Friday, 26 February 2010

Microvesicles Important Mediators of Cell-to-cell Communication

Study offering hope for tissue regeneration and sheds new light on how body repairs itself when organs become diseased Friday, 26 February 2010 Researchers at Rhode Island Hospital have discovered how cells communicate with each other during times of cellular injury. The findings shed new light on how the body repairs itself when organs become diseased, through small particles known as microvesicles, and offers hope for tissue regeneration. The paper is published in the March 2010 edition of the journal Experimental Hematology and is now available online in advance of publication. Lead author Jason Aliotta, MD, a physician researcher in the pulmonary/critical care and haematology/oncology departments at Rhode Island Hospital, and his colleagues focused their work on the microvesicles. These particles are several times smaller than a normal cell and contain genetic information such as messenger ribonucleic acid (RNA), other species of RNA and protein. The paper shows a novel mechanism by which the cells communicate with each other through these microvesicles. During times of cellular injury or stress, or with certain diseases like cancer, infections and cardiovascular disease, these particles are shed and then taken up by other cells in the body. The genetic information and protein in the microvesicles helps to reprogram the accepting cell to behave more like the cell from which the particle was derived. Aliotta is also an assistant professor of medicine at The Warren Alpert Medical School of Brown University and a physician with University Medicine Foundation, Inc. He says: "What we attempted to understand is how cells within the bone marrow are able to repair organs that are unrelated to those bone marrow cells, such as the lung. Our work suggests that when the lung is injured or diseased and cells within the lung are stressed or dying, they shed microvesicles. Those microvesicles are then consumed by cells within the bone marrow, including stem cells, which are present in small numbers within the circulatory system. Those bone marrow cells then turn into lung cells." Other researchers have reported similar findings over the last couple of years, however, microvesicles have been known about for over 40 years and have often been considered irrelevant. "We are now recognizing the relevance of microvesicles: They are important mediators of cell-to-cell communication. What is unique to our research is the finding that microvesicles not only supply information to stem cells with lung injury, but this process also occurs in other organs as well, like the heart, liver and brain," Aliotta adds. The researchers report unique findings, noting that the change in those stem cells that have consumed microvesicles made by injured lung cells is very stable – the change appears to be permanent. Stem cells are reprogrammed due to the transfer of microvesicle-based transcription factors. These factors cause cells to behave atypically. "This would be relevant to any type of disease – if you want to repair damaged tissue, these microvesicles potentially provide a durable fix, and the hope is that it would be fixed forever," Aliotta says. The study is part of ongoing stem cell research at Rhode Island Hospital under the direction of Peter Quesenberry, MD, director of haematology/oncology at Rhode Island Hospital, who is a co-author on the paper. He is the principal investigator for a recent $11 million Center of Biomedical Research Excellence (COBRE) grant to Rhode Island Hospital from the National Center for Research Resources of the National Institutes of Health (NIH). "We believe this research presents a novel finding in the understanding of stem cells and signifies practical implications for the world of medicine. These microvesicles can change the basic nature of adjoining cells, and that presents a world of possibilities in tissue restoration efforts." Quesenberry says. Quesenberry, who is a physician with University Medicine Foundation, Inc., also holds the Paul Calabresi, MD, professorship in oncology and is director of the division of haematology/oncology at Alpert Medical School. Among the practical implications from their findings is an understanding of the mechanism of tissue repair and determining whether or not microvesicles can be used in a therapeutic fashion. "If you have an injured organ, our hope is that if we were to deliver large numbers of microvesicles to that injured organ, it would help the repair process," Aliotta explains. Based on their findings, the researchers also hypothesize that microvesicles could potentially be mediators of cancer metastasis. It is known that in cancer, there are higher levels of circulating microvesicles, and these microvesicles may be responsible for transferring the traits of the cancer to other organs. "If we can define the microvesicles that are shed from cancer cells, we can identify unique characteristics, which might help us to block their uptake into normal cells. This could, in theory, stop the metastasis of cancer," Aliotta notes. "Our work explained in this paper and the work still to come from our COBRE grant hold great promise in terms of future treatment of tissue repair and cancer," Quesenberry concludes. ......... ZenMaster

