This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.To define the influence of specific extracellular matrix components and placental mesenchymal cells on trophoblast differentiation from human embryonic stem cells.Our laboratory has established an in vitro model to study trophoblast differentiation. Human embryonic stem cells (hESC) will consistently differentiate to trophoblasts and secrete high levels of placental hormones when allowed to form embryoid bodies (EBs), and are transferred into a three-dimensional (3D) extracellular matrix (Matrigel) environment (ECM). Hormone secretion was enhanced in this 3D system in comparison with planar trophoblast outgrowths in standard adherent culture.To further explore the spatial and mechanical effects of 2D vs. 3D culture, an alternative approach utilizing collagen I, a simple ECM, was employed. The mechanical features of the ECM were changed by allowing gels containing EBs to either maintain adherence to the culture dish rendering a rigid gel, or to float in the culture media rendering a flexible gel. In these experiments, EBs were used that had been maintained in suspension for 8 days and therefore already had a differentiated trophoblast layer. These EBs were then mixed with collagen I forming a 3D environment or alternatively, the EBs were plated on top of the collagen I gel forming a 2D environment. After the gels had formed, a pipette tip was used to lift the outer edges of the gels and 2 ml of media was added to 'float' the flexible gels. Half of the gels were 'floated' and half were left attached to the culture dish. HCG secretion was assayed to determine trophoblast differentiation through 30 days of culture.Preliminary experiments revealed significantly different levels of hCG secretion between experimental conditions on particular days of culture. EBs in 3D-Rigid gels secreted significantly higher levels of hCG on days 15 and 20 of culture compared to EBs in 3D-Flexible gels. Also, 2D-rigid gels secreted significantly higher levels of hCG on days 8 and 10 compared to 3D-Flexible. In addition, hCG levels were significantly higher for EBs in 2D-Flexible gels compared to 3D-Flexible gels on days 8, 10, 15, 20 and 2D-Rigid gels on day 10. Interestingly, EBs in 2D-Flexible conditions did not differ from the 3D-Rigid conditions. This preliminary data suggests that although some differences were observed at limited time points during culture, the most sustained difference in hCG secretion (~2 weeks) was between EBs in 2D-Flexible gels and in 3D-Flexible gels. This implies that a cell-ECM dimensional dynamic optimizes trophoblast differentiation. Further investigation is necessary to identify if the spatial, as well as matrix-interactions encountered by EBs will induce similar signaling that is found during in vivo differentiation. This research used WNPRC Research Services, including federally approved human ES cell lines.
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