In the first trimester of human pregnancy specialised placental cells, termed cytotrophoblasts differentiate into extravillous trophoblasts (EVTs), which grow out from the placenta and invade into the maternal decidua, acting to physically attach the placenta to the decidua, and adapt the uterine spiral arteries to support pregnancy. A proportion of these EVTs also temporarily occlude the spiral arteries for approximately the first 10 weeks of gestation, preventing maternal blood flow to the placenta and creating a low oxygen environment in which placental and fetal development occur. The processes of trophoblast outgrowth and invasion, and the establishment of a low oxygen environment, are essential for the success of pregnancy, and inadequate trophoblast invasion into the uterus has been associated with recurrent miscarriage, pre-eclampsia and fetal growth restriction. However, the exact mechanisms that control the differentiation of cytotrophoblasts down the extravillous trophoblast lineage are poorly understood. Since the extent of this invasive process is unique to human implantation, animal models are of limited value in studying trophoblast invasion. Existing in vitro models have major limitations in that many are very difficult to quantify while others many not study the correct trophoblast population. The research in this thesis has focussed on the development of novel models by which to study cytotrophoblast differentiation, and the use of these models to further understand cytotrophoblast differentiation down the EVT lineage, and the regulation of EVT outgrowth by oxygen. Methods Outgrowth from first trimester villous explants was characterized using immunohistochemistry. Explant viability was investigated using dual staining with chloromethylfluorescin diacetate (CMFDA) and ethidium bromide, by examining DNA laddering, and by immunostaining sectioned explants over 96 hours of culture. The extended viability of cytotrophoblasts in multilayered cell islands in villous tips was exploited to isolate these cells using sequential trypsin digests of cultured villous explants. The trophoblast population obtained were characterized by immunohistochemistry. Finally, villous explants were cultured in either 1.5% or 8% oxygen and the frequency and area of outgrowths was quantified in order to determine the effect of gestation and oxygen on EVT outgrowth. Results Approximately 1/4 of explants cultured in 20% oxygen produced EVT outgrowth. Outgrowth formation and expansion resulted from proliferation of cells in the tips of anchoring villi, and EVTs within the outgrowth did not proliferate. The percentage of explants producing outgrowth declined as gestation increased from 8 to 12 weeks. Dual staining with CMFDA and ethidium bromide revealed degeneration of the syncytiotrophoblast by non-apoptotic mechanisms within 4 hours of culture, but this syncytiotrophoblast layer was able to be regenerated. The majority of cytotrophoblasts died within one week of culture, but despite this explants were able to produce EVT outgrowth for up to 3 weeks due to the extended survival of a specific set of cytotrophoblasts located in cell islands in the tips of anchoring villi. These surviving cells were able to differentiate into EVTs, but not regenerate the surrounding syncytiotrophoblast in the villus tip. Trypsinization of first trimester villi after extended explant culture resulted in the isolation of a viable population of ‘putative EVT progenitors’ that did not syncytialise in culture, but were able to proliferate. 20% of these cells differentiated down the EVT lineage within 96 hours of culture. The putative EVT progenitors expressed markers previously localised to cytotrophoblasts in cell islands of anchoring villi, including αvβ6 integrin and FGFR-2. The addition of exogenous FGF-4 did not affect the differentiation of these cells into EVTs, nor did FGF-4 alter the frequency of EVT outgrowth from explants. Culture in 1.5% oxygen significantly reduced the frequency and area of outgrowths in comparison to 8% oxygen. HLA-G and α1 integrin were both expressed throughout outgrowths with no difference in expression of these proteins between oxygen concentrations. Gestation influenced the response of explants to oxygen, with a significant differential response to oxygen concentration in placentae under 11 weeks of gestation but no differential response in placentae of 11 or 12 weeks. Conclusions In the first trimester, oxygen and gestational age regulate extravillous trophoblast outgrowth in both an independent and interdependent manner. The cytotrophoblast population in the first trimester does not consist of one homogenous bipotent population. Rather there are at least two separate populations: 1) EVT progenitors that exist in the tips of potential anchoring villi that are likely to be committed to EVT differentiation and 2) monolayer villous cytotrophoblasts which are likely to be committed to syncytiotrophoblast differentiation. The second population is easily isolated by traditional enzymatic digestion methods whereas the first much smaller population can be isolated by exploiting their prolonged survival in explant culture.
| Identifer | oai:union.ndltd.org:ADTP/275413 |
| Date | January 2006 |
| Creators | James, Joanna |
| Publisher | ResearchSpace@Auckland |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated., http://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm, Copyright: The author |
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