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New UK Group ‘Amplifies’ Proteomics/Stem Cell Research Into Cardiovascular Repair

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The newly formed Vascular Proteomics Group at the James Black Center at King’s College in London will use a portion of a £9 million ($17.7 million) grant from the British Heart Foundation to ratchet up its work looking into the role stem cells may play in cardiovascular repair — one of the comparatively few projects marrying proteomics with stem cell research.
 
It was not immediately clear how much of the grant, awarded in December, the group will use, though it comes in addition to a £600,000 grant it received from the BHF last month. It will use the smaller award to hire three additional people, bringing the total number of researchers and technicians it employs to 10, Manuel Mayr, the head of the group, told ProteoMonitor.
 
The group opened last June at a cost of about £900,000, paid for by a private foundation that Mayr did not want identified, as well as the BHF and the university, and has since been conducting proteomic research into cardiovascular disease. The two new grants will enable it to “amplify” those efforts, Mayr said.
 
Armed with a Thermo Fisher Scientific LTQ XL mass spectrometer with electron transfer dissociation capability, and an LTQ Orbitrap XL, Mayr’s group is studying whether cardiovascular stem cells can repair damaged vessels and tissues.
 
Thermo Fisher is collaborating with Mayr’s group, providing hands-on training for the mass specs, but no funding, a company official said.
 
“We’re not a core facility,” Mayr said of his group. “We’re not doing any kind of the routine stuff. We only use these instruments for cardiovascular research for very dedicated questions to solve cardiovascular problems.”
 
In particular, the group is researching paracrine signaling in stem cells and the role it may play in tissue repair.
 
Currently, clinical trials are being conducted in Europe in which patients with myocardial infarction are being injected with bone marrow progenitor cells. While many of the cells are immediately lost after injection with additional cell loss occurring in the months that follow, patients appear to clinically benefit from the injections. Why that is so is not fully understood, Mayr said.
 

“If the current markers are not sufficient to deliver this kind of analysis, we have to use proteomics to find new markers to really ensure that all of these cells are differentiated and that they are safe to be used either for grafts or for tissue repair.”

It has been hypothesized that the stem cell-mediated clinical benefits are not the result of “defective stem cell differentiation, but are actually triggered by paracrine factors,” released by stem cells into damaged tissues, he said.
 
The goal of Mayr’s group, he said, is to investigate how stem cells from different people differ. Most studies have been limited to isolating stem cells based on the expression of surface markers, “but this doesn’t tell us much about the cell … just that these cells express at this surface marker,” Mayr said. “It does not tell us anything about the activation state of the cell,” or why a stem cell injected in one patient will be activated but the same stem cell injected into another patient will not. 
 
But if the stem cells can be isolated and the paracrine signaling can be analyzed, “we get a more complete picture of what type of cells we are injecting into the patients,” Mayr said. While standard ELISAs can look at molecules one at a time, mass spectrometry can enable the scientists to “actually measure all of the molecules above a certain threshold in one go.
 
“And because the secretome is quite limited in its complexity, we can actually get down to levels of nanograms per milliliter, so we can measure cytokines, [and] we can measure chemokines, which would be very difficult to achieve in a complex proteome such as plasma,” he said.
 
Because proteomics allows for broad protein detection, if a researcher found unusual secreted proteins in a stem cell preparation, he “might actually think twice before” injecting those stem cells into a patient, Mayr said.
 
As it continues its research, Mayr’s group will isolate stem cells from patients, characterize their secreted factors, and try to determine which stem cells deliver benefits and which don’t.
 
“In the long-term, what we might want to achieve is not to give stem cells to patients because there are a lot of problems, a lot of kind of ethical problems and logistical problems,” he said. “But if you can identify certain factors [with] immediate benefits from a pharmacological point of view, it would be much better to deliver these proteins or these factors than the cells.”
 
ETD to Look at PTMs
 
The work of Mayr’s group is one of the comparatively few projects marrying proteomics with stem cell research. This summer, recognizing the potential opportunity for proteomics in the research area, the Human Proteome Organization and the International Society for Stem Cell Research kicked off an initiative to explore proteomic methods and technologies for the evaluation of stem cells in drug development work [See PM 10/11/07 and 06/21/07].
 
Last year, Mayr and his colleagues described a method they developed using CyDye and biotin labeling for the semi-quantitative comparison of membrane protein expression in different cell lines.
 
“Proteomics can be really helpful [in stem-cell research] because we’re not looking at one or two membrane proteins, and they’re not just relying on the classification of a handful of protein markers,” Mayr said. “We actually want to see that the proteomic profile of a stem cell-derived cell is similar to the mature cell, so that we can only use mature cells in grafts or in tissues.”
 
His group is working both with adult and embryonic stem cells. With the latter, they have to make sure that during differentiation all of the cells become differentiated: a single undifferentiated cell can result in a tumor.
 
“If the current markers are not sufficient to deliver this kind of analysis, we have to use proteomics to find new markers to really ensure that all of these cells are differentiated and that they are safe to be used either for grafts or for tissue repair,” Mayr said.
 
In addition to identifying proteins, his group is looking at post-translational modifications. 2D gels allows researchers to see the post-translational modifications and separate different isoforms of a molecule, he said, but in a shotgun proteomic approach, the modifications, or shifts, cannot be identified. In fact, most post-translational modifications are lost at the time of analysis on the mass spec, Mayr said.
 
With the ETD capability on the LTQ XL mass spec, however, his group hopes to assign post-translational modifications to protein spots on the 2D gel and then explain the shifts that occur under certain disease conditions.
 
“We know that they are occurring, but it’s sometimes very difficult to say that this particular modification caused the shift,” Mayr said. “In some cases, you may find a modification, but it still doesn’t prove that this modification is responsible for the shift because many other modifications [might have been] lost during the fragmentation process.”
 
According to Martin Hornshaw, Thermo Fisher’s European commercial marketing manager for proteomics and metabolomics life sciences mass spectrometry, Mayr will also have the chance to run some samples on the LTQ Orbitrap with ETD capability in April in the company’s Bremen, Germany, facility, making him one of the first people in the world to “get his hands” on the platform, which is still in development.
 
A physician by training, Mayr said that he sees proteomics moving away from merely searching for biomarkers to identifying biological functions.
 
“One of the key improvements for proteomics is trying to get away from biomarker research. I’ve been to so many proteomic conferences, and I’ve heard these biomarker talks over and over again,” Mayr said. “I think there’s a lot to be gained from actually using proteomics as a tool in biology and for medical research. And this is where we need not only mass spectrometry looking for biomarkers in plasma, but we also need biologists or medics using proteomics as a tool in their medical research.”

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