Reversible 3-D Cell Culture Gel Invented

Thixotropic three-dimensional gel to revolutionize cell culture Sunday, 28 September 2008 Singapore's Institute of Bioengineering and Nanotechnology (IBN), which celebrates its fifth anniversary this year, has invented a unique user-friendly gel that can liquefy on demand, with the potential to revolutionize three-dimensional (3D) cell culture for medical research. As reported in Nature Nanotechnology, IBN's novel gel media has the unique ability to liquefy when it is subjected to a moderate shear force and rapidly resolidifies into a gel within one minute upon removal of the force. This phenomenon of reverting between a gel and a liquid state is known as thixotropy. IBN's thixotropic gel is synthesized from a nano-composite of silica and polyethylene glycol (PEG) under room temperature, without special storage conditions. This novel material facilitates the safe and convenient culture of cells in 3D since cells can be easily added to the gel matrix without any chemical processes. According to IBN Executive Director Jackie Y. Ying, Ph.D.: "Cell culture is conventionally performed on a flat surface such as glass slides. It is an essential process in biological and medical research, and is widely used to process cells, synthesize biologics and develop treatments for a large variety of diseases.” "Cell culture within a 3D matrix would better mimic the actual conditions in the body as compared to the conventional 2D cell culture on flat surfaces. 3D cell culture also promises the development of better cell assays for drug screening," Dr. Ying added. Another key feature of IBN's gel is the ease with which researchers can transfer the cultured cells from the matrix by pipetting the required amount from the liquefied gel. Unlike conventional cell culture, trypsin is not required to detach the cultured cells from the solid media. As trypsin is an enzyme that is known to damage cells, especially in stem cell cultures, the long-term quality and viability of cells cultured using IBN's thixotropic gel would improve substantially without the exposure to this enzyme. Researchers are also able to control the gel's stiffness, thus facilitating the differentiation of stem cells into specific cell types. "Ways to control stem cell differentiation are important as stem cells can be differentiated into various cell types. Our gel can provide a novel method of studying stem cell differentiation, as well as an effective new means of introducing biological signals to cells to investigate their effect in 3D cultures," said Shona Pek, IBN Research Officer. Andrew Wan, Ph.D., IBN Team Leader and Principal Research Scientist, said: “Another interesting property of the gel is its ability to support the extracellular matrix (ECM) secretions of cells. Gel stiffness is modulated by ECM secretions, and can be used to study ECM production by cells responding to drug treatments or disease conditions.” "The thixotropic gel may then provide new insights for basic research and drug development," Dr. Wan added. About Institute of Bioengineering and Nanotechnology (IBN): IBN, a member of Singapore's Agency for Science, Technology and Research (A*STAR), was established in 2003. Massachusetts Institute of Technology (MIT) Professor Jackie Yi Ru Ying, 42, was hand-picked by then A*STAR Chairman Philip Yeo to lead the institute as its Executive Director. She has been on MIT's Chemical Engineering faculty since 1992, and was promoted to professor in 2001. She is among the youngest to be promoted to this rank at MIT. Under her direction, IBN conducts research at the cutting-edge of bioengineering and nanotechnology. Its programs are geared towards linking multiple disciplines across all fields in engineering, science and medicine to produce research breakthroughs that will improve healthcare and our quality of life. IBN's research activities are focused in the following areas:

• Drug and Gene Delivery, where the controlled release of various therapeutics involve the use of functionalized polymers and hydrogels for targeting diseased cells and organs, or for responding to specific biological stimuli.
• Cell and Tissue Engineering, where biomimicking materials, stem cell technology and bioimaging are combined to develop novel approaches to regenerative medicine and artificial organs.
• Pharmaceuticals Synthesis and Nanobiotechnology, which encompass the efficient catalytic synthesis of chiral pharmaceuticals, and new materials for sustainable technology and alternative energy generation.
• Biosensors and Biodevices, which involve nanotechnology and microfabricated platforms for the detection and treatment of diseases, and the synthesis and screening of biologics.

IBN's innovative research is aimed at creating new knowledge and intellectual properties in the emerging fields of bioengineering and nanotechnology, to attract top-notch researchers and business partners to Singapore. Since 2003, IBN researchers have produced a total of 436 papers published/in press, of which 177 were published in journals with impact factor greater than 3. IBN's work on hybrid magnetic-fluorescent nanoparticles, published in the Journal of the American Chemical Society in 2005, has received over 100 citations in three years. IBN also plays an active role in technology transfer and spinning off companies, linking the research institute and industrial partners to other global institutions. As of July 2008, IBN has filed 578 patent applications on its inventions and the Institute is currently looking for partners for collaboration and commercialization of its portfolio of technologies. IBN's current staff strength totals about 170 scientists, engineers and doctors. With its multinational and multidisciplinary research staff, the institute is geared towards generating new biomaterials, devices, systems, equipment and processes to boost Singapore's economy in the fast-growing biomedical sector. IBN is also committed to nurturing young minds, and the institute acts as a training ground for PhD students and undergraduates. In October 2003, IBN initiated a Youth Research Program to open its doors to university students, as well as students and teachers from various secondary schools and junior colleges. It has since reached out to more than 23,000 students and teachers from over 190 local and overseas schools and institutions. In 2008, IBN celebrates 5 years of innovative research. Reference: A Thixotropic Nanocomposite Gel for Three-Dimensional Cell Culture Y. Shona Pek, Andrew C. A. Wan, Asha Shekaran, Lang Zhuo & Jackie Y. Ying Nature Nanotechnology, Published online: 28 September 2008, doi:10.1038/nnano.2008.270 ......... ZenMaster

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Why US Standing In the World Are Declining

A short, provocative, outside view on US political future Sunday, 28 September 2008 When will you, American’s, realize that the 700-billion-dollar-bailout is related to the trillions of dollars in war spending Bush’s has demanded during his eight years in power! Moreover, all of them borrowed from the Chinese and Saudi’s and other oil rich dictators! China has used the last seven years to prepare for the Olympics, its presence in space and becoming the world’s production house for ‘everything’! During the same time, the US has shadow-fought al-Qaida in Afghanistan and Iraq, pretty much as Don Quixote de la Mancha was fighting windmills. In addition, Bush still did not gain any oil revenue’s from the Iraqi oil fields as he expected! While China was building new subways, air terminals, highways, high-speed railways, parks and space rockets for manned flights, paid by its domestic production, the US was building metal detectors and UAV’s and poorly armoured vehicles for its military personnel, paid by borrowed money. What else could you expect, than the present meltdown of your economic system, and finally loosing your status as super-power. Soviet Union needed a star-wars race to be exposed as the bankrupt economy and political system it was, US only needed two high-rise buildings to fall. ZenMaster

