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The manipulation of human embryonic stem cells (hESCs) has enormous potential for drug screening, as well as for treating cancers and a variety of other diseases. Yet much remains to be learned about how to optimize the expansion and differentiation of hESCs into specific cell types. Andre Choo at the A*STAR Bioprocessing Technology Institute and co-workers at the Netherlands Proteomics Centre have now quantified early signaling events triggered by activation of a growth factor that is important for maintaining cultured hESCs in an undifferentiated state.
The culture supplement fibroblast growth factor-2 (FGF-2) is known to activate receptor tyrosine kinases, which are key players in the complex molecular circuitry that controls whether stem cells self-renew indefinitely or differentiate into any of the cell types found in the adult body. Dynamic changes in the way that protein signaling intermediates are tagged with phosphate groups on serine, threonine or tyrosine residues are known to control cell proliferation and development by ultimately changing patterns of gene expression and protein activity. However, quantitatively determining the sites and regulation of phosphorylation remains a major challenge. In addition, whereas previous studies have paid much attention to proteome-wide analysis of serine and threonine phosphorylation, relatively little work has been done on tyrosine phosphorylation.
Choo and his co-workers used mass spectrometry to show that exposure of FGF-2-starved cells to FGF-2 rapidly changed levels of phosphorylation at 735 tyrosine residues on 430 proteins. “We have done this at a scale unmatched by prior studies of tyrosine phosphorylation in stem cells,” says Choo. Besides known targets of FGF-2 signaling, such as the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3-K) pathways, they detected the activation of several other tyrosine kinase receptors such as epidermal growth factor, insulin, ephrin and vascular endothelial growth factor receptors. Moreover, increased phosphorylation of Src kinase substrates supports an emerging role of reorganization of the actin cytoskeleton in controlling pluripotency, and implicates FGF-2 in controlling this process.
“The routine use of hESC-based therapies will very likely require the development of chemically defined culture systems that direct hESC differentiation with sufficient efficiency to ensure near-homogenous preparations of differentiated cells,” explains Choo. “Reaching this goal will almost certainly depend on a better understanding of the environment required for sustained proliferation of undifferentiated hESCs.” By illuminating the poorly understood role of tyrosine phosphorylation in maintaining pluripotency, these new findings will help identify rational approaches to optimize differentiation protocols that may provide high yields of therapeutically important cell types.
The A*STAR-affiliated researchers contributing to this research are from the Bioprocessing Technology Institute.
