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Researchers Report Most Extensive Phosphoproteome Study of Human Embryonic Stem Cells

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This story originally ran on Aug. 12.

By Tony Fong

Using phosphoproteomic technologies and methods, researchers in California have completed what they consider to be the most extensive study of the phosphoproteome of human embryonic stem cell tissue, providing a roadmap to better understand the mechanisms that control differentiation in stem cells and a basis for continued research leading to potentially new clinical therapies.

Described in a study published in the Aug. 7 issue of Cell Stem Cell, the research, which took four years, resulted in a catalog of 2,546 phosphorylation sites on 1,602 phosphoproteins, a several-fold increase over previous numbers. Prior to the study, the number of phosphorylation sites and phosphoproteins for human embryonic stem cells, or hESCs, numbered a "few dozen," Laurence Brill, first author on the study and senior scientist at the proteomics facility at the Burnham Institute for Medical Research, told ProteoMonitor this week.

In their study, Brill and his co-authors said that hESCs are "a model developmental system that may have potential clinical value for mitigating diseases," but the mechanisms involved in deciding whether stem cells divide or differentiate are "not well defined."

In addition to transcriptional and translational regulation, protein phosphorylation controls cell fate determination, but protein phosphorylation has not been well-characterized in pluripotent cells, which, because they are undifferentiated and capable of being manipulated into different cell types, are of the greatest interest for the purposes of clinical therapeutic development.

To address this, Brill and his colleagues performed a multidimensional liquid chromatography, mass spec-based phosphoproteomic analysis of undifferentiated hESCs and their differentiated derivatives. What they found was that a large number of regulators including epigenetic and transcription factors are phosphorylated in hESCs, suggesting proteins may play a crucial role in the fate of stem cells.

Although some proteins have previously been implicated in hESC renewal, their functions were unclear. By significantly increasing the number of known phosphorylation sites, "our results expanded the repertoire of pathways that facilitate hESC culture and support the suggestion that multiple signaling inputs are needed to maintain undifferentiated hESCs," the authors wrote.

While Brill said that stem cell-based therapeutics are still several years away, "as phosphoproteins controlling pluripotent behavior are understood better, methods for developing model systems with stem cells, and potential therapeutic applications, may become increasingly clear."

First Large-Scale Analysis

While stem cell research has been hyped as a new potential avenue for therapeutic development, the field has been relatively untouched by proteomics. In 2007, in an effort to get two fields that have largely ignored each other to work more collaboratively and to develop standards, the Human Proteome Organization and the International Society for Stem Cell Research jointly created an initiative called Proteome Biology of Stem Cells [see PM 06/29/07 and 10/11/07].

The work by Brill and his colleagues is the first comparative large-scale analysis of hESCs and their differentiated derivatives, and was enabled by advances in technology made only within the past few years, Brill said. Previously, people had studied one or a few specific proteins, "and then they looked at a few of the phosphorylation sites on those proteins and typically previously known phosphorylation sites as well."

But with improvements in separation technologies and in mass spectrometers — especially instruments with electron-transfer dissociation capability — interest has shifted toward more large-scale phosphoproteomics research. Still, because of the difficulty involved, only a small handful of groups are capable of doing large-scale phosphoproteomics "well," Brill said.

Phosphoproteomics is "highly challenging," he said, as is culturing human embryonic stem cells. "So putting the two disciplines together has been a multidisciplinary team effort that's taken a lot of work."

Brill and his co-researchers initially undertook a tyrosine phosphorylation-focused strategy that used antiphosphotyrosine immunoprecipitation. Tyrosine phosphorylation is a "dominant player" in cell signaling "and tends to be at the top of the hierarchy with the receptor tyrosine kinases and related proteins that they interact with and phosphorylate," Brill said. He and his colleagues had succeeded with that strategy with other cell lines, he added, but on the hESC line H1 used for their CSC study, "basically no data" was retrieved, though it remains unclear why.

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So instead, he and his colleagues turned to a total phosphoproteome approach, in which they took the total cellular complement of proteins and digested them with trypsin and then performed a multidimensional LC-MS analysis.

To their surprise, with that approach the researchers were still able to discover a vast amount of tyrosine phosphorylations even without enriching specifically for tyrosine phosphorylation in undifferentiated cells, implying that a variety of growth factors are important in the cells.

"And this is what some other groups are finding more and more recently as well, that a complex interaction of growth factors really maintains the undifferentiated cells in their pluripotent state," Brill said.

The researchers were also the first to find that the JNK pathway, an important signal transduction protein downstream of many receptor tyrosine kinases, was phosphorylated in undifferentiated hESCs, they said, and in hESC cultures, inhibition of JNK signaling led to differentiation.

Of the 2,546 non-redundant phosphorylation sites identified by the researchers, they report that 472 were on proteins containing more phosphorylation site identifications in undifferentiated hESCs, and 726 were on proteins containing more phosphorylation site identifications in differentiated hESC derivatives.

Of the 1,602 phosphoproteins identified, 382 contained more phosphorylation sites in undifferentiated hESCs, while 540 contained more phosphorylation sites in differentiated hESC derivatives. And among the 1,602 phosphoproteins were "hundreds" of novel ones whose presence in hESCs had been unknown, "providing a rich resource for further investigation," the authors wrote.

All the data from the research is available for free at the Proteomics Identifications Database, or PRIDE.

'More Rational Way'

The value of the study is that it provides a jumping-off point for more in-depth, targeted research, Brill said. And while therapies using stem cell technologies may be the golden fruit, at this point "we would hope to facilitate rational application rather than anything clinical per se," he said.

"The message [we're] trying to get across is that this is a big step closer, and this is a way to do it in a more rational way rather than a serendipitous approach. This gives us a lot of hypotheses about what proteins may be driving the cells," he said. "There's a lot more work to be done, but this should be a nice step toward clinical trials."

He said that some potential areas for follow-up research include the use of the data to improve the culturing of undifferentiated hESCs, and research into known phosphoproteins and specific differentiation pathways that could potentially control that differentiation.

Also, progress has been made recently in controlling undifferentiated cells into a more uniform population of differentiated cells, such as neuron cells, cardiac cells, and muscle cells. The data from the current study could be used to compare cells from a differentiated derivative with undifferentiated cells to investigate what proteins are involved in "driving more specific differentiation," Brill said.

He and his colleagues are working on applying the technology to cell types other than stem cells. "Protein phosphorylation is so critical to a variety of cell types and cellular behaviors," he said. However, they don't plan to do extensive follow-up work on hESCs until they can get additional funding. When the study began, there were restrictions on public funding for stem cell research, so it was funded privately.

The change in the White House administration has changed that, though. "I do feel more hopeful, tremendously so," about the chance of landing some federal grants for continuing work, Brill said.

He said that there still remain many phosphorylation sites and phosphoproteins to be discovered and other technologies that were not used in the study, such as TiO2 chromatography and soluble dendrimers, could be useful in expanding the phosphoproteome.

Brill's group at the Burnham Institute is "striving" to get a Thermo Fisher Scientific Orbitrap mass spec with ETD, Brill said.

"Proteomics is really an analytical chemists' domain and the biologist tends to understand it very poorly, if at all, but they're extremely interested in the data that it can yield," he said, adding he expects the CSC study to generate a buzz among stem cell biologists to "start targeting those proteins [of interest] in various hypothesis-driven experiments on their cells."

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