Gene Therapy Shrink Brain Tumours

Animal study supports creating zone of resistance in surrounding normal tissue. Thursday, 25 September 2008 Massachusetts General Hospital (MGH) researchers are investigating a new approach to gene therapy for brain tumours – delivering a cancer-fighting gene to normal brain tissue around the tumour to keep it from spreading. An animal study published in the journal Molecular Therapy, the first to test the feasibility of such an approach, found that inducing mouse brain cells to secrete human interferon-beta suppressed and eliminated growth of human glioblastoma cells implanted nearby. "We had hypothesized that genetically engineering normal tissue surrounding a tumour could create a zone of resistance – a microenvironment that prevents the growth or spread of the tumour," says Miguel Sena-Esteves, PhD, of the MGH Neuroscience Center, the study's senior author. "This proof of principle study shows that this could be a highly effective approach, although there are many additional questions that need to be investigated." Glioblastoma is the most common and deadly form of brain tumour. Human clinical trials of other gene therapies have not significantly reduced tumour progression. One problem has been that patients' immune systems target the viral vectors used to deliver cancer-eliminating genes. Another issue has been inefficient gene delivery, due in part to the inherent cellular diversity found within an individual patient's tumour as well as among tumours from different patients. In addition, if tumour cells are successfully induced to express an anticancer protein, production of that protein will drop as the tumour dies, allowing any cells that did not receive the gene to resume growing. In the current study the MGH team examined whether expression of a therapeutic gene in normal brain cells could form a stable and effective anti-tumour reservoir. The researchers first pre-treated immune-deficient mice by delivering a gene for human interferon-beta – a protein being tested against several types of cancer – into the animals' brains using adeno-associated virus vectors known to effectively deliver genes to neurons in the brain without the immune reaction produced by other vectors. Two weeks later, human glioblastoma cells were injected into the same or adjacent areas of the animal's brains. After only four days, mice expressing interferon-beta had significantly smaller tumours than did a control group pre-treated with gene-free vector. Two weeks after the glioblastoma cells were introduced, the tumours had completely disappeared from the brains of the gene-therapy-treated mice. Several additional experiments verified that the anti-tumour effect was produced by expression of interferon-beta in normal tissue. The same tumour growth suppression was seen when the genes were delivered to one side of the brain and tumour cells were injected into the other. Using a specialized vector that allows genes to be expressed only in neuronal cells and not the glial cells from which glioblastomas originate also produced similar results. While other gene therapy studies that have induced tumour regression in mouse models required several vector injections, these experiments were able to suppress growth and eliminate the implanted tumour with a single injection of the interferon-beta-encoding vector, underscoring the approach's effectiveness. "These results are particularly important as we build on our understanding of the microenvironments that allow tumours to grow and spread," explains Sena-Esteves, an assistant professor of Neurology at Harvard Medical School. "The therapeutic principle of genetically engineering normal brain tissue could be used to manipulate proteins required for that microenvironment, preventing tumours from migrating within the patient’s brain and escaping other therapies." The same zone-of-resistance approach could also be applied to the treatment of other solid tumours, he notes. Since interferon-beta treatment is known to have side effects, it will be important to identify any toxicity caused by long-term secretion of the protein in the brain and develop preventive strategies, such as turning off the introduced genes. Next, the MGH team is planning to test this strategy on glioblastomas that occur naturally in dogs, which could not only generate additional data supporting human trials but also develop veterinary treatments for canine patients. Reference: Preventing Growth of Brain Tumors by Creating a Zone of Resistance Casey A Maguire, Dimphna H Meijer, Stanley G LeRoy, Laryssa A Tierney, Marike LD Broekman, Fabricio F Costa, Xandra O Breakefield, Anat Stemmer-Rachamimov and Miguel Sena-Esteves Molecular Therapy (2008) 16 10, 1695–1702 doi:10.1038/mt.2008.168 ......... ZenMaster

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Novel Inhibitor of Human microRNA

Discovery points to new avenue for cancer treatment Thursday, 25 September 2008 Scientists at The Wistar Institute and their colleagues have identified, for the first time, a molecule that can regulate microRNAs – short strands of RNA that play a vital role in gene expression and are closely associated with cancer. The discovery points the way to the development of a new generation of cancer drugs. The research team identified a small molecule that blocks the pathway of a particular miRNA, called miR-21, which is implicated in brain cancer, as well as lung, colon, breast, and ovarian cancer. With further development, the molecule has the potential to boost patient response to existing chemotherapies, as well as to become a stand-alone cancer drug, says Wistar's Qihong Huang, M.D., Ph.D., co-senior author of the study. Although miRNAs were discovered less than two decades ago, their importance in regulating human development and disease is already clear. While the human genome is thought to contain 800 to 1,000 miRNAs, only a few hundred have been described. Thus, miRNAs represent a largely unexplored class of targets for the development of therapeutics and diagnostics, says Huang, an assistant professor in Wistar's Molecular and Cellular Oncogenesis Program. "This is a totally novel target," he says. "It's very understudied, and still in its infancy, but its potential is tremendous. Because miRNAs have the ability to shut down genes and prevent their expression, they may ultimately provide a target for therapies that are more selective than conventional chemotherapy drugs and have fewer side effects." Alexander Deiters, Ph.D., of North Carolina State University, co-directed the study. In regulating the molecular mechanisms behind gene expression, miRNAs can control the way in which whole chromosomes, or regions of chromosomes, are activated or deactivated. They are thought to directly regulate the expression of at least 30 percent of all human protein-encoding genes. miRNAs regulate protein synthesis by binding to the messenger RNAs that provide the recipe for protein construction. In doing so, the miRNAs repress the relevant protein's production. Misregulation of miRNAs can result in genes being over- or under-expressed, leading to cancer and other diseases. Huang notes that one sizeable hurdle in harnessing the power of miRNAs is getting "the right molecule into the right place at the right time" to regulate their function. "In terms of developing therapeutic agents for cancer, for example, we need to identify small molecules that can get into the bloodstream and get into the cells," he says. "The problem is, to date, no one had been able to show that such miRNA inhibitors exist." Huang and his colleagues developed a method to identify inhibitors of miRNA pathways in live human cells. The researchers created screening assays, or tests, to look for small molecules or compounds that selectively repress miRNA. They selected miR-21 as the target agent due to its documented role in preventing cell death – thereby allowing the unchecked cell proliferation associated with cancer – and its elevated levels in various cancers. The team designed an assay that contained the DNA binding sequence complementary to miR-21, bound to luciferase, the protein fireflies use to create light. Because miRNAs inhibit protein production, when miR-21 is functioning normally, it binds with the complementary sequence and inhibits the translation of luciferase, thus reducing the intensity of the light signal. "The idea was that when we add small molecules that inhibit the function of miR-21, the light signal will increase," Huang says. The scientists then screened a "library" of 1,000 compounds and found one molecule that inhibited miR-21 in the assay. The molecule, diazobenzene 2, decreased miR-21 levels by 80 percent and produced a nearly five-fold increase in the intensity of the light signal from the firefly protein. Groups of control cells and untreated cells showed no such signal when treated with the small-molecule inhibitor. The findings were published in the September 15 issue of the scientific journal Angewandte Chemie. Preliminary data from the researchers' ongoing studies suggest that the inhibitor could be used in combination with other chemotherapy drugs to provide a synergistic effect, Huang says. The researchers also will evaluate its potential as a stand-alone cancer drug. Huang and his colleagues are now conducting studies in mice to assess the inhibitor's effectiveness against brain, breast, and colon tumours, and they are working to modify the molecule to make it even more efficient. The screening test developed by the researchers provides a unique tool that can be used to advance investigations of miRNAs and their involvement in various diseases, Huang says. "The cell-based assay that we have established can potentially be used to screen for additional small-molecule inhibitors that can block miRNA," he notes. ......... ZenMaster

