Elucidating the Metabolic Rewiring of Pluripotent Stem Cells during Differentiation into Adult Progenitors Using an Updated and Enzyme-Constrained Human Genome-Scale Model

Pluripotent stem cells (PSCs) offer new potentials for treating multiple diseases via gene therapy and regenerative medicine due to their unique regenerative abilities. PSCs differentiate into many distinct germ layer cells and produce various human organs via various transcriptional, epigenetic and metabolic changes. While the transcriptional and epigenetic changes during stem cell differentiation is widely researched, the metabolic changes of PSCs during differentiation remain largely uncovered, except the well-known switch from aerobic glycolysis into oxidative phosphorylation for energy generation. Therefore, we utilize a systematic approach combining transcriptomic data and the human genome-scale model (GEM) to examine the metabolic signatures of pluripotent stem cells, germ layer cells, and adult stem cells in comparison to various mature cells. Here, it should be highlighted that although the availability of several human GEMs has substantially improved our understanding on the molecular basis of various diseases and phenotypes, we still observed the variability in internal metabolic fluxes predicted by the models, across conditions due to the limited constraints typically used in constraints-based flux analysis. To address this critical issue, here, we present the most comprehensive and biochemically consistent human GEM to-date, Recon3E, and included the necessary kinetome information to enable its ready use during the simulations, thereby improving the phenotype predictions further. Recon3E accounts for 3463 open reading frames, 7182 reactions corresponding to 987 unique enzymes, and 3023 unique metabolites. Importantly, Recon3E contains the kinetic information, i.e. k_cat, for 672 of the total 987 enzymes where 74% of these values are human-specific. Using Recon3E, we were able to predict the metabolic rewiring which occurs during the differentiation of PSCs into adult progenitors, emphasizing the role of unsaturated lipids, amino acid derivatives and one-carbon metabolites in maintaining self-renewal and pluripotency, in addition to the commonly cited glycolysis and oxidative phosphorylation. (Funding: SSAC# PJ01334605, Republic of Korea)