Browsing by Subject "naïve"

The extraembryonic placenta is composed of trophoblast cells consisting of the proliferative cytotrophoblasts (CTB) and its differentiated subtypes syncytiotrophoblast (SCT) and extravillous trophoblast (EVT). A normal trophoblast development is important as disruptions can lead to pregnancy complications such as pre-eclampsia. Therefore, it is crucial to investigate the underlying causes behind these abnormalities to discover treatments for patients suffering from pregnancy related disorders. Previously placental research was conducted largely on animal models and despite shared conservative pathways with humans, there are differences that exist. Only recently have researchers managed to successfully isolate and culture primary trophoblast stem cells (TSC)s by creating a TSC medium. Due to limited access to placental cells, pluripotent stem cells (PSC)s can be differentiated to TSCs by using the TSC medium. Naïve and primed states are described to be PSCs in different developmental stages, the former representing the pre-implantation state and the latter the post-implantation state. There lacks a consensus on whether both PSC states can be used to generate TSCs that correspond to primary trophoblasts. It has been argued that naïve cells possess more potential to differentiate into TSCs compared to the primed ones. The primed cells have been induced with the bone morphogenic protein (BMP) 4 to generate TSCs. This method is controversial as some suggest the induction resulting in other than TSCs, such as amniotic cells. Therefore, the aim of this thesis was to investigate whether both PSC states could be used to generate TSCs and its subtypes, if at all. Further, the effect of BMP4 was examined in the prime- derived differentiation protocol. The generated cells were then characterized and analyzed using imaging, immunocytochemistry (ICC) and quantitative reverse transcription PCR (RT-qPCR). The thesis found that although TSCs and its subtypes could be successfully generated from both PSC states, differences were observed. In addition to morphological differences, the most significant finding was the expression of the HLA-G gene, an EVT-specific marker, in the prime-derived TSCs (TSC(BMP4)). HLA-G was also significantly more expressed in the prime-derived EVTs (EVT(p)) compared to the naïve-derived EVTs (EVT(n)). Further, MMP2 which is also an EVT specific marker, was significantly more expressed in the EVT(n) compared to the EVT(p). As a result, the research question regarding the validity of the TSCs using both methods and the effect of BMP4 remains open. Further studies are required including single-cell RNA sequencing to obtain a better and broader view of the trophoblast profile and functional assays for subtype differentiation. Additionally, the role of BMP4 should be investigated in more depth.