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Cloned Pigs with Cystic Fibrosis

Model to mimic human disease Thursday, 25 September 2008 In a first, researchers at the University of Iowa and the University of Missouri (MU) have developed a pig model for Cystic Fibrosis (CF) that appears to closely mimic the disease in human infants. The striking similarities between disease manifestations in the CF piglets and human newborns with CF suggest that this new model will help improve understanding of the disease and may also speed discovery of new treatments. The study is published in the Sept. 26 issue of Science. CF is a common hereditary disease that affects multiple organ systems, including the intestines, pancreas, and lung. Mice with CF-causing mutations have helped researchers learn more about this disease, however, differences in physiology and biology mean that mice with CF mutations do not develop many of the typical symptoms that affect humans with CF. Cystic Fibrosis (CF) continues to be a lethal disease for humans despite the identification of the problematic gene two decades ago. Many humans born with CF – the most common genetic disease in Caucasians - often die because of a lung disease developed later. Scientists have been unable to develop an animal model that develops the fatal lung disease. Now, a University of Missouri researcher is producing pigs born with cystic fibrosis that mimic the exact symptoms of a newborn with CF. The researchers are hopeful that these pigs will continue to mimic the human symptoms so the fatal lung disease can be studied and ultimately treated. "Right now, if you want to do experiments to find treatments or therapies for the lung disease that is fatal for people with CF, you would have to experiment on kids that have CF," said Randy Prather, distinguished professor of reproductive biotechnology in the MU College of Agriculture, Food and Natural Resources. "When the genetic mutation is introduced into mice, they do not display the symptoms of CF. That's why these new swine models are so important. We have been able to get them through the initial stages of the disease, which they display just like humans, and now we are just waiting for them to grow and potentially develop the lung disease so we can start experimenting in ways that have never been possible." Prather collaborated with Michael Welsh from the Howard Hughes Medical Institute at the University of Iowa. To create the genetic defect in pigs, a team led by Welsh made genetic modifications in pig cells. Prather's group then generated the genetically modified pigs from the cells using a process known as nuclear transfer. The pigs – called founder animals - that were produced carried only one copy of the mutated gene. Prather bred the pigs naturally and now many piglets have been born with CF. Once a litter is born, the piglets are immediately flown to Iowa where physicians who perform the corrective surgery on human newborns with CF do the same for the pigs. Meanwhile, MU researchers perform analysis during the transit to determine which piglets have the mutations "So far, all the mutations in the pigs have exactly mimicked the problems in humans born with CF," Prather said. "The whole cellular physiology of the pig is similar to humans. That's why having this break- through model is so exciting for the potential it has to move research on cystic fibrosis forward." "Lack of a better model has hampered our ability to answer long-standing questions in CF," explained Christopher Rogers, Ph.D., a former postdoctoral fellow in internal medicine at the UI Roy J. and Lucille A. Carver College of Medicine, and one of the study's lead authors. "The CF pig provides a unique opportunity to study one of the most common genetic diseases, and we hope to translate this new knowledge into better therapies and preventions." In addition to Rogers, co-lead authors of the study were David Stoltz, M.D., Ph.D., UI assistant professor of internal medicine, and David Meyerholz, D.V.M., Ph.D., UI assistant professor of pathology. The senior study author was Michael Welsh, M.D., UI professor of internal medicine and molecular physiology and biophysics, who holds the Roy J. Carver Chair of Internal Medicine and Physiology and Biophysics. Welsh also is a Howard Hughes Medical Institute investigator. CF occurs when a person inherits two mutated copies of the CFTR gene leading to loss of ion channel function that adversely affects many organs. To create the CF pigs, the researchers used gene targeting to disrupt one copy of the normal gene in pig cells. They then cloned these altered cells to produce pigs with only one good copy of the gene. Like human CF-carriers, these animals did not show disease symptoms. The pigs were then bred naturally, and about one in four of the piglets were born with two disrupted copies of the gene. The researchers established that piglets lacking CFTR have the abnormal ion channel activity that is a hallmark of CF disease. They also showed that the CF piglets develop the same disease characteristics that are commonly seen in newborn humans with CF, including a bowel obstruction known as meconium ileus, which often is the first sign of CF in humans. The pigs also have an abnormal pancreas, liver, and gall bladder, similar to CF patients. "Thus far, the clinical, physiological and age-related appearance of disease in the pigs, as well as the organs involved, mimic CF seen in people," Stoltz said. A primary cause of death and disability in patients with CF is lung disease. However, many questions remain about how infection and inflammation leads to lung damage. In the study, the lungs of the newborn CF pigs appeared similar to the lungs of their normal littermates and had no sign of infection or inflammation, possibly shedding some initial insight on the process. As the CF pigs mature and are exposed to airborne bacteria and viruses, the researchers hope to learn more about how and why lung disease develops in patients with CF. "Researchers can now begin to study the disease progression as it is happening, something not possible in humans," Meyerholz said. Reference: Disruption of the CFTR Gene Produces a Model of Cystic Fibrosis in Newborn Pigs Christopher S. Rogers, David A. Stoltz, David K. Meyerholz, Lynda S. Ostedgaard, Tatiana Rokhlina, Peter J. Taft, Mark P. Rogan, Alejandro A. Pezzulo, Philip H. Karp, Omar A. Itani, Amanda C. Kabel, Christine L. Wohlford-Lenane, Greg J. Davis, Robert A. Hanfland, Tony L. Smith, Melissa Samuel, David Wax, Clifton N. Murphy, August Rieke, Kristin Whitworth, Aliye Uc, Timothy D. Starner, Kim A. Brogden, Joel Shilyansky, Paul B. McCray, Jr., Joseph Zabner, Randall S. Prather, Michael J. Welsh Science 26 September 2008, Vol. 321. no. 5897, pp. 1837 – 1841, DOI: 10.1126/science.1163600 New Pig Model Could Improve Understanding of Cystic Fibrosis HHMI NEWS - September 26, 2008 ......... ZenMaster

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What to Do With Leftover Embryos in the US?