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Friday, 19 February 2010

NIH Stem Cell Guidelines Should Be Modified

NIH Stem Cell Guidelines Should Be Modified Friday, 19 February 2010 Bernard Lo, MD..A UCSF team, led by bioethicist Bernard Lo, MD, recommends that the National Institutes of Health ethics guidelines for embryonic stem cell research be modified to better protect the rights of individuals donating egg or sperm to patients undergoing in vitro fertilization. The recommendation is reported in the February 19, 2010 issue of Science. Third parties frequently donate sperm and egg, or “gametes,” for patients attempting to create embryos in the in vitro fertilization clinic. Under current practice in the United States, gamete donors sign a form giving the IVF patient unrestricted legal authority to determine how to dispose of any embryos that may be leftover following fertility treatments. Donor banks and IVF clinics are not required to brief gamete donors about the various options for disposition, which include donating the embryos for stem cell research, thereby enabling scientists to derive new human embryonic stem cell lines; discarding the embryos, or donating them to other IVF patients. While many state, national, and international scientific committees and agencies have recommended that third-party gamete donors give formal “informed consent” for stem cell research with embryos remaining after infertility treatment, the NIH did not stipulate this requirement in its guidelines issued in March 2009. As these guidelines determine which human embryonic stem cell (hESC) lines may be studied under NIH research grants – which are expected to play a growing role in funding stem cell research – the ethical implications are significant, says Lo, chair of the UCSF Gamete, Embryonic Stem Cell Research Committee, members of which published the Science paper. “We urge the NIH to revise its guidelines to require that gamete donors be advised that embryos containing their sperm or egg could be used for embryonic stem cell research, before they grant dispositional authority over embryos to the IVF patient,” he says. “Because some gamete donors may not approve of embryonic stem cell research, we consider this the ethically appropriate position.” In their paper, the team recommends a process that is less complex than the detailed “informed consent” process carried out when IVF patients donate embryos for research. They suggest the disclosure to gamete donors may be made through oral discussion or brochures before donors sign a form authorizing the IVF patient to determine the disposition of embryos. Importantly, says Lo, the gamete donors’ instructions would not disrupt the IVF process. IVF patients would learn of a gamete donor’s restrictions in advance of selecting embryos for IVF treatment, and could select other gamete donors if not satisfied with the donors’ disposition restrictions. The recommendation is consistent with that of the National Academy of Sciences and the International Society for Stem Cell Research says Lo, a member of the ethics committee of the ISSCR, and the co-chair of the Standards Working Group of the California Institute for Regenerative Medicine. “It would be highly desirable to have consistency among standards and regulations,” he says. “If such harmonization were achieved, many university Institutional Review Boards and other research oversight bodies would likely allow NIH-eligible human embryonic stem cell lines to be used for any otherwise acceptable hESC research.” “It’s critical that we consider all parties involved in the creation of embryos and honor their wishes,” says co-author Arnold Kriegstein, MD, PhD, director of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF. “The field of human embryonic stem cell research offers enormous promise for patients suffering from devastating diseases. We want to build this field on an ethical foundation of which we can be proud.” Exceptions to the guideline could be justified for hESC lines already in existence if there were strong scientific reasons to use the cell lines and the third-party gamete donor had granted rights to the IVF patient to determine disposition of the embryos. Reference: NIH Guidelines for Stem Cell Research and Gamete Donors Bernard Lo, Lindsay Parham, Marcelle Cedars, Susan Fisher, Elena Gates, Linda Giudice, Dina Gould Halme, William Hershon, Arnold Kriegstein, Radhika Rao, Clifford Roberts, and Richard Wagner Science 19 February 2010, Vol. 327. no. 5968, pp. 962 – 963, DOI: 10.1126/science.1180725 ......... ZenMaster

