Feeding the Schwanns: New technique could bring cell therapy for nerve damage a step closer

ScienceDaily (Oct. 11, 2012) — A new way to grow cells vital for nerve repair, developed by researchers from the University of Sheffield, could be a vital step for use in patients with severe nerve damage, including spinal injury (1).
Schwann cells are known to boost and amplify nerve growth in animal models, but their clinical use has been held back because they are difficult, time-consuming and costly to culture.
The Sheffield team, led by Professor John Haycock, has developed a new technique with adult rat tissue which overcomes all these problems, producing Schwann cells in less than half the time and at much lower cost.
"The ability of Schwann cells to boost nerve growth was proved many years ago in animals, but if you want to use this technique with patients, the problem is: where do you get enough cells from?" says Professor Haycock, from the University's Department of Materials Science and Engineering.
"To reduce immune rejection, the cells have to be grown from the patient's own tissue. Of course, you want to take the smallest amount of tissue necessary, so the technique must be efficient. It must also be fast, so treatment can begin as soon as possible after injury. For clinical use, it must also provide pure Schwann cells. And finally, to make it viable, it has to be at a reasonable cost."
Existing methods for growing Schwann cells from adult tissue promote the growth of another type of cell, called fibroblasts, which swamp the Schwann cells, reducing the speed they grow and their numbers. This means that large amounts of tissue are needed at the outset, to grow sufficient cells for therapeutic use. It also requires extra purification stages added to the process, making it slow and costly -- taking up to 3 months to complete.
Professor Haycock and his team have come up with a very simple solution: feed the Schwann cells but starve the fibroblasts. The research, published October 11 in Nature Protocols
, uses an amino acid that only the Schwann cells can break down and feed off, and are able to produce a 97 per cent pure population of Schwann cells in a much shorter space of time -- just 19 days -- from a small sample of adult tissue.
Professor Haycock is confident the technique can be replicated in humans. His team are trialling same method using human nerve tissue, with results expected within the next 6 months.
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The above story is reprinted from materials provided by University of Sheffield, via EurekAlert!, a service of AAAS.
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Journal Reference:
Rossukon Kaewkhaw, Andy M Scutt, John W Haycock. Integrated culture and purification of rat Schwann cells from freshly isolated adult tissue. Nature Protocols, 2012; 7 (11): 1996 DOI: 10.1038/nprot.2012.118
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New glow for electron microscopy: Protein-labeling technique allows high-resolution visualization of molecules inside cells

ScienceDaily (Oct. 22, 2012) — The glowing green molecule known as green fluorescent protein (GFP) has revolutionized molecular biology. When GFP is attached to a particular protein inside a cell, scientists can easily identify and locate it using fluorescence microscopy. However, GFP can't be used with electron microscopy, which offers much higher resolution than fluorescence microscopy.

Chemists from MIT have now designed a GFP equivalent for electron microscopy -- a tag that allows scientists to label and visualize proteins with unprecedented clarity.

"With things that may appear only a few pixels across by fluorescence microscopy -- for example, a mitochondrion -- you can't make out any of the internal features. But with electron microscopy it's very easy to discern the intricate internal structures," says Jeff Martell, a graduate student in chemistry at MIT and lead author of a paper describing the new tag in the Oct. 21 online edition of Nature Biotechnology.

The new tag could help scientists pinpoint the locations of many cell proteins, providing new insight into those proteins' functions, according to the researchers.

Improving on nature

Dubbed APEX, the new tag is similar to naturally occurring proteins that have been tried as imaging labels for electron microscopy. Horseradish peroxidase (HRP) is one commonly used tag, but it works only in a few compartments of a cell. Other recently developed tags work throughout a cell but are technically challenging to use because they require light to be shined on the sample and oxygen to be bubbled through it.

To improve on these methods, the researchers started with a protein similar to HRP, called ascorbate peroxidase (APX). APX is more versatile than HRP because it can function within a cell's cytosol, in the main cavity of a cell.

Both HRP and APX belong to a class of enzymes called peroxidases, which remove an electron and a proton from other molecules in a process known as oxidation. Every peroxidase has different targets, and one of HRP's main targets is a molecule called DAB, which when oxidized can be visualized with electron microscopy. The researchers genetically engineered APX so that it would also target DAB.

To use this new APEX tag (for "engineered APX"), the researchers deliver, into a living cell, a small ring of DNA containing the APEX gene joined to the gene for the protein they plan to image. The cell then produces the target protein, bound to the APEX protein.

Next, the researchers need to deliver DAB, which is not normally found in cells. This delivery takes place during the process of "fixing," or stabilizing cells, which must be done before they can be imaged with electron microscopy.

When the APEX protein oxidizes DAB, it generates radicals that rapidly clump together into a tarlike polymer. That polymer can be detected through electron microscopy, allowing the researchers to pinpoint the location of the target protein.