What to Do With Leftover Embryos in the US? Thursday, 25 September 2008 The majority of infertility patients are in favour of using leftover embryos for stem cell research and would also support selling leftover embryos to other couples, according to a recent survey. The survey is published in two related studies in the September issue of the journal Fertility and Sterility. The researchers surveyed 1,350 women who presented for infertility at a large, university hospital-based fertility centre in Illinois. The survey included 24 questions on patient demographics, obstetric and infertility history, and opinions about using extra embryos for stem cell research and selling extra embryos to other couples. Assisted reproductive technology has resulted in the creation and cryopreservation of extra embryos at fertility centres across the country. It was estimated in 2002 that 396,526 embryos were in storage at US fertility clinics, according to previously published research. These embryos may be used for future pregnancy attempts, donated to other couples or agencies, given to researchers, or discarded. Because infertility patients are the gatekeepers of these leftover embryos, it is important to understand their opinions, according to Dr. Tarun Jain, University of Illinois at Chicago assistant professor of reproductive endocrinology and infertility, clinical IVF director, and lead author of the study. When asked if using leftover embryos for stem cell research should be allowed, 73 percent of the 636 respondents who stated a definitive opinion answered yes. "Infertility patients, in general, are altruistic, and it makes sense that they would try to advance medicine and help others," said Jain. African Americans and Hispanics were less likely to approve of using leftover embryos for stem cell research, compared with Caucasians. Patients younger than 30, Protestant, less wealthy and single were also less likely to support using leftover embryos for stem cell research. The researchers also asked infertility patients if they would be willing to sell their extra embryos to other couples, a practice that is considered ethically unacceptable by the American Society for Reproductive Medicine and the American College of Obstetricians and Gynecologists. There is an emerging demand from infertility patients who cannot conceive using their own oocytes, or eggs, to purchase leftover, pre-existing embryos because it is a more cost-effective option than using an egg donor, according to the authors. When asked if selling leftover embryos to other couples should be allowed, 56 percent of the 588 respondents who stated a definitive opinion answered yes. Hispanics were less likely to approve of selling extra embryos when compared with Caucasians, but all East Indian respondents approved of the practice. Women who had never been pregnant were also less likely to approve, according to the study. The authors say this is the first survey to examine the opinions of a general infertility population related to the use of leftover embryos and to analyze the results based on the patients' socio-demographic and reproductive backgrounds. "Given the potential for a significant increase in the commoditizing of spare embryos, medical societies and policy makers may need to pay close attention to this controversial area," conclude Jain and co-author Stacey Missmer from Brigham and Women's Hospital and Harvard Medical School. ......... ZenMaster

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Well Protected Olympics!

Well Protected Olympics!

Outside the Olympic tennis court. © CellNEWS All rights reserved.

ZenMaster

Breakthrough in Spinal Injury Treatment

Breakthrough in Spinal Injury Treatment Friday, 19 September 2008 Researchers in Rochester, N.Y., and Colorado have shown that manipulating stem cells prior to transplantation may hold the key to overcoming a critical obstacle to using stem cell technology to repair spinal cord injuries. Research from a team of scientists from the University of Rochester Medical Center and the University of Colorado Denver School of Medicine, published today in the online Journal of Biology, may lead to improved spinal cord repair methods that pave the way for victims of paralysis to recover the use of their bodies without the risk of transplant-induced pain syndromes. The research focuses on a major support cell in the central nervous system called astrocytes. When nerve fibres are injured in the spinal cord, the severed ends of the nerve fibres fail to regenerate and reconnect with the nervous system circuitry beyond the site of the injury. During early development, astrocytes are highly supportive of nerve fibre growth, and scientists believe that if properly directed, these cells could play a key role in regenerating damaged nerves in the spinal cord. The Rochester team – which consists of biomedical geneticists Chris Proschel, Ph.D., Margot Mayer-Proschel, Ph.D., and Mark Noble, Ph.D. – are pioneers in manipulating stem cells to generate nervous system cells that can be used for therapeutic treatments. Rather than transplanting naïve stem cells, the team has adopted an approach of pre-differentiating stem cells into better defined populations of brain cells. These are then selected for their ability to promote recovery. Here glial restricted precursor (GRP) cells – a population of stem cells that can give rise to several different types of brain cell – were induced to make two different astrocyte sub-types using different growth factors that promote cell formation during normal development. Although these astrocytes are made from the same stem cell population, they apparently have very distinct characteristics and functions "These studies are particularly exciting in addressing two of the most significant challenges to the field of stem cell medicine – defining the optimal cell for repair and identifying means by which inadequately characterized stem cell approaches may actually cause harm," said Noble, who is also co-director of the New State Center of Research Excellence in Spinal Cord Injury, one of the primary funders of the research. The research team in Colorado, which consisted of Stephen Davies, Ph.D. and Jeannette Davies, Ph.D., transplanted the two types of astrocytes into the injured spinal cords of rats and found dramatically different outcomes. One type of astrocyte was remarkably effective at promoting nerve regeneration and functional recovery, with transplanted animals showing very high levels of new cell growth and survival, as well as recovery of limb function. However, the other type of astrocyte not only failed to promote nerve fibre regeneration or functional recovery but also caused neuropathic pain, a severe side effect that was not seen in rats treated with the beneficial astrocytes. Moreover, transplantation of the precursor cells themselves, without first turning them into astrocytes, also caused pain syndromes without promoting regeneration. Using signal molecules known to be involved in the generation of embryonic astrocytes during spinal cord development, the researchers were able to make pure cultures of two different types of astrocytes from the glial restricted precursor (GRP) cells. When the research team in Colorado, transplanted these two types of astrocytes into the injured spinal cord, they had dramatically different effects. One type of astrocyte called GDAsBMP was remarkably effective at promoting nerve regeneration and recovery of limb motion when transplanted into spinal cord injuries. However, the other type of astrocyte cell generated called GDAsCNTF, not only failed to promote nerve fibre regeneration or functional recovery but also caused neuropathic pain, a severe side effect that was not seen in rats treated with GDAsBMP. "To our knowledge, this is the first time that two distinct sub-types of astrocytic support cells generated from a common stem cell-like precursor have been shown to have robustly different effects when transplanted into the injured adult nervous system," said Mayer-Proschel. Transplantation of the stem cell-like precursor cells without first turning them into astrocytes, also caused pain syndromes and no spinal repair, Davies said. "It has long been a concern that therapies that promote growth of nerve fibres in the injured spinal cord would also cause sprouting in pain circuits," said Stephen Davies. "However by using the right astrocytes to repair spinal cord injuries we can have all the gains without the pain, while these other cell types appear to provide the opposite – pain but no gain." "These results emphasize the importance of astrocytes in controlling the outcome of neurological disease processes," said Proschel. "In addition, because transplants of undifferentiated stem cells harbour the risk of making deleterious astrocytes, it is important to understand their properties and how they might form. By being able to study different types of astrocytes derived from a common neural precursor, we are now underway to finding means of preventing the formation of the deleterious astrocyte type in the first place." The research teams considered the distinction between the effects of GDAsBMP, GDAsCNTF and GRP cells a "breakthrough" that might change the way stem cell technologies are used to repair spinal cord injuries. Controlling the development of stem cells immediately before transplanting them into injured spinal cords is essential because doctors cannot rely on the injured tissues of the body to create the right types of cells from "naïve" stem cells. Co-author Mark Noble said: "These studies are particularly exciting in addressing two of the most significant challenges to the field of stem cell medicine – defining the optimal cell for tissue repair and identifying means by which inadequately characterized approaches may actually cause harm.' To that end, the researchers are developing a safe, efficient and cost-effective way to make human GDAsBMP with an eye toward testing this new stem cell technology in humans.” The research teams in Denver and Rochester consider the dramatically dissimilar outcomes between the different astrocyte transplants a development that can change the way stem cell technologies are used to repair spinal cord injuries. To that end, the researchers are in the process of developing a safe, efficient and cost-effective way to use this approach to better define the optimal human astrocytes with an eye toward use for clinical trials. Refeerence: Transplanted astrocytes derived from BMP- or CNTF-treated glialrestricted precursors have opposite effects on recovery and allodynia after spinal cord injury Jeannette E Davies, Christoph Proschel, Ningzhe Zhang, Mark Noble, Margot Mayer-Proschel and Stephen JA Davies Journal of Biology 2008, 7:24, doi:10.1186/jbiol85 ......... ZenMaster

