Purine nucleotides are critical precursors for DNA and RNA synthesis and play essential roles in cell signaling and embryonic development. Nucleotides are synthesized through the de novo synthesis and salvage pathways. The de novo synthesis pathway is more active in proliferating cells, whereas differentiated tissues primarily utilize the salvage pathway. Isotope tracing reveals rapid glucose-dependent purine synthesis in the placenta and embryo during a midgestation metabolic transition phase (gestational days 10.5–11.5) in mice. Interestingly, although the embryo exhibits a higher capacity for de novo synthesis, purine levels are overall higher in the placenta. Pregnant mice also show increased circulating hypoxanthine, suggesting the placenta may rely on the salvage pathway to sustain a larger purine pool and not compete with nutrient resources with the embryo.

To determine which pathway is used by the placenta during development, we compared de novo purine synthesis between trophoblast stem cells (hTSCs) and differentiated syncytiotrophoblasts (STBs) under hypoxanthine-replete and hypoxanthine-depleted conditions. hTSCs exhibit flexibility in using both pathways, switching between them based on the availability of hypoxanthine in the medium. However, de novo purine synthesis is significantly attenuated upon differentiation into STBs, and cells predominantly depend on the salvage pathway to maintain purine levels.

Notably, guanosine monophosphate (GMP) levels remain high throughout hTSC differentiation to STBs. We tested the requirement for GMP synthesis during STB differentiation using mycophenolic acid (MPA). Inhibition of GMP synthesis via blockade of IMPDH (inosine monophosphate dehydrogenase) suppresses STB differentiation. Mechanistically, reduced GMP synthesis leads to downregulation of Rheb, a key activator of mTOR signaling. We confirm that mTOR activation is essential during the early stage of STB differentiation in hTSCs and in patient-derived term placental organoids.