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Too Hot for FGF? – Thermal Stability of FGF Protein is a Determinant Factor in Regulating Self-Renewal, Differentiation and Reprogramming in Human Pluripotent Stem Cells

From Stem Cells 
By Stuart P. Atkinson

Amongst the factors that are know to maintain pluripotent cell types, factors in the FGF pathway are perhaps the most important, as they have been implicated in cell survival, proliferation, pluripotency, and lineage determination during differentiation (Eisellova et alLanner and RossantLevenstein et alVallier et al and Xu et al). FGF2 is most commonly used to maintain self-renewal and pluripotency of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) (Akopian et al). However, there are several unknowns, such as the mechanisms behind FGF2 function and why high concentrations of FGF2 are required, questions which have been addressed in a study from the laboratory ofJames A. Thomson at the University of Wisconsin, USA (Chen and Gulbranson et al (2012)).

ERK1/2 phosphorylation was used as a sensitive assay for the effect of FGFR activation by FGF, signalling, which alongside TGFβ/NODAL pathway signaling is sufficient for pluripotency in humans (Chen et al (2011) and Vallier et al).FGF1, 2, 4, 6 and 9, but not TGFβ, led to ERK1/2 phosphorylation after short term incubation (15 minutes), and also to an upregulation in NANOG mRNA expression. However, FGF1 only functioned over this short time period and had no effect at 24 hours and it was demonstrated that pre-incubation of FGF1 (37oC for 6 hours) led to the loss of ERK1/2 phosphorylation activity and NANOG expression, while other FGFs retained this ability. This suggests that at 37oC FGF1 is not stable and further suggests that perhaps other FGF proteins are also unstable, when exposed to longer periods of 37oC. Indeed, it was next found that FGF2, important for the cultivation of hESCs in vitro, lost most of its activity after a 24 hour pre-incubation period at 37oC. Addition of Heparin, also known to aid the maintenance of pluripotency, was found to preserve FGF2 activity during this pre-incubation step, acting as an FGF2 protector rather than being able to recover lost activity. The authors next demonstrated that the loss of FGF2 activity was not due to degradation but in fact the aggregation of FGF2.

FGF1 effect on hESCs was then used as a model system to analyse the effects of thermal stability of proteins. FGF1 treatment every 24 hours was not sufficient to support hESC self-renewal, but more frequent feeding significantly improved short-term cell culture, suggesting that FGF1 stability, and not receptor specificity plays a major role in its function. However, addition of Heparin did not protect FGF1 function. Next, truncated FGF1 derivatives were studied and thermal stability of these derivatives was found to be improved by mutations at several amino acids (Q40, S47, and H93) while maintaining FGF activity (Zakrzewska et al). FGF1 proteins with different combinations of point mutations were studied, with all having the ability to sustain ERK phosphorylation in hESCs over a 24 hour period, whereas the wild-type FGF1 could not. Pre-incubation (37oC for 24 hours) led to the loss of function in the WT and single mutant FGF1-K112N, the loss of function in the triple mutant FGF1 (FGF1-3X) in the absence of Heparin (similar to FGF2) while the quadruple mutant FGF1 (FGF1-4X) was thermally stable independent of heparin. Indeed, incubation of FGF1-3X or FGF1-4X in place of FGF2 allowed for the sustained growth of hESCs with normal karyotype FGF2 and the FGF1-derivatives were also shown to significantly enhance reprogramming efficiency of iPSCs from fibroblasts.

Overall, this study provides evidence that thermal stability contributes to FGF specificity in stem cell pluripotency, differentiation and reprogramming, an important and relatively unstudied area.   High levels of expensive factor such as FGF2, required for ESC pluripotency, could be replaced by lower levels of FGF derivatives, allowing for better control of ESC growth and a reduction in running costs in ESC culture.

 

References

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