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Different Stem Cell Types Defined by Exclusive Combinations of Genes Working Together

Singapore scientists report findings in Cell Stem Cell Thursday, 18 September 2008 In the new issue of Cell Stem Cell, scientists report that the same transcription factor, which is crucial for the survival of different stem cell types, can behave differently. This study clearly showed for the first time that exclusive combinations of genes working together define different types of stem cells, and this is under the influence of a single key stem cell factor (called Sall4). The finding is timely since other researchers have recently revealed that specific genetic recipes can be used to turn non-stem cells into different stem cells that can be useful clinically. This finding reveals important insights about how scientists may be able to manipulate and engineer different stem cells for the treatment of human degenerative disorders. Understanding the behaviour of transcription factors, a class of gene regulators, helps pave the way for important advancements in stem cell technology and clinical research. Stem cells are important for the cell-based therapy of many degenerative tissue disorders. Each type of body tissue has its own unique type of stem cells whose behaviour is controlled by different sets of genes. Given the enormous complexity of each stem cell type and the underlying genetic bases for their unique purpose, it has been a major challenge for scientists to unravel the similarities and differences between the different stem cells. The latest research, led by Bing Lim, Senior Group Leader at the Genome Institute of Singapore (GIS), focused on identifying and understanding the functions of powerful genetic molecules, also known as "stem cell factors". Dr. Bing Lim said: "This new discovery has provided us with important new leads and ideas on how to grow and expand various stem cells for clinical research and treatment needs." Dr. Daniel Tenen, Professor of Medicine at Harvard Medical School, and Director for Cancer Research Centre of Excellence at the National University of Singapore, said: "These studies are of great significance, as they provide important clues as to how a single transcription factor might regulate different targets in different stem cells." Interestingly, this stem cell factor also appeared to be associated with certain diseases, particularly leukaemia. Dr. Li Chai, Instructor at the Department of Pathology at the Harvard Medical School, added that, "as Sall4 plays an important role in both normal hematopoietic stem cell function and in leukaemia stem cells, these findings may have clinical relevance; they may lead to understanding differences between normal and cancer stem cells." Genome Institute of Singapore (GIS): GIS is a member of the Agency for Science, Technology and Research (A*STAR). It is a national initiative with a global vision that seeks to use genomic sciences to improve public health and public prosperity. Established in 2001 as a centre for genomic discovery, the GIS will pursue the integration of technology, genetics and biology towards the goal of individualized medicine. The key research areas at the GIS include Systems Biology, Stem Cell & Developmental Biology, Cancer Biology & Pharmacology, Human Genetics, Infectious Diseases, Genomic Technologies, and Computational & Mathematical Biology. The genomics infrastructure at the GIS is utilized to train new scientific talent, to function as a bridge for academic and industrial research, and to explore scientific questions of high impact. Agency for Science, Technology and Research: The Agency for Science, Technology and Research, or A*STAR, is Singapore's lead agency for fostering world-class scientific research and talent for a vibrant knowledge-based Singapore. A*STAR actively nurtures public sector research and development in Biomedical Sciences, Physical Sciences and Engineering, with a particular focus on fields essential to Singapore's manufacturing industry and new growth industries. It oversees 14 research institutes and supports extramural research with the universities, hospital research centres and other local and international partners. At the heart of this knowledge intensive work is human capital. Top local and international scientific talent drive knowledge creation at A*STAR research institutes. The agency also sends scholars for undergraduate, graduate and post-doctoral training in the best universities, a reflection of the high priority A*STAR places on nurturing the next generation of scientific talent. Reference: Sall4 regulates distinct transcription circuitries in different blastocyst-derived stem cell lineages. Chin Yan Lim, Wai-Leong Tam, Jinqiu Zhang, Haw Siang Ang, Hui Jia, Leonard Lipovich, Huck-Hui Ng, Chia-Lin Wei, Wing Kin Sung, Paul Robson, Henry Yang and Bing Lim Cell Stem Cell, 10.1016/j.stem.2008.08.004 ......... ZenMaster

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RNA Interference Plays Bigger Role Than Previously Thought

IBM and Genome Institute of Singapore report in Nature Thursday, 18 September 2008 In a paper published online in the journal Nature, IBM and the Genome Institute of Singapore (GIS) reported findings from a joint research study that provides new information on how stem cell differentiation is controlled by microRNAs. The two teams have shown that microRNAs — small molecules that are an important regulatory component in the machinery of living cells — have roles that go well beyond what was previously thought. In 2006, IBM scientists developed a mathematical model that led to a conjecture about an expanded role for microRNAs. The team decided to test the hypothesis by focusing on mouse stem cells. IBM used computation to guide the experimental effort that GIS carried out. The work is expected to provide new insights on stem cell differentiation as well as on the role of microRNAs in cell process regulation and the onset of cancer, neurodegenerative disorders, diabetes and other diseases. The research is also expected to suggest future avenues for novel diagnostics and the development of therapeutics. "We have made yet another step towards understanding the intricate nature of microRNAs and the roles they play in the regulation of cellular processes," said Isidore Rigoutsos, manager of the Bioinformatics Group in IBM Research's Computational Biology Center. "The finding that microRNAs can extensively target locations in the amino acid coding regions of a transcript is an exciting discovery and reveals another important aspect of microRNA activity." GIS Senior Group Leader Bing Lim added: "We learn from this study that the targeting of coding regions by microRNAs can also have a real impact on cells. We observed that a single microRNA forced into the powerful embryonic stem cell can impose differentiation. This is exciting because one could envisage using microRNAs as a small molecule to control the differentiation of stem cells, or to make new stem cells. The fun part of this research was the visualization of a trend of thought from computational prediction all the way to cell transformation." Details of discovery: For more than a decade, microRNAs were assumed to interact primarily with their targets through the 3' un-translated region (3'UTR) of the targets' mRNA. The nucleotide sequences of the targeted locations were believed to be generally conserved across different organisms whereas interactions with mRNA regions beyond the 3'UTR were thought to be atypical. Some of the new research findings suggest that microRNA targets in the amino acid coding region (CDS) of a gene's mRNA may in fact be as frequent as those in the mRNA's 3'UTR, providing experimental evidence to a conjecture put forth in an earlier publication by the two teams. It also shows that a gene's CDS serves as template of microRNA targeting activity, in addition to its coding for the corresponding protein's amino acid sequence. Working with three microRNAs whose expression increases upon differentiation of mouse embryonic stem cells (ESCs), the teams showed that Nanog, Oct4 and Sox2, three transcription factors that are central to maintaining the pluripotency of mouse ESCs and determining the initiation of differentiation, are controlled through their CDS region by the three studied microRNAs. By introducing mutations at the identified target locations, the two teams showed that they could prevent the down-regulation of these transcription factors and delay stem cell differentiation. For the majority of the validated microRNA targets, their sequence is not conserved in the rhesus monkey and mouse counterparts of Nanog, Oct4 and Sox2. This suggests that seeking putative microRNA targets by aligning the instances of a gene across different organisms will underestimate the number of bona fide microRNA targets. Additionally, the studied microRNAs generally have multiple targets in the CDS region of the same gene possibly suggesting an underlying need for redundancy that can ensure the down-regulation of the intended target. Finally, several of the studied targets stride exon-exon junctions: this finding suggests that microRNAs play a role in the selective targeting of a gene's splice variants. "This discovery has vast implications for the role that computational models can play in biological science," said Ajay Royyuru, senior manager for the Computational Biology Center at IBM Research. "Computational biology allows scientists to develop theories using powerful computers and even preliminarily prove those theories prior to conducting experiments in wet labs – which reduce the time spent on trial and error throughout the process of scientific discovery." GIS Executive Director Edison Liu said: "This work is a great example of how future medical discovery will progressively require the joint efforts of computer scientists working in conjunction with biologists. The complexity of the control of human cells through regulatory networks demands computational modelling in order to decipher the signals from the noise. But in the end, it still boils down to doing the lab experiment." About IBM Research: IBM Research is the world's largest information technology research organization, with about 3,000 scientists and engineers globally. About Genome Institute of Singapore (GIS): GIS, a member of the Agency for Science, Technology and Research (A*STAR), is a national initiative with a global vision that seeks to use genomic sciences to improve public health and public prosperity. Established in 2001 as a centre for genomic discovery, the GIS pursues the integration of technology, genetics and biology towards the goal of individualized medicine. Key research areas include systems biology, stem cell & developmental biology, cancer biology & pharmacology, human genetics, infectious diseases, genomic technologies, and computational & mathematical biology. GIS' genomics infrastructure is utilized to train new scientific talent, function as a bridge for academic and industrial research and explore scientific questions of high impact. Reference: miRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation Yvonne Tay, Jinqiu Zhang, Andrew M. Thomson, Bing Lim & Isidore Rigoutsos Nature, 17 September 2008, doi:10.1038/nature07299 ......... ZenMaster

