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Researchers identify phosphorylated signaling proteins in human embryonic stem cells
LA JOLLA, Calif.—A team of researchers from the Burnham Institute for Medical Research and The Scripps Research Institute (TSRI) have completed what is believed to be the first comparative, large-scale phosphoproteomic analysis of human embryonic stem cells (hESCs) and their derivatives.
The resulting data may provide stem cell researchers with an understanding of the mechanisms that determine whether stem cells divide or differentiate, what types of cells they become and how to control those complex mechanisms to facilitate development of new therapies.
The study was published in the journal Cell Stem Cell.
"This research will be a big boost for stem cell scientists," says Dr. Laurence M. Brill, senior scientist at Burnham's proteomics facility. "The protein phosphorylation sites identified in this study are freely available to the broader research community, and researchers can use these data to study the cells in greater depth and determine how phosphorylation events determine a cell's fate."
Protein phosphorylation, the biochemical process that modifies protein activities by adding a phosphate molecule, is central to cell signaling.
Using sophisticated phosphoproteomic analyses, the team of Brill, Drs. Sheng Ding, associate professor at TSRI, and Evan Y. Snyder, professor and director of Burnham's Stem Cell and Regenerative Biology program, cataloged 2,546 phosphorylation sites on 1,602 phosphoproteins.
Brill says the data provides focused information for use by the worldwide stem cell community for development and testing of hypotheses on which proteins control the ability of stem cells to differentiate into all cell types in the body.
"It suggests which proteins could help control specific differentiation of the cells to desired cell types. It also suggests proteins that are possible drug targets in order to help control cellular proliferation specific differentiation and other cellular behaviors," he says.
Prior to this research, protein phosphorylation in hESCs was poorly understood. Identification of these phosphorylation sites provides insights into known and novel hESC signaling pathways and highlights signaling mechanisms that influence self-renewal and differentiation.
The researchers performed large-scale, phosphoproteomic analyses of hESCs and their differentiated derivatives using multi-dimensional liquid chromatography and tandem mass spectrometry. The researchers then used the phosphoproteomic data as a predictive tool to target a sample of the signaling pathways that were revealed by the phosphorylated proteins in hESCs, with follow-up experiments to confirm the relevance of these phosphoproteins and pathways to the cells.
The study showed that many transcription regulators such as epigenetic and transcription factors, as well as a large number of kinases are phosphorylated in hESCs, suggesting that these proteins may play a key role in determining stem cell fate. Proteins in the JNK signaling pathway were also found to be phosphorylated in undifferentiated hESCs, which suggested that inhibition of JNK signaling may lead to differentiation, a result that was confirmed in hESC cultures.
These methods were extremely useful to discover novel proteins relevant to the human embryonic stem cells. For example, the team found that phosphoproteins in receptor tyrosine kinase (RTK) signaling pathways were numerous in undifferentiated hESCs.
Follow-up studies used this unexpected finding to show that multiple RTKs can support hESCs in their undifferentiated state.
This research shows that phosphoproteomic data can be a powerful tool to broaden understanding of hESCs and how their ultimate fate is determined. With this knowledge, stem cell researchers may be able to develop more focused methods to control hESC differentiation and move closer to clinical therapies.
Brill points out that initially, the stem cells for the study were cultured at the Scripps Research Institute, and he carried out the phosphoproteomic analysis was carried out largely while at the Genomics Institute of the Novartis Research Foundation (GNF). Brill subsequently moved to the Burnham Institute.
"Further experiments involving more stem cell culture, to provide additional validation of the phosphoproteomic analyses, were performed at The Burnham Institute," he adds. "The Burnham Institute/TSRI team that carried out the research is multidisciplinary, with expertise in analytical chemistry, biology, and bioinformatics."
The scientists at TSRI and The Burnham Institute are continuing to collaborate, Brill points out.
"In addition, the San Diego (now named the Sanford) Consortium for Regenerative Medicine, in which the Burnham Institute, TSRI, the Salk Institute and UCSD have—and will continue to have—a long-standing collaborative relationship, including sharing a new building that is currently under construction," he says.
Brill says some of the next steps are to target additional phosphorylated proteins that have already been identified, and the pathways that these proteins participate in for promotion of improved stem cell culture.
"Similar studies will use stem cells that have been differentiated to more homogeneous populations of cells in order to understand which phosphorylated proteins work together to control that differentiation," he says. "The results should guide procedures to improve specific stem cell differentiation, as well as lead toward an improved ability to make the cells differentiate more completely. Complete differentiation should decrease potential risks from residual, undifferentiated cells."