Biological question resolved

To demonstrate the usefulness of their new tag, the researchers set out to resolve an open question regarding the location of a calcium channel protein discovered last year. Two research groups identified the protein and reported that it is located within mitochondria, but they had conflicting theories as to its precise location and orientation. Using the new imaging technique, the MIT-led team labeled the protein and determined that it is embedded in the inner mitochondrial membrane and faces into the innermost part of mitochondria, the mitochondrial matrix.

The team also showed that the new tag can label proteins throughout the cell -- not only within mitochondria but also in the nucleus, the endoplasmic reticulum and the cytosol.

Martell and Alice Ting, the Ellen Swallow Richards Associate Professor of Chemistry at MIT and senior author of the Nature Biotechnology paper, invented the new technology. Other authors who helped to test the tag and explore biological applications are Mark Ellisman, Thomas Deerinck and Gina Sosinsky of the University of California at San Diego, Yasemin Sancak and Vamsi Mootha of Harvard Medical School, and Thomas Poulos of the University of California at Irvine.

In current studies, the researchers are working on filling entire cells, such as neurons, with their imaging agent. This allows certain neurons in an electron microscope image to stand out, making it easier to trace the connections they make with other neurons. For that project, the MIT researchers are collaborating with Joshua Sanes, a professor of molecular and cellular biology at Harvard University, who says he believes the new labeling technology will be very useful.

"We want to find the exact connections that these cells are making, and APEX is a good way to label cells for electron microscopy. We can label specific types of cells and figure out how they fit into the neural circuitry," Sanes says.

Ting and Martell have filed for a patent on their imaging technology and are now working on making the APEX molecule more stable and better able to bind heme (an iron atom embedded in an organic compound), which is necessary for it to function properly.

The research was funded by the National Institutes of Health.

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The above story is reprinted from materials provided by Massachusetts Institute of Technology. The original article was written by Anne Trafton.

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Journal Reference:

Jeffrey D Martell, Thomas J Deerinck, Yasemin Sancak, Thomas L Poulos, Vamsi K Mootha, Gina E Sosinsky, Mark H Ellisman, Alice Y Ting. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nature Biotechnology, 2012; DOI: 10.1038/nbt.2375

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Breakthrough technique images breast tumors in 3-D with great clarity, reduced radiation

ScienceDaily (Oct. 22, 2012) — Like cleaning the lenses of a foggy pair of glasses, scientists are now able to use a technique developed by UCLA researchers and their European colleagues to produce three-dimensional images of breast tissue that are two to three times sharper than those made using current CT scanners at hospitals. The technique also uses a lower dose of X-ray radiation than a mammogram.

These higher-quality images could allow breast tumors to be detected earlier and with much greater accuracy. One in eight women in the United States will be diagnosed with breast cancer during her lifetime.

The research is published the week of Oct. 22 in the early edition of the Proceedings of the National Academy of Sciences.

The most common breast cancer screening method used today is called dual-view digital mammography, but it isn't always successful in identifying tumors, said Jianwei (John) Miao, a UCLA professor of physics and astronomy and researcher with the California NanoSystems Institute at UCLA.

"While commonly used, the limitation is that it provides only two images of the breast tissue, which can explain why 10 to 20 percent of breast tumors are not detectable on mammograms," Miao said. "A three-dimensional view of the breast can be generated by a CT scan, but this is not frequently used clinically, as it requires a larger dose of radiation than a mammogram. It is very important to keep the dose low to prevent damage to this sensitive tissue during screening."

Recognizing these limitations, the scientists went in a new direction. In collaboration with the European Synchrotron Radiation Facility in France and Germany's Ludwig Maximilians University, Miao's international colleagues used a special detection method known as phase contrast tomography to X-ray a human breast from multiple angles.

They then applied equally sloped tomography, or EST -- a breakthrough computing algorithm developed by Miao's UCLA team that enables high-quality image-reconstruction -- to 512 of these images to produce 3-D images of the breast at a higher resolution than ever before. The process required less radiation than a mammogram.

In a blind evaluation, five independent radiologists from Ludwig Maximilians University ranked these images as having a higher sharpness, contrast and overall image quality than 3-D images of breast tissue created using other standard methods.

"Even small details of the breast tumor can be seen using this technique," said Maximilian Reiser, director of the radiology department at Ludwig Maximilians University, who contributed his medical expertise to the research.

The technology commonly used today for mammograms or imaging a patient's bones measures the difference in an X-ray's intensity before and after it passes through the body. But the phase contrast X-ray tomography used in this study measures the difference in the way an X-ray oscillates through normal tissue rather than through slightly denser tissue like a tumor or bone. While a very small breast tumor might not absorb many X-rays, the way it changes the oscillation of an X-ray can be quite large, Miao said. Phase contrast tomography captures this difference in oscillation, and each image made using this technique contributes to the overall 3-D picture.

