BACKGROUND: The placenta is a multifunctional organ whose primary functions are to nourish and protect the fetus throughout gestation. The immune response of the placenta plays an important part in gestational outcome. Microbial infection during pregnancy can be detrimental to both maternal health and fetal development, increasing the risk for miscarriage, preterm birth, and congenital abnormalities. However, evaluating immunological response has been an on-going challenge for scientists and clinicians due to the complexity of the maternal-fetal interface. Research has been done to understand the mechanisms by which pathogens activate placental immune response, but our understanding is still lacking in many areas due to the dynamic changes that occur in immunology over the gestational timeline. The primary challenge faced by researchers is the availability of placental tissue, which is limited by donors and their finite viability in culture once harvested. Additionally, legal restrictions placed on fetal-tissue research have severely limited advancement in the field. Human induced pluripotent stem cells (hiPSCs) present a unique tool to study the differentiation of trophoblasts and maternal-placental immunology without the need of fetal tissue. OBJECTIVE: The goal of this project is to develop an in vitro model for studying placental immunology and pathogenesis using human induced pluripotent stem cell (hiPSC)-derived trophoblasts. Our aim is to report a robust protocol for producing hiPSC-derived trophoblasts and to characterize them against primary trophoblasts using both gene and protein expression detection techniques. Successful modeling of human trophoblasts would allow us the unique opportunity to investigate the cellular interface between the maternal and fetal systems without needing to isolate primary human trophoblasts. Once we produce and fully characterize several hiPSC cell clones from multiple normal individuals, we will demonstrate the use of these cells as a model for infections at the maternal-fetal interface by exposing them to viral pathogens known to target the placenta. METHODS: Earlier publications have reported the differentiation of embryonic stem cells into trophoblasts in culture by using bone morphogenetic protein-4 in conjunction with inhibitors of activin A and FGF2-signaling (BMP4/A83-01/PD173074; BAP-treatment). We applied this approach to hiPSC lines from two different lineage origins and characterized the outcome against known trophoblast markers. We also developed a novel approach to maintain proliferative trophoblast stem cells in culture long term. Two viral pathogens, a recombinant vesicular stomatitis virus strain engineered to express a green fluorescent protein (rVSV-GFP) and a strain of Zika virus (ZIKV-PRVABC59), were used to determine if it is possible to infect hiPSC-derived trophoblasts in culture. RESULTS: Using this approach, hiPSC readily differentiate into trophoblasts by day 8 of culture. These cells demonstrate formation of multinuclear syncytium, invasive capacities, and secretion of placental hormones. Further characterization using quantitative real-time PCR and immunofluorescent staining indicates that these cells express a number of trophoblast markers at levels comparable to those expressed by primary first-trimester trophoblasts. We were also able to maintain a putative CT population which retains the capacity to double and give rise to terminal cell types. HiPSC-derived trophoblasts infected with rVSV-GFP and ZIKV-PRVABC59 tested positive for viral infection by 72 hours post-infection (HPI), demonstrating the use of these cells as an in vitro model for studying placental pathogens at the maternal-fetal interface. / 2022-06-08T00:00:00Z

Made available in DSpace on 2016-08-15T20:31:21Z (GMT). No. of bitstreams: 1 FerdinandoVFB_DISSERT.pdf: 10097957 bytes, checksum: aaf72cc9003c689e5792192fd220aef8 (MD5) Previous issue date: 2014-02-28 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The subplacenta is considered an ideal model for comparative studies of trophoblastic processes in humans. Thus, the aim of this study was made a morphological description of the development of the Spix s yellow-toothed cavy. To do this, 12 females were distributed in three groups in a proportion of 1 male to 4 females, kept in pickets of 5m2. Thereafter, vaginal cytology examinations were made daily, to verify if the females were copulated and to separate them from the other females. Then, the collection of subplacentas was made in the 15, 23, 30, 45, 53 and 55 days of gestation, by slaughtering the pregnant females using a specific anesthesic protocol. The samples were processed to standard histological techniques, cytochemistry and immunohistochemistry, and to transmission electronic microscopy. In the 15th day of gestation, the subplacenta was formed by a single cytotrophoblastic layer surrounded by a vacuolized syncytium with maternal lacunae which presented invasive characteristic in the portions apart of the cytotrophoblastic layer. In the 23th day of gestation, the subplacenta did not presented a well-defined shape, however, it was organized in lobules composed of predominantly of cytotrophoblast e its syncytium was related to the regions where maternal lacunae previously appeared. In the 30th day of gestation, the subplacenta appeared as a compact organ with a well-defined shape and with an evident mesenchymal capilarization; the lobules was composed by syncytiotrophoblast in the centre, and by a mesenchyme surrounded by cytotrophoblast. From the 45th day of gestation, the degeneration of the subplacental tissue was evident and the syncytiotrophoblast was more abundant than the cytotrophoblast; moreover, the syncytium was markedly vacuolized and presented signs of cellular death. Near to gestation term (53-54thday), the subplacenta was in an advanced degeneration stage, with evident signs of cellular death and reduction of subplacental tissue. The presence of fetal circulation was characteristic from the 23th day of gestation, evidenced by the positive reaction to vimentin; positive reaction to cytokeratin was observed during entire gestation. The proliferative activity of the