Thermal-stable FGF1 mutant obtained ability to support ERK phosphorylation and pluripotency. (A): FGF1 is unstable at 37°C. Media with FGF1 were incubated with heparin at 37°C, and then applied for 15 minutes on FGF-starved embryonic stem cells (ESCs) at specific time points, before proteins were collected to analyze for ERK phosphorylation. (B): FGF1 protein was mutated to increase thermal stability or heparin affinity. The mutated amino acid was colored with red letters [29, 30]. (C): Mutations improved maintenance of ERK phosphorylation for 24 hours. H1 ESCs were plated into basic media (E8 media without TGFβ) with different FGFs (100 ng/ml) for 24 hours, and proteins were then collected to detect ERK phosphorylation. (D): FGF1 mutant FGF1-4X (3X with K112N) is thermally stable, while FGF1-3X and FGF2 were stabilized by heparin. Media with different FGFs were preincubated at 37°C for 24 hours, and then applied for 15 minutes on FGF-starved ESCs, before proteins were collected to analyze for ERK phosphorylation. (E): Mutated FGF1 supports ESC growth. H1 cells were maintained in E8 media with different FGFs, and cells were counted after 96 hours. (F): Mutated FGF1 supports pluripotency of human ESCs. H1 cells were maintained in E8 media without TGFβ and different FGFs, and OCT4 expression was analyzed after two passages. Different FGFs were also tested in regular ESC media in the presence of TGFβ, and similar results were obtained (Supporting Information Fig. S2E). Abbreviations: ERK, extracellular-signal-regulated kinase; FGF, fibroblast growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TGF, transforming growth factor; WT, wild type.