The computational algorithm EST developed by Miao's UCLA team is a primary driver of this advance. Three-dimensional reconstructions, like the ones created in this research, are produced using sophisticated software and a powerful computer to combine many images into one 3-D image, much like various slices of an orange can be combined to form the whole. By rethinking the mathematic equations of the software in use today, Miao's group developed a more powerful algorithm that requires fewer "slices" to get a clearer overall 3-D picture.

"The technology used in mammogram screenings has been around for more than 100 years," said Paola Coan, a professor of X-ray imaging at Ludwig Maximilians University. "We want to see the difference between healthy tissue and the cancer using X-rays, and that difference can be very difficult to see, particularly in the breast, using standard techniques. The idea we used here was to combine phase contrast tomography with EST, and this combination is what gave us much higher quality 3-D images than ever before."

While this new technology is like a key in a lock, the door will only swing open -- bringing high-resolution 3-D imaging from the synchrotron facility to the clinic -- with further technological advances, said Alberto Bravin, managing physicist of the biomedical research laboratory at the European Synchrotron Radiation Facility. He added that the technology is still in the research phase and will not be available to patients for some time.

"A high-quality X-ray source is an absolute requirement for this technique," Bravin said. "While we can demonstrate the power of our technology, the X-ray source must come from a small enough device for it to become commonly used for breast cancer screening. Many research groups are actively working to develop this smaller X-ray source. Once this hurdle is cleared, our research is poised to make a big impact on society."

These results represent the collaborative efforts of senior authors Miao, Bravin and Coan. Significant contributions were provided by co-first authors Yunzhe Zhao, a recent UCLA doctoral graduate in Miao's laboratory, and Emmanuel Brun, a scientist working with Bravin and Coan. Other co-authors included Zhifeng Huang of UCLA and Aniko Sztrókay, Paul Claude Diemoz, Susanne Liebhardt, Alberto Mittone and Sergei Gasilov of Ludwig Maximilians University.

The research was funded by UC Discovery/Tomosoft Technologies; the National Institute of General Medical Sciences, a division of the National Institutes of Health; and the Deutsche Forschungsgemeinschaft-Cluster of Excellence Munich-Centre for Advanced Photonics.

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The above story is reprinted from materials provided by University of California - Los Angeles. The original article was written by Melody Pupols.

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Journal Reference:

Yunzhe Zhao, Emmanuel Brun, Paola Coan, Zhifeng Huang, Aniko Sztrókay, Paul Claude Diemoz, Susanne Liebhardt, Alberto Mittone, Sergei Gasilov, Jianwei Miao, and Alberto Bravin. High-resolution, low-dose phase contrast X-ray tomography for 3D diagnosis of human breast cancers. Proceedings of the National Academy of Sciences, 2012; DOI: 10.1073/pnas.1204460109

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New technique for sorting live cells may expedite biomedical research

ScienceDaily (Oct. 17, 2012) — Researchers from North Carolina State University and University of North Carolina at Chapel Hill have developed a new technique that uses sound waves to rapidly separate selected collections of cells for use in biomedical research.

"We think this is important because it will make it faster and easier for researchers to sort out the live cells they need for research ranging from disease study to drug development," says Dr. Xiaoning Jiang, an associate professor of mechanical and aerospace engineering and adjunct professor of biomedical engineering at NC State and co-author of a paper on the work.

Biomedical research often focuses on how specific cell types respond to various chemicals or environmental factors. These cells are often grown in a liquid medium and on top of a collection of "micropallets," which are essentially small plastic platforms that sit on the substrate at the bottom of the container. Researchers then select the cells they want and detach the relevant micropallets, which can be removed for additional experimentation or analysis.

Current techniques for removing these micropallets rely on lasers or physical manipulation to separate the pallets from the substrate. But each approach has its drawbacks. Physical manipulation is a slow process, while the energy produced by lasers to release larger micropallets (e.g., a micropallet 500 micrometers in diameter) can inadvertently kill a significant number of the cells. Neither technique is efficient at detaching a significant number of large micropallets quickly.

The new technique from NC State uses ultrasound technology to release the micropallets. Specifically, it uses focused, relatively high-frequency sound waves that are translated into a wave of pressure within the substrate itself. When that wave of force hits a targeted micropallet, the pallet is lifted off the substrate and can be removed, together with its attached cells, for further study.

Using this technique, micropallets can be selectively released in less than a millisecond. This is not as fast as laser-based techniques, but is much faster than physical manipulation. However, the ultrasound technique has a viability rate of better than 90 percent, meaning that more than 90 percent of live cells survive the process. This is significantly better than existing techniques for the release of large-sized pallets, which can have viability rates of less than 50 percent.

Video: https://www.youtube.com/watch?v=pVIeabhU1OY

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The above story is reprinted from materials provided by North Carolina State University.

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Journal Reference:

Sijia Guo, Yuli Wang, Nancy Allbritton and Xiaoning Jiang. Ultrasound-induced release of micropallets with cells. Applied Physics Letters, Volume 101 / Issue 16 (2012) DOI: 10.1063/1.4757648

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