Human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) can proliferate without limit and have the potential to generate cell types in all three germ layers [1
]. These properties make them ideal model systems for studies of human embryogenesis, for drug discovery, and for clinic applications.
Human ESCs and iPSCs closely resemble mouse epiblast stem cells (EpiSCs) in embryogenesis [7
] and are maintained in cell culture by the six growth factors that activate fibroblast growth factor (FGF), TGF/Nodal, and Insulin/IGF pathways [9
]. The FGF pathway, in particular, has been implicated in all phases of mammalian ESC culture, such as cell survival, proliferation, pluripotency, and lineage determination during differentiation [10
]. Among the 18 FGF proteins in mammals [16
], FGF2 is most commonly used to maintain self-renewal and pluripotency of human ESCs and iPSCs [18
] and mouse EpiSCs [16
The FGF pathway is activated through the binding of FGF ligands to FGF receptors, which in turn may trigger the activation of various downstream signaling pathways such as the rat sarcoma (RAS)-mitrogen-activated protein (MAP) kinase pathway, the P-I-3 kinase-AKT pathway, and the phospholipase C (PLC)γ
]. Multiple factors may contribute to the differential regulation of various FGF proteins in vivo. First, each FGF protein has a different affinity to each of the four FGF receptors (FGFRs) that activate specific pathways [20
]. Second, the combination of differential expression of FGFs and FGFRs leads to specific physiological roles in specific tissues [17
]. However, even the existence of these multiple factors is not sufficient to explain how specific FGFs regulate certain processes in human ESC culture. To better modulate human pluripotent stem cells through FGF pathways, it is important for us to understand all the critical mechanisms potentially involved in determining FGF function.
FGF2’s ability to support pluripotency is just one of the unknowns of FGF regulation in stem cell culture. The high concentrations of FGF2 used to support pluripotency in defined long-term culture—up to 100 ng/ml—is also of interest because these concentrations are higher than those used on other cell types, which usually range from 1 to 10 ng/ml [18
]. It has also been suggested that FGF signaling is dosage-dependent, and that a high level of FGF2 is probably required to satisfy a specific signaling threshold [24
] or to prevent inhibitions such as protein degradation [15
]. Furthermore, heparin and heparan sulfate were reported to promote pluripotency [25
]. It remains unclear how heparin and heparan sulfate directly function through the FGF pathway to regulate human ESCs and iPSCs.
Given the importance of the FGF pathway to pluripotency and differentiation in human ESCs, we decided to perform a systematic study of FGF proteins. Our goal was to identify novel regulatory mechanisms that contribute to the function of FGF proteins in human ESCs and iPSCs. In addition to identifying a specific set of FGFs that activate FGFR in human ESCs, we found that thermal stability is another deciding factor in determining a specific FGF’s capacity to support self-renewal. We demonstrated that modulating FGF stability with heparin or point mutation could significantly impact FGF’s ability to control various aspects of stem cell culture, ranging from pluripotency and differentiation to reprogramming. Our studies thus provide a new strategy: manipulating human ESCs by altering FGF thermal stability.