Fig. 2: Polyunsaturated lipids are a metabolic signature of early differentiating ESCs and TSCs.

ESCs and TSCs of the same background (129/black 6) were seeded in Tx defined culture medium. After 24 h, medium was changed to fresh medium with (“Undiff”) or without (“Early Diff”) factors that support self-renewal. Their metabolome was examined after 48 h, using 0.5 × 10⁶ cells per sample. The second principal component (PC2) of a partial least squares discriminant analysis (PLS-DA; A) shows discrete metabolome of the differentiating SCs. R² = 0.93, Q² = 0.83 (n = 6). A heat map representation of the abundance of identified differential polyunsaturated phospholipids of the dataset shown in (A), following variable importance in projection (VIP; > 1) analysis (B). The abundance of each metabolic feature was normalized to the sum intensity of peaks of each chromatogram. The colors of quadrants in the heat maps represent the relative abundance of the metabolites, after normalization to the mean abundance of each metabolite across all samples (n = 6). Following hydrolysis of lipids in ESC extracts, total abundance of linoleic acid (C) and arachidonic acid (D) was measured by GC-MS and normalized to the sum of peaks area. The biosynthesis of PUFAs starts from essential fatty acids with two double bonds, and is catalyzed by D6D and D5D (E). D6D and D5D expression is upregulated in early differentiating ESCs (F) and TSCs (G), as measured by RT-PCR (n = 6). ESCs and TSCs (129/black 6 background) were treated by the D6D inhibitor SC-26196 (0.2 µM) or DMSO control for 48 h in feeder free culture without 2i/LIF or FGF4/TGFβ/heparin, respectively, and the expression of stem cell markers was measured by RT-qPCR (H, I; n = 4). Values are the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.