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Human Skin Cells Turned Into Insulin-producing Cells

Human Skin Cells Turned Into Insulin-producing Cells Thursday, 18 September 2008 Researchers at the University of North Carolina at Chapel Hill School of Medicine have transformed cells from human skin into cells that produce insulin, the hormone used to treat diabetes. The breakthrough may one day lead to new treatments or even a cure for the millions of people affected by the disease, researchers say. The approach involves reprogramming skin cells into pluripotent stem cells, or cells that can give rise to any other foetal or adult cell type, and then inducing them to differentiate, or transform, into cells that perform a particular function – in this case, secreting insulin. Several recent studies have shown that cells can be returned to pluripotent state using "defined factors" (specific proteins that control which genes are active in a cell), a technique pioneered by Dr. Shinya Yamanaka, a professor at Kyoto University in Japan. However, the UNC study is the first to demonstrate that cells reprogrammed in this way can be coaxed to differentiate into insulin-secreting cells. Results of the study are published online in the Journal of Biological Chemistry. "Not only have we shown that we can reprogram skin cells, but we have also demonstrated that these reprogrammed cells can be differentiated into insulin-producing cells which hold great therapeutic potential for diabetes," said study lead author Yi Zhang, Ph.D., Howard Hughes Medical Institute investigator, professor of biochemistry and biophysics at UNC and member of the Lineberger Comprehensive Cancer Center. "Of course, there are many years of additional studies that are required first, but this study provides hope for a cure for all patients with diabetes," said John Buse, M.D., Ph.D., president of the American Diabetes Association and professor and chief of the endocrinology division in the UNC School of Medicine's department of medicine. About 24 million Americans suffer from diabetes, a disease that occurs when the body is unable to produce or use insulin properly. Virtually all patients with type I diabetes, the more severe of the two types, must rely on daily injections of insulin to maintain their blood sugar levels. Recent research exploring a possible long-term treatment – the transplantation of insulin-producing beta cells into patients – has yielded promising results. However, this approach faces its own challenges, given the extreme shortage of matched organ donors and the need to suppress patients' immune systems. The work by Zhang and other researchers could potentially address those problems, since insulin-producing cells could be made from diabetic patients' own reprogrammed cells. Zhang is collaborating with Buse to obtain skin samples from diabetes patients. He said he hoped his current experiments will take this approach one step closer to a new treatment or even a cure for diabetes. Reference: Generation of insulin-secreting islet-like clusters from human skin fibroblasts Keisuke Tateishi, Jin He, Olena Taranova, Gaoyang Liang, Ana C. D'Alessio, and Yi Zhang J. Biol. Chem, 10.1074/jbc.M806597200 ......... ZenMaster

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Scientists Identify Genes Capable of Regulating Stem Cell Function

Animal model provides insight on pathways used for adult tissue maintenance and regeneration Wednesday, 17 September 2008 Scientists from The Forsyth Institute, Boston, MA, and the Howard Hughes Medical Institute at the University of Utah School of Medicine have developed a new system in which to study known mammalian adult stem cell disorders. This research, conducted with the flatworm planaria, highlights the genetic similarity between these invertebrates and mammals in the mechanisms by which stem cell regulatory pathways are used during adult tissue maintenance and regeneration. It is expected that this work may help scientists pursue pharmacological, genetic, and physiological approaches to develop potential therapeutic targets that could repair or prevent abnormal stem cell growth that can lead to cancer. In recent years, planarians have been recognized as a powerful model system in which to molecularly dissect conserved stem cell regulatory mechanisms in vivo. This research reveals that planaria are also a great model in which to study the molecular relationship between stem cells and cancer. The gene characterized in this study (PTEN) is one of the most commonly mutated genes in human cancers. As in human beings, genetic disturbance of the gene in planarians led to miss-regulation of cell proliferation resulting in cancer-like characteristics. These results indicate that some of the pattern control mechanisms that enable regeneration of complex structures may go awry in cancer. Abnormal stem cell proliferation in planarians is induced by genetic manipulation of conserved cellular signalling pathways. These abnormal cells can be specifically targeted without disturbing normal stem cell functions that support adult tissue homeostasis and regeneration. Importantly, this type of analysis could not be achieved in more traditional adult invertebrate model systems such as the fruit fly Drosophila and the nematode C. elegans. This research was published in the journal Disease Models & Mechanisms online on August 30. According to the paper's lead author, Dr. Néstor J. Oviedo, an Assistant Research Investigator in the Forsyth Center for Regenerative and Developmental Biology, this work provides new opportunities to expand knowledge of this regulatory molecule and the role it plays in cancer and tissue regeneration. "Our findings demonstrate that important signalling pathways regulating adult stem cell proliferation, migration and differentiation are evolutionarily and functionally conserved between planarians and mammals. Planarians are poised to not only advance the understanding of how diverse adult tissues are functionally maintained in vivo, but also will enhance our capabilities to identify, prevent, and remediate abnormal stem cell proliferation." Summary of Study The scientists have identified two genes, Smed-PTEN-1 and Smed-PTEN-2, capable of regulating stem cell function in the planarian Schmidtea mediterranea. Both genes encode proteins homologous to the mammalian tumour suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Inactivation of Smed-PTEN-1and -2 by RNA interference (RNAi) in planarians disrupts regeneration, and leads to abnormal outgrowths in both cut and uncut animals followed soon after by death (lysis). The resulting phenotype is characterized by hyperproliferation of neoblasts (planarian stem cells), tissue disorganization and a significant accumulation of post-mitotic cells with impaired differentiation capacity. Further analyses revealed that rapamycin selectively prevented such accumulation without affecting the normal neoblast proliferation associated with physiological turnover and regeneration. In animals in which PTEN function is abrogated, the HHMI/University of Utah and Forsyth researchers also detected a significant increase in the number of cells expressing the planarian Akt gene homolog (Smed-Akt). However, functional abrogation of Smed-Akt in Smed-PTENRNAi-treated animals does not prevent cell overproliferation and lethality, indicating that functional abrogation of Smed-PTEN is sufficient to induce abnormal outgrowths. Altogether, the data reveal roles for PTEN in the regulation of planarian stem cells that are strikingly conserved to mammalian models. In addition, the results implicate this protein in the control of stem cell maintenance during the regeneration of complex structures in planarians.

