Thermal Stability of Fibroblast Growth Factor Protein Is a Determinant Factor in Regulating Self-Renewal, Differentiation, and Reprogramming in Human Pluripotent Stem Cells

Additional Supporting Information may be found in the online version of this article.

Filename	Format	Size	Description
STEM_1021_sm_SuppFig1.pdf		566K	Figure S1. FGF activity and heparin (A) FGF18 has a weaker capacity to induce ERK phosphorylation compared to FGF2. FGF18 and FGF2 (10 ng / ml) were applied on FGF-starved ES cells for 15 minutes, and protein was harvested to detect ERK phosphorylation by western blot. (B) Dosage dependent ERK phosphorylation by FGF2. A serial of human and zebrafish FGF2 were applied on FGF-starved ES cells for 15 minutes, and protein was harvested to detect ERK phosphorylation by western blot. Human FGF2 has a similar ability to zebrafish FGF2 in activating ERK phosphorylation. (C) Heparin maintained zebrafish FGF2 during 37°C incubation. FGF2 was treated with different conditions before it was applied on FGF-starved ES cells for 15 minutes, and protein was harvested to detect ERK phosphorylation by western blot. Heparin maintained FGF2 activity, while BSA and β-mecaptoethanol could not. (D and E) Additional reagents also can maintain FGF2 stability. Human FGF2 was treated with different reagents at 37°C for 24 hours, and was then applied to FGF-starved cells for ERK phosphorylation detection. Dextran Sulfate and Cyclodextrin Sulfate also helped maintain FGF activity. (F) In order to maintain FGF2 stability, heparin has to be present during incubation. Zebrafish FGF2 was treated under different conditions at 37°C for 24 hours, and then applied on FGF-starved ES cells for 15 minutes, and protein was harvested to detect ERK phosphorylation by western blot. In treatment 4, FGF2 media was incubated at 37°C without heparin, and media was then mixed with heparin to treat cells; in treatment 5, FGF2 media contained heparin during 37°C treatment. (G) Heparin prevents loss of zebrafish FGF2 monomer/dimer. Zebrafish FGF2 (50 ng / ml) was treated under different conditions, then separated on PAGE-gel, and stained with coomassic blue. DTT was added into samples to demonstrate that same amount of total FGF2 was used in the treatment. Heparin increased FGF2 dimers, but the total amount of dimer and monomer was not changed after 37°C treatment. In control and BME treatment, most of monomer was lost after 37°C treatment, and protein aggregates were observed in corresponding wells.
STEM_1021_sm_SuppFig2.pdf		762K	Figure S2. Stabilized FGF proteins supports ES cell pluripotency. (A) Frequent addition of FGF1 (100 ng / ml) helped maintain ES cell morphology. H1 cells were cultured in specific conditions for 5 days before photo-imaging. (B) Full-length and truncated FGF1s lost their activity after 6-hour incubation at 37°C, and heparin failed to preserve the activity. Media with full-length FGF1 or truncated FGF1 were treated with or without heparin at 37°C for 6 hours, and were then applied to FGF-starved ES cells for 15 minutes. Protein was harvested to measure ERK phosphorylation. (C) FGF2 and FGF1 derivatives were purified from E. Coli. The image shows coomassie staining after PAGE electrophoresis. (D) Mutations did not significantly affect FGF activity. Same amount of FGFs (10 ng / ml) were applied onto FGF-starved cells for 15 minutes, and protein was harvested to measure ERK phosphorylation. (E) In E8 (TGFβ) media, FGF1-3X sustained long-term ES cell culture. °CT4 staining was performed after 3 passages. ES cells were maintained in FGF1-3X media for more than 10 passages, and cells maintained normal karyotype. (F and G) FGF2 Heparin binding-site was mutated (K128N), and this mutation stabilized FGF2 proteins during 37°C treatment.
STEM_1021_sm_SuppFig3.pdf		234K	Figure S3. Dynamic regulation to maintain FGF pathway activity at relatively low level. (A) ERK phosphorylation tunes down after initial activation. FGF2 (100 ng / ml) was added to FGF-starved ES cells, and proteins were collected at specific time points for western blot. ERK phosphorylation was significantly lower than previous time points. (B) In the same set of experiments, growth media was collected from the cell culture and was applied to FGF-starved cells for 15 minutes, and proteins were collected for western blot. There was no significant loss of FGF2 activity in media at 12 hours. (C) ERK phosphorylation is controlled at consistently moderate level in continuous FGF culture. FGF2 was applied onto FGF-starved and FGF-primed cells, and cells were harvested at specific time points.
STEM_1021_sm_SuppFig4.pdf		129K	Figure S4. Specific FGF affects reprogramming efficiency in fibroblasts. Foreskin fibroblasts were reprogrammed with viral-free approach as previously described (Chen et al., 2011). Different FGFs (100 ng / ml) were used to replace FGF2 in culture media at each reprogramming stage. iPS colonies were scored 30 days after the transfection of reprogramming factors.

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