Acetyl-CoA metabolism and human trophoblast differentiation

Cytotrophoblasts are trophoblast stem cells that undergo rapid self-renewal and differentiation during early pregnancy. Disruptions in this process can lead to impaired placentation or placental dysfunction leading to complications in pregnancy. This thesis investigates the role of ATP citrate lyase (ACLY), an enzyme that links glucose metabolism to histone acetylation by producing acetyl-CoA, a substrate for epigenetic modifications. The hypothesis was that ACLY is essential for maintaining trophoblast stemness by providing a localised supply of acetyl-CoA for histone acetylation, thus leading to a transcriptionally permissive state associated with trophoblast self-renewal. The role of cell culture medium on trophoblast behaviour was initially investigated as standard cell culture media are composed of unphysiological concentrations of nutrients and metabolites. Given the link between metabolism and epigenetics, which is the focus of this thesis, the composition of culture medium may impact not only the metabolic profile but also epigenetic processes. Cytotrophoblast metabolism, histone acetylation and trophoblast differentiation were compared in cells cultured in the physiologically-relevant medium (Plasmax) versus standard medium (DMEM-F12). Compared to standard medium, the physiologically-relevant medium was demonstrated to promote cytotrophoblast self-renewal, improved differentiation capacity and increased metabolic activity and histone acetylation. This finding suggested that the use of the physiologically-relevant medium more accurately models the trophoblast behaviour and was thus used for the remainder of this thesis. The progenitor cytotrophoblasts exhibit high glycolytic activity as well as higher acetyl-CoA levels which declines with differentiation into syncytiotrophoblast or extravillous trophoblasts. It was further demonstrated that during trophoblast differentiation, changes in histone acetylation precede changes in markers associated with differentiation, suggesting that metabolic and epigenetic processes may be driving trophoblast fate. Additionally, ACLY was predominantly localised in the nucleus of cytotrophoblasts but translocates to the cytoplasm upon differentiation, suggesting a change in nuclear acetyl-CoA production as trophoblasts differentiate. Knockdown of ACLY led to global decreases in several histone acetylation marks with the most pronounced effect on H3K27Ac. ACLY knockdown also led to the downregulation of stemness markers while syncytialization markers were upregulated, indicating premature differentiation. Single-nucleus RNA sequencing confirmed these effects, revealing a reduction in stemness-associated gene expression and an increase in gene signatures associated with STB-like cells. Notably, ACLY knockdown did not result in substantial changes to glycolytic or TCA cycle intermediates, nor mitochondrial respiration, suggesting that the phenotypic changes were due to impaired epigenetic regulation rather than metabolic deficits. Moreover, ACLY depletion impaired the ability of cytotrophoblasts to form trophoblast organoids, underscoring its essential role in trophoblast self-renewal and placental development. Lastly, it was demonstrated that the regulatory regions of trophoblast stemness genes were highly enriched in H3K27Ac and that loss of ACLY resulted in reduced H3K27Ac binding to these regions. These results identify ACLY as a key mediator linking cellular metabolism to epigenetic regulation during early placental development. By regulating acetyl-CoA levels for histone acetylation, ACLY supports the transcription of stemness genes, maintaining trophoblast stem cell function and preventing premature differentiation. Loss of ACLY function leads to precocious differentiation, particularly toward the STB lineage, which may compromise placental formation and function during early stages. This study suggests that targeting ACLY and metabolic-epigenetic pathways could provide novel therapeutic strategies to enhance trophoblast stem cell function and improve pregnancy outcomes. These findings contribute to a deeper understanding of how metabolic and epigenetic mechanisms shape placental development and pave the way for future research on interventions, such as periconceptional supplementation, to mitigate early metabolic disruptions and improve pregnancy health.