subplacenta was assessed by PCNA and AgNOR procedures, and demonstrated to be higher in the beginning of the gestational period, decreasing progressively during the gestation. Ultrastructurally, the subplacenta presented cellular and syncytial trophoblastic characteristics. The development of the Spix s yellow-toothed cavy subplacenta starts around the 15th day of gestation, reaching maximum development in the 30th day and becomes necrotic in the end of gestation. Moreover, it presented an organization and structure similar to the subplacenta of other cavidae / A subplacenta é considerada um modelo ideal para o estudo comparativo dos processos trofoblásticos em humanos. Dessa maneira objetivou-se descrever morfologicamente o desenvolvimento da subplacenta no preá. Para isto foram utilizadas 12 fêmeas desta espécie que foram distribuídas em três grupos numa relação de um macho para quatro fêmeas, mantidos em boxes de 5m2. Após formação dos grupos, exames de citologia vaginal eram realizados diariamente, para verificação da cópula, separando-se dos grupos as fêmeas que eram cobertas. A partir da ocorrência da cópula programaram-se as coletas das subplacentas nos dias 15, 23, 30, 45, 53, e 55 da gestação e estas foram realizadas mediante sacrifício das fêmeas gestantes com a utilização de protocolo anestésico específico. O material, então, era processado segundo técnicas para histologia convencional, citoquímica, imunohistoquímica e microscopia eletrônica de transmissão. No 15° dia de gestação observou-se que a subplacenta era constituída por uma monocamada citotrofoblástica envolta por sincício que apresentava vacúolos e lacunas maternas e que ao se afastar da camada de citotrofoblasto apresentava características invasivas. Aos 23 dias de gestação a subplacenta apresentou-se ainda sem uma forma característica definida, porém, mostrava uma conformação em lóbulos compostos predominantemente por citotrofoblasto e seu sincício esteve relacionado com as regiões onde apareciam as lacunas de origem materna. Aos 30 dias de gestação a subplacenta mostrou-se como um órgão compacto de forma definida e com a capilarização do mesênquima bastante evidente, os lóbulos eram constituídos por citotrofoblasto que envolvia o mesênquima e em seu centro possuía sinciciotrofoblasto. A partir dos 45 dias de gestação a degeneração do tecido subplacentário era evidente e a quantidade de sinciciotrofoblasto supera a de citotrofoblasto, alem disto o sincício apresenta uma grande quantidade de vacúolos e sinais de morte celular podiam ser visualizados. Aproximando-se ao termo da gestação (53 e 55 dias) a subplacenta encontrava-se em avançada degeneração e os sinais de morte celular assim como a diminuição de seu tecido eram evidentes. A presença da circulação fetal foi característica a partir do 23° dia de gestação podendo ser destacada pela reação positiva a vimentina, e a reação a citoqueratina foi positiva durante toda a gestação especialmente no citotrofoblasto. A atividade proliferativa do tecido subplacentário, avaliada pelas técnicas de PCNA e AgNOR, demonstrou ser maior no inicio da gestação e decair com o avançar desta. Ultraestruturalmente evidenciou-se as características do trofoblasto celular e sincicial. O desenvolvimento da subplacenta do preá inicia-se por volta do 15° dia de gestação, tem seu auge aos 30 dias e a termo é necrótica. Além disto, é muito semelhante a subplacenta de outros cavídeos, quanto a sua organização e estrutura

The outer surface of the human placenta, the syncytiotrophoblast, results from the fusion of many cytotrophoblast cells such that many nuclei are contained in this layer. It is possible for these nuclei to cluster forming syncytial nuclear aggregates (SNAs). SNAs have been linked to pathology with increased numbers and earlier formation of SNAs in preeclampsia and fetal growth restriction (FGR). SNAs can be grouped into subtypes including bridges, knots and sprouts, dependent on morphology and attachment to surrounding placental villi. Little is known about SNA formation, but the pyknotic appearance of nuclei within SNAs has led to development of a hypothesis that SNAs are the terminal point of nuclear turnover in the syncytiotrophoblast. Some cytoskeletal proteins have been associated with SNAs indicating their potential involvement in SNA formation. This project aimed to uncover differences between SNA subtypes, whether the degenerate nuclear morphology represents apoptosis and to understand which mechanisms drive nuclear collection into SNAs. Experimental approaches included a review of an electron micrograph archive and application of immunohistochemical techniques to ex vivo placental tissue. A long-term explant model was developed to examine SNA development in vitro; these experiments were further explored using an isolated primary cytotrophoblast model. Nuclei within SNAs were more frequently pyknotic and less frequently eukaryotic than nuclei dispersed in the syncytiotrophoblast. However, few SNAs were positive for the cytokeratin-M30 neoepitope, a caspase dependent breakdown product of cytokeratin-18 and no subtype of SNA showed greater M30 staining than general areas of syncytiotrophoblast. There were increased syncytial knots and decreased syncytial bridges in placentas from women with preeclampsia compared to controls and FGR. While cytoskeletal proteins are seen surrounding SNAs, inhibition of actin and tubulin had no effect on SNA turnover or stability. Very limited nuclear movement was recorded from in vitro culture indicating that syncytiotrophoblast nuclei move far less than had been expected. These data suggest that cell death was not prominent within SNAs but different prevalence of subtypes were present in preeclampsia indicating that SNAs might represent larger changes in placenta structure. As nuclei moved less and SNAs were more static than expected it is suggests that SNAs are more stable than previously thought. Overall, the hypothesis that SNAs are highly active in preeclampsia is questioned and new hypotheses of the role of SNAs are considered in the light of these experimental findings, including whether they form by chance and represent changes in cell turnover of the syncytiotrophoblast.