expression of CD45 a marker common to lymphoid lineage hematopoietic stem cells and differentiation of lymphoid lineage precursors are affected by in-vitro exposure to DES. Methods: Murine ES cells were cultured using a variety of techniques: an OP9 co-culture system, and formation of embryoid bodies (EBs) in a liquid medium and hanging drop system. The OP9 co-culture system did not appear to give rise to well differentiated lymphoid lineage cells during 12 days of differentiation. The hanging drop EB culture system, previously shown to promote differentiation of endothelial and lymphoid precursor cells, was chosen for further studies of ES cell differentiation. ES cells were harvested at five time points: undifferentiated (day 0), and differentiated (days 3, 8, 12 and 16). Differentiating ES cells were treated with DES beginning on day 3. The synthetic estrogen, DES, was chosen as a treatment because of its similar potency to 17β estradiol and documented association with neoplasia in women exposed in-utero. Surface marker expression, measured by real-time RT-PCR amplification, was recorded using fluorogenic TaqMan(R) probes designed specifically for the surface proteins of interest: oct4, c-Kit, Flk1, ERα, ERβ, CD45, Flt1, & VE-cadherin. Analysis & Results: Changes in surface marker gene expression between day 0 and day 16 of differentiation were analyzed using the RT-PCR threshold counts (CT) and the comparative threshhold cycle method. The expression of each target mRNA was normalized internally to a housekeeping gene (18s rRNA) and calculated relative to day 0. ANOVA (Type 3 fixed-effects analysis, SAS) was performed using the unexponentiated ΠΠCT values. The effects of DES, time, and the interaction between DES and time were evaluated for days 8, 12 and 16. Additionally, the effects of DES on the expression of each marker were evaluated for day 16. Expression of estrogen receptor receptor α & β (ERα & β) in the EBs was established, and did not appear to be affected at any time by treatment with DES. ERα was expressed in significant levels on day 16, while ERβ was expressed in low levels throughout the period of differentiation. The expression of the cell surface marker, c-Kit was significantly (P<0.0001) altered by the presence of DES between the three time points sampled. The expression of the VEGF receptor, Flt1, and the adhesion molecule, VE-cadherin, markers of endothelial cells, were also significantly (P<0.026) altered by treatment with DES on day 16 of differentiation. Treatment with DES appeared to have no effect on the expression of CD45, a marker common to lymphoid precursor cells. Conclusions: These results indicate the presence of estrogen receptors in differentiating ES cells as early as day three in-vitro (ERβ) until day 16 (ERα). DES alters expression of common hemangioblastic and hematopoietic precursor, as well as endothelial lineage markers, but has no effect on expression of the marker of lymphoid lineage development before day 16. These effects coincided with the expression of ERα. The enduring effects of DES exposure in-utero may not be manifest in this ES model, or may occur at later stages of differentiation or in selected subpopulations of CD45+ cells.en_US
dc.publisherVirginia Techen_US
dc.rightsI hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Virginia Tech or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.en_US
dc.subjectEmbryonic Stem Cellsen_US
dc.subjectimmune system developmenten_US
dc.titleEffects of Diethylstilbestrol on Murine Early Embryonic Stem Cell Differentiation Using an Embryoid Body Culture Systemen_US
dc.contributor.departmentLarge Animal Clinical Sciencesen_US
dc.description.degreeMaster of Scienceen_US
thesis.degree.nameMaster of Scienceen_US
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen_US
thesis.degree.disciplineLarge Animal Clinical Sciencesen_US
dc.contributor.committeememberBuechner-Maxwell, Virginia A.en_US
dc.contributor.committeememberEyestone, Willard H.en_US
dc.contributor.committeecochairHuckle, William R.en_US
dc.contributor.committeecochairWitonsky, Sharon G.en_US

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