Researchers identify phosphorylated signaling proteins in human embryonic stem cells

LA JOLLA, Calif.—A team of researchers from the BurnhamInstitute for Medical Research and The Scripps Research Institute (TSRI) havecompleted what is believed to be the first comparative, large-scalephosphoproteomic analysis of human embryonic stem cells (hESCs) and theirderivatives.

The resulting data may provide stem cell researchers with anunderstanding of the mechanisms that determine whether stem cells divide ordifferentiate, what types of cells they become and how to control those complexmechanisms 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 cellscientists," says Dr. Laurence M. Brill, senior scientist at Burnham'sproteomics facility. "The protein phosphorylation sites identified in thisstudy are freely available to the broader research community, and researcherscan use these data to study the cells in greater depth and determine howphosphorylation events determine a cell's fate."

Protein phosphorylation, the biochemical process thatmodifies protein activities by adding a phosphate molecule, is central to cellsignaling.

Using sophisticated phosphoproteomic analyses, the team ofBrill, 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 bythe worldwide stem cell community for development and testing of hypotheses onwhich proteins control the ability of stem cells to differentiate into all celltypes in the body.

"It suggests which proteins could help control specificdifferentiation of the cells to desired cell types. It also suggests proteinsthat are possible drug targets in order to help control cellular proliferationspecific differentiation and other cellular behaviors," he says.

Prior to this research, protein phosphorylation in hESCs waspoorly understood. Identification of these phosphorylation sites providesinsights into known and novel hESC signaling pathways and highlights signalingmechanisms that influence self-renewal and differentiation.

The researchers performed large-scale, phosphoproteomicanalyses of hESCs and their differentiated derivatives using multi-dimensionalliquid chromatography and tandem mass spectrometry. The researchers then usedthe phosphoproteomic data as a predictive tool to target a sample of thesignaling pathways that were revealed by the phosphorylated proteins in hESCs,with follow-up experiments to confirm the relevance of these phosphoproteinsand pathways to the cells.

The study showed that many transcription regulators such asepigenetic and transcription factors, as well as a large number of kinases arephosphorylated in hESCs, suggesting that these proteins may play a key role indetermining stem cell fate. Proteins in the JNK signaling pathway were alsofound to be phosphorylated in undifferentiated hESCs, which suggested thatinhibition of JNK signaling may lead to differentiation, a result that wasconfirmed in hESC cultures.

These methods were extremely useful to discover novelproteins relevant to the human embryonic stem cells. For example, the teamfound that phosphoproteins in receptor tyrosine kinase (RTK) signaling pathwayswere numerous in undifferentiated hESCs.

Follow-up studies used this unexpected finding to show thatmultiple RTKs can support hESCs in their undifferentiated state.

This research shows that phosphoproteomic data can be apowerful tool to broaden understanding of hESCs and how their ultimate fate isdetermined. With this knowledge, stem cell researchers may be able to developmore focused methods to control hESC differentiation and move closer toclinical therapies.

Brill points out that initially, the stem cells for thestudy were cultured at the Scripps Research Institute, and he carried out thephosphoproteomic analysis was carried out largely while at the GenomicsInstitute of the Novartis Research Foundation (GNF). Brill subsequently movedto the Burnham Institute.

"Further experiments involving more stem cell culture, toprovide additional validation of the phosphoproteomic analyses, were performedat The Burnham Institute," he adds. "The Burnham Institute/TSRI team thatcarried out the research is multidisciplinary, with expertise in analyticalchemistry, biology, and bioinformatics."

The scientists at TSRI and The Burnham Institute arecontinuing to collaborate, Brill points out.

"In addition, the San Diego (now named the Sanford)Consortium for Regenerative Medicine, in which the Burnham Institute, TSRI, theSalk Institute and UCSD have—and will continue to have—a long-standingcollaborative relationship, including sharing a new building that is currentlyunder construction," he says.

Brill says some of the next steps are to target additionalphosphorylated proteins that have already been identified, and the pathwaysthat these proteins participate in for promotion of improved stem cell culture.

"Similar studies will use stem cells that have beendifferentiated to more homogeneous populations of cells in order to understandwhich phosphorylated proteins work together to control that differentiation,"he says. "The results should guide procedures to improve specific stem celldifferentiation, as well as lead toward an improved ability to make the cellsdifferentiate more completely. Complete differentiation should decreasepotential risks from residual, undifferentiated cells."