TP53 is required in trophoblast lineage development from human expanded potential stem cells

Abstract

p53 is a well-studied tumour suppressor gene that plays a role in various biological processes. Its importance in early development and human fertility has been suggested, although the exact mechanisms are not fully understood. The placenta is a transient organ crucial for supporting foetus growth during gestation, as it connects the embryo to the mother and mediates the supply of nutrients and oxygen to the conceptus. Defects in placental development can result in foetal growth restriction and pregnancy-related diseases which would lead to miscarriage in severe cases. Trophoblasts serve as the major primary building blocks of the placenta, and their extensive proliferation and differentiation are essential for normal placental development. However, the molecular requirements underpinning normal trophoblast differentiation remain remarkably underexplored. Human pluripotent stem cells including expanded potential stem cells (hESPCs) provide a cell resource for studying trophoblast development, as they possess the differentiation potential towards both embryonic and extraembryonic lineages, including trophoblasts, in vitro and in vivo. Using hEPSCs, a novel role of p53 in human trophoblast development was uncovered. Heterogeneous p53 expression in hEPSCs was initially observed by immunofluorescence. Subsequently, this led to the construction of a p53 reporter hEPSCs line, which helped reveal enhanced trophoblast differentiation capacity in p53-high hEPSCs. A significant increase in p53 expression was observed during the early differentiation of hEPSCs into trophoblasts. Inactivation of p53 in hEPSCs via the CRISPR technology resulted in impaired trophoblast gene expression during their differentiation towards trophoblast stem cells (TSCs), leading to trophoblast defects such as compromised pregnancy hormone secretion and failure in the in vitro implantation assays. Genomics and proteomics analyses identified hyperactivation of ERK/MAPK signalling in TP53-/- hEPSCs. Downregulation of ERK/MAPK signalling by specific ERK inhibitors restored the trophoblast differentiation defects caused by p53 deficiency. Using high throughput sequencing following chromatin immunoprecipitation of p53, DUSP5 and DUSP6 were identified as a potential p53 binding target involved in the regulation of ERK/MAPK signalling in hEPSCs and in trophoblast development. Overexpression of DUSP5 failed to rescue the trophoblast differentiation defects from TP53KO-hEPSCs, while DUSP6 overexpression partially promotes trophoblast differentiation potential. The uncomplete rescue suggests other potential mechanisms behind p53 regulating human trophoblast differentiation. Moreover, using mouse early pre-implantation embryonic development as an in vivo model, it has been demonstrated that inhibition of p53 activity from 4-cell stage severely compromised mouse embryonic development, especially the specification of trophectoderm lineage. These findings suggest a conserved role of p53 in the commitment of trophoblast lineage. Additionally, TP53-/- hEPSCs could form teratomas when injected to NOD-SCID mice, but containing immature neural rosettes, reflecting the functions of p53 in promoting neural lineage commitment from hESCs. In contrast, interestingly, it was demonstrated that p53 depletion promotes haematopoiesis from hEPSCs. The research findings of this thesis identify the critical role of p53 in early embryo development and carry important implications in clinical applications.