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The planarian Schmidtea mediterranea. Credit: A. Sánchez Alvarado.

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The PTEN molecules were originally identified and characterized in the laboratory of Dr. Alejandro Sanchez Alvarado, HHMI investigator and Professor of Neurobiology and Anatomy at the University of Utah School of Medicine. Dr. Sánchez Alvarado's is the paper's senior author. His laboratory is engaged in the identification of the molecular and cellular basis of animal regeneration. His laboratory's work on planarians has led to the establishment of this organism as an important model system to study stem cells, regeneration and tissue homeostasis. The Forsyth research team is led by Michael Levin, Ph.D., Senior Member of the Staff in The Forsyth Institute and the Director of the Forsyth Center for Regenerative and Developmental Biology. Through experimental approaches and mathematical modelling, Dr. Levin and his group examine the processes governing large-scale pattern formation and biological information storage during animal embryogenesis. The lab investigates mechanisms of signalling between cells and tissues that allow a living system to reliably generate and maintain a complex morphology. The Levin team studies these processes in the context of embryonic development and regeneration, with a particular focus on the biophysics of cell behaviour. ......... ZenMaster

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Embryonic Stem Cells Reduce Transplantation Rejection

Embryonic Stem Cells Reduce Transplantation Rejection Monday, 15 September 2008 Researchers have shown that immune-defence cells influenced by embryonic stem cell-derived cells can help prevent the rejection of hearts transplanted into mice, all without the use of immunosuppressive drugs. The University of Iowa and the Iowa City Veterans Affairs (VA) Medical Center finding has implications for possible improvements in organ and bone marrow transplantation for humans. The study results appeared Friday in the online journal PLoS ONE, published by the Public Library of Science. People who need bone marrow or solid organ transplantation must take immunosuppressive drugs that can cause side effects nearly as severe as the disease they have. They also can experience graft-versus-host disease, which can cause death. These problems are spurring researchers to develop methods to reduce transplantation rejection, said the study's principal investigator Nicholas Zavazava, M.D., Ph.D., professor of internal medicine and director of transplant research at the University of Iowa Carver College of Medicine. "The idea behind the study is to 'prep' a recipient's immune system to make it receptive to the eventual organ or bone marrow donor's genetic make-up," said Zavazava, who also is a staff physician with the Iowa City VA Medical Center. "The approach involves taking embryonic stem cells with the same genetic background as the donor from which the organ or bone marrow ultimately will come and adapting them into another type of stem cell that can be injected into the recipient." Specifically, the team treated mouse embryonic stem cells with a "cocktail" of growth factors, causing them to become blood stem cells. These cells express very low levels of so-called "transplantation antigens" and are therefore protected from immunological rejection. The researchers then injected the blood stem cells into the recipient mouse's blood circulation. These stem cells found their way into the recipient mouse's thymus, where, as happens in humans, the recipient's own bone marrow cells typically migrate and develop into immune-defence cells known as T-cells. With the donor-related blood stem cells now present in the thymus, the mouse recipient's own T-cells learned to recognize them as part of itself and therefore caused no rejection. These now 'donor-friendly' T-cells then circulated within the recipient mouse's blood, Zavazava explained. "When we then transplanted into the recipient mouse a donor mouse heart that had the same genetic make-up as the previously injected stem cells, the T-cells didn't reject the heart because they recognized it as compatible," Zavazava said. "If we could eventually use this approach for organ transplantation in humans, it would be a huge advantage over the method we're currently using," he added. In addition to its potential for organ transplantation treatment, the embryonic stem cell-based method might also have implications for treating bone marrow diseases such as leukaemia. Because a mouse is so small, it was not possible in the study to remove the animal's existing heart and replace it with another. Thus, to test for transplant success, the study approach involved leaving the original heart intact, transplanting a second functional heart into the abdomen and then linking the transplanted heart to the aorta. The UI study built on previous research led by Zavazava that focused on the concept of using embryonic stem cells as an alternative source of cells for traditional bone marrow transplantations. The current study was supported by grants from the National Heart, Lung and Blood Institute, a VA Merit Review and the Roche Organ Transplantation Research Foundation. Reference: ES-Cell Derived Hematopoietic Cells Induce Transplantation Tolerance Sabrina Bonde, Kun-Ming Chan, Nicholas Zavazava PLoS ONE 3(9): e3212. doi:10.1371/journal.pone.0003212 ......... ZenMaster

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Embryonic Stem Cells Repair Congenital Heart Defect

Potential therapy for inherited conditions Friday, 12 September 2008 Mayo Clinic investigators have demonstrated that stem cells can be used to regenerate heart tissue to treat dilated cardiomyopathy, a congenital heart defect. Publication of the discovery was expedited by the editors of Stem Cells and appeared online on the journal's Web site. The study expands on the use of embryonic stem cells to regenerate tissue and repair damage after heart attacks and demonstrates that stem cells also can repair the inherited causes of heart failure. "We've shown in this transgenic animal model that embryonic stem cells may offer an option in repairing genetic heart problems," says Satsuki Yamada, M.D., Ph.D., cardiovascular researcher and first author of the study. "Close evaluation of genetic variations among individuals to identify optimal disease targets and customize stem cells for therapy opens a new era of personalized regenerative medicine," adds Andre Terzic, M.D., Ph.D., Mayo Clinic cardiologist and senior author and principal investigator. How They Did It The team reproduced prominent features of human malignant heart failure in a series of genetically altered mice. Specifically, the "knockout" of a critical heart-protective protein known as the K-ATP channel compromised heart contractions and caused ventricular dilation or heart enlargement. The condition, including poor survival, is typical of patients with heritable dilated cardiomyopathy. Researchers transplanted 200,000 embryonic stem cells into the wall of the left ventricle of the knockout mice. After one month the treatment improved heart performance, synchronized electrical impulses and stopped heart deterioration, ultimately saving the animal's life. Stem cells had grafted into the heart and formed new cardiac tissue. Additionally, the stem cell transplantation restarted cell cycle activity and halved the fibrosis that had been developing after the initial damage. Stem cell therapy also increased stamina and removed fluid build-up in the body, so characteristic in heart failure. The researchers say their findings show that stem cells can achieve functional repair in non-ischemic (cases other than blood-flow blockages) genetic cardiomyopathy. Further testing is underway. Reference: Embryonic Stem Cell Therapy of Heart Failure in Genetic Cardiomyopathy Satsuki Yamada, Timothy J. Nelson, Ruben Crespo, Carmen Terzic, Xiao Ke Liu, Takashi Miki, Susumu Seino, Atta Behfar, Andre Terzic Stem Cells, published online July 31, 2008 ......... ZenMaster

