BioWorld International Correspondent

LONDON - The fields of tissue engineering and drug discovery are both set to benefit from a new method of surveying all the proteins activated in a cell by a given stimulus, such as a growth factor.

The technique, which involves analyzing which proteins undergo phosphorylation following delivery of a stimulus, could bring scientists closer to their goal of differentiating adult stem cells into various types of mature cells.

It could allow researchers to analyze exactly how existing drugs influence cellular metabolism. Because it also should shed light on what proteins and signalling pathways are responsible for changing the fate of a cell - whether it undergoes apoptosis or not, and whether it differentiates or not - the novel method also could reveal many targets for drugs to treat a range of diseases.

Matthias Mann, director of the Center for Experimental Bioinformatics at the University of Southern Denmark in Odense, told BioWorld International: "We think this work is a milestone. For the first time, we have been able to read out a control point of cell differentiation - how a stem cell decides to become a bone cell or not. We were then able to intervene and control the fate of the cell by manipulating that control point. This is what you want to be able to do with a drug."

Mann and his colleagues reported their study in the June 3, 2005, issue of Science in a paper titled "Mechanism of Divergent Growth Factor Effects in Mesenchymal Stem Cell Differentiation."

For their work, the group used human mesenchymal stem cells - stem cells from bone marrow. They do not give rise to blood cells, but in culture they can be stimulated using growth factors to differentiate into osteoblasts (bone cells), adipocytes (fat cells) and chondrocytes (cartilage cells).

Mann's team used a quantitative mass spectrometry technique based on a method called stable isotope labelling by amino acids in cell culture, or SILAC, to examine all the proteins in the human mesenchymal stem cells that were phosphorylated by tyrosine kinase when growth factors were added to the culture.

They found that the stem cells would differentiate into osteoblasts when epidermal growth factor (EGF) was added, but not when platelet-derived growth factor (PDGF) was added. The researchers identified 113 proteins that were phosphorylated by tyrosine kinase in response to the two growth factors, but fewer than 10 percent of those had undergone phosphorylation in response to just EGF or PDGF.

Further analysis showed that PDGF, but not EGF, activated the phosphatidylinositol 3-kinase (PI3K) pathway. Mann said: "Often in systems biology, people have made very complex models involving hundreds of differential equations to describe the differences in these sorts of pathways, but in this case, there was a stark difference - a relatively simple control point."

To check their results, they cultured the stem cells as normal before adding an inhibitor of the PI3K pathway at the same time adding PDGF. They found that, as they had expected, the cells behaved as though they had added EGF.

Future work by the team will involve improving the technique so that it can be carried out faster, and adapting it so that all the phosphorylation that takes place in a cell can be monitored - not just a subset, as in the current work.

Mann predicted that the method will have potential in many areas of biology, pharmacology and tissue engineering. He said: "It will be of great interest to take a drug like Gleevec, Herceptin or any drug that interferes with growth factor signalling and find out exactly what molecules in the cell it interferes with. You will be able to see how similar one drug is to another drug in terms of what signalling pathways it turns on or off."

Ultimately, he pointed out, drug discovery is about changing the fate of the cell. For drugs against cancer, for example, a helpful strategy would be to switch cells into the pathway toward apoptosis. Mass spectrometry proteomics now make it possible to identify exactly which signalling pathways are involved in apoptosis, and which drugs will redirect a cancer cell to commit suicide. "We can ask how a cancer cell evades the signal to undergo apoptosis," Mann added.

More immediately, the technique should provide information to make tissue engineering more successful. "Adult stem cells are already being used, in limited circumstances, for bone regeneration in the clinic. The discovery in this paper could boost the success of such strategies," Mann said.