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Keeping Nerve Axons on Target

A second career for a growth factor receptor Friday, 12 September 2008 Neurons constituting the optic nerve wire up to the brain in a highly dynamic way. Cell bodies in the developing retina sprout processes, called axons, which extend toward visual centres in the brain. They are lured by attractive cues and making U-turns when they take the wrong path. How they find targets so accurately is a central question of neuroscience today. Using the mouse visual system, a team of Salk Institute for Biological Studies investigators led by Dennis O'Leary, Ph.D., identified an unanticipated factor that helps keep retinal axons from going astray. They report in the Sept. 11 issue of Neuron that p75, a protein previously known to regulate whether neurons live or die, leads a double life as an axon guidance protein. "Historically, we thought that factors that mediate cell survival and those controlling axon guidance were part of two separate processes," says O'Leary, a professor in the Molecular Neurobiology Laboratory. "But in this study we show a direct interaction between these two systems." Collaborating with Kuo-Fen Lee, Ph.D., professor in the Clayton Foundation Laboratories for Peptide Biology, the O'Leary team observed a defect in mice genetically engineered to lack p75. Through their synaptic connections, retinal axons develop a two-dimensional map of the retina in their targets in the brain. In the mice lacking p75, retinal axons stopped short of their final target and formed a map that was shifted forward to the superior colliculus, a major visual centre in the brain. Such a defect in p75-null mice was puzzling: researchers have studied p75 for decades and found it associated with activities as varied as neuronal growth, survival, and degeneration. Axonal migration was not among them. Todd McLaughlin, Ph.D., a senior research associate in the lab and co-first author, says that insight came in an eureka moment: "We realized that what we were observing in these mice was similar to what would happen if you deleted a gene called ephrin-A from the retina." Unlike p75, ephrin-A was a well-characterized sender and receiver of axon guidance signals, but it lacked appendages normally seen on proteins controlling axon migration. p75, however, displayed those elements, suggesting that the proteins could pair up — one receiving the migration signal and the other transmitting it. The research team then turned to biochemical analyses and with the added expertise of Tsung Song, a research associate in Dr. Lee's lab, obtained evidence that supported this hypothesis. The group found that ephrin-A and p75 complexes in axonal membranes and showed that when activated they could generate the signals required to guide axons and develop their map in the brain.

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When immature neurons are placed on a microscopic running track, where flanking lanes are carpeted with repellent factors, their growing axons remain in their lanes (top). Neurons from mice lacking p75 are unreceptive to repulsive cues: when placed on the track, their axons meander all over the field, crossing lanes and running down repellent-covered stripes (bottom). Credit: Courtesy of Dr. Yoo-Shick Lim, Salk Institute for Biological Studies.

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But the clincher was the "stripe assay," a classical screen for guidance molecules that repel growing axons. In it, an immature neuron is placed on a microscopic running track, just as it starts to develop an axon. When flanking lanes are carpeted with repellent factors, the sprouting axon bursts from the block but remains in its lane like a well-coached runner, avoiding neighbouring tracks. Constructing tracks made from the repulsive factor sensed by ephrin-A, the researchers confirmed that axons from normal retinal neurons stayed in their lanes when flanked by the repellent. But neurons from mice lacking p75 were unreceptive to repulsive cues: when placed on the track their axons meandered all over the field, crossing lanes and running down repellent-covered stripes. Why retinal neurons missed the target in the p75-minus mice became clear: they lacked the cellular machinery to respond to critical repellent signals encountered in the brain and stopped migrating prematurely. Among its myriad functions, p75's new role is a critical one. "Repulsion is probably the dominant force in axon guidance and a stronger influence than attraction," explains McLaughlin, noting that providing axons with a lot of options is not the way to build a brain. "Attraction is like finding the best seat in an empty movie theatre, but repulsion is like picking the lone empty seat in a full theatre." "We have shown that ephrin-A cannot transduce an intracellular signal by itself and instead requires the co-receptor p75," summarizes Yoo-Shick Lim, Ph.D., a postdoctoral fellow in the O'Leary lab and co-first author. "This interaction could operate in numerous events in neural development." O'Leary believes that identifying mechanisms underlying developmental events is fundamental to understanding the basis of any biological disorder. "These studies establish that two distinct molecular systems, neurotrophins and axon guidance, both critical for neural development directly collaborate to develop neural connectivity”. “Findings such as these lend critical insight into how one might repair damage to the nervous system due to genetic defects, tumours or wounds to the brain or spinal cord," he says. "We hope one day to be able to repair these defects and get cells to form functional connections again." About The Salk Institute: The Salk Institute for Biological Studies in La Jolla, California, is an independent non-profit organization dedicated to fundamental discoveries in the life sciences, the improvement of human health and the training of future generations of researchers. Jonas Salk, MD, whose polio vaccine all but eradicated the crippling disease poliomyelitis in 1955, opened the Institute in 1965 with a gift of land from the City of San Diego and the financial support of the March of Dimes. Reference: p75NTR Mediates Ephrin-A Reverse Signaling Required for Axon Repulsion and Mapping Yoo-Shick Lim, Todd McLaughlin, Tsung-Chang Sung, Alicia Santiago, Kuo-Fen Lee, and Dennis D.M. O'Leary Neuron, Vol 59, 746-758, 11 September 2008 ......... ZenMaster

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China Produce Its First IVF Monkeys

Plan to engineer gene-modified monkeys later Thursday, 11 September 2008 Chinese scientists have created the country's first test-tube monkeys, which are said to be the first step to engineer gene-modified monkeys. "Our next step is to bring about more test-tube monkeys and eventually make gene-modified monkeys benefiting for medical research," said Dr. Sun Qiang, at the Shanghai Institute of Brain Functional Genomics (IBFG), East China Normal University in Shanghai. Sun's team capitalized on a few new technologies on stimulating more eggs from female monkeys and collecting semen and mammalian oocytes, as well as new ways of in vitro fertilization and embryo transplantation. The new technologies "can significantly improve the pregnancy rate and live birth of healthy baby monkeys," the scientist said. All seven new born monkeys are healthy, Sun said, with the oldest Lele, or Happiness, being aged at one and half years. Reference: Efficient reproduction of cynomolgus monkey using pronuclear embryo transfer technique Qiang Sun, Juan Dong, Wenting Yang, Yujuan Jin, Mingying Yang, Yan Wang, Philip L. Wang, Yinghe Hu and Joe Z. Tsien PNAS September 2, 2008 vol. 105 no. 35 12956-12960 ......... ZenMaster

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