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Sunday, 7 February 2010

Virus-free Technique Easily Makes Pluripotent Stem Cells

Virus-free Technique Easily Makes Pluripotent Stem Cells Sunday, 07 February 2010 Tiny circles of DNA are the key to a new and easier way to transform stem cells from human fat into induced pluripotent stem cells for use in regenerative medicine, say scientists at the Stanford University School of Medicine. Unlike other commonly used techniques, the method, which is based on standard molecular biology practices, does not use viruses to introduce genes into the cells or permanently alter a cell's genome. It is the first example of reprogramming adult cells to pluripotency in this manner, and is hailed by the researchers as a major step toward the use of such cells in humans. They hope that the ease of the technique and its relative safety will smooth its way through the necessary FDA approval process. "This technique is not only safer, it's relatively simple," said Stanford surgery professor Michael Longaker, MD, and co-author of the paper. "It will be a relatively straightforward process for labs around the world to begin using this technique. We are moving toward clinically applicable regenerative medicine." The Stanford researchers used the so-called mini-circles - rings of DNA about one-half the size of those usually used to reprogram cell - to induce pluripotency in stem cells from human fat. Pluripotent cells can then be induced to become many different specialized cell types. Although the researchers plan to first use these cells to better understand - and perhaps one day treat-human heart disease, induced pluripotent stem cells, or iPS cells, are a starting point for research on many human diseases. "Imagine doing a fat or skin biopsy from a member of a family with heart problems, reprogramming the cells to pluripotency and then making cardiac cells to study in a laboratory dish," said cardiologist Joseph Wu, MD, PhD. "This would be much easier and less invasive than taking cell samples from a patient's heart." Wu is the senior author of the research, which will be published online Feb. 7 in Nature Methods. Research assistant Fangjun Jia, PhD is the lead author of the work. Longaker is the deputy director of Stanford's Institute for Stem Cell Biology and Regenerative Medicine and director of children's surgical research at Lucile Packard Children's Hospital. Wu is an assistant professor of cardiology and of radiology, and a member of Stanford's Cardiovascular Institute. A third author, Mark Kay, MD, PhD, is the Dennis Farrey Family Professor in Pediatrics and professor of genetics. The finding brings together disparate areas of Stanford research. Kay's laboratory invented the mini-circles several years ago in a quest to develop suitable gene therapy techniques. At the same time, Longaker was discovering the unusual prevalence and developmental flexibility of stem cells from human fat. Meanwhile, Wu was searching for ways to create patient-specific cell lines to study some of the common, yet devastating, heart problems he was seeing in the clinic. "About three years ago Mark gave a talk and I asked him if we could use mini-circles for cardiac gene therapy," said Wu. "And then it clicked for me, that we should also be able to use them for non-viral reprogramming of adult cells." The mini-circle reprogramming vector works so well because it is made of only the four genes needed to reprogram the cells (plus a gene for a green fluorescent protein to track mini-circle -containing cells). Unlike the larger, more commonly used DNA circles called plasmids, the mini-circles contain no bacterial DNA, meaning that the cells containing the mini-circles are less likely than plasmids to be perceived as foreign by the body. The expression of mini-circle genes is also more robust, and the smaller size of the mini-circles allows them to enter the cells more easily than the larger plasmids. Finally, because they do not replicate they are naturally lost as the cells divide, rather than hanging around to potentially muck up any subsequent therapeutic applications. The researchers chose to test the reprogramming efficiency of the mini-circles in stem cells from human fat because previous work in Wu and Longaker's lab has shown that the cells are numerous, easy to isolate and amenable to the iPS transformation, probably because of the naturally higher levels of expression of some reprogramming genes. They found that about 10.8 percent of the stem cells took up the mini-circles and expressed the green fluorescent protein, or GFP, versus about 2.7 percent of cells treated with a more traditional DNA plasmid. When the researchers isolated the GFP-expressing cells and grew them in a laboratory dish, they found that the mini-circles were gradually lost over a period of four weeks. To be sure the cells got a good dose of the genes, they reapplied the mini-circles at days four and six. After 14 to 16 days, they began to observe clusters of cells resembling embryonic stem cell colonies - some of which no longer expressed GFP. They isolated these GFP-free clusters and found that they exhibited all of the hallmarks of induced pluripotent cells: they expressed embryonic stem cell genes, they had similar patterns of DNA methylation, they could become multiple types of cells and they could form tumours called teratomas when injected under the skin of laboratory mice. They also confirmed that the mini-circles had truly been lost and had not integrated into the stem cells' DNA. Altogether, the researchers were able to make 22 new iPS cell lines from adult human adipose stem cells and adult human fibroblasts. Although the overall reprogramming efficiency of the mini-circle method is lower than that of methods using viral vectors to introduce the genes (about 0.005 percent vs. about 0.01-0.05 percent, respectively), it still surpasses that of using conventional bacterial-based plasmids. Furthermore, stem cells from fat, and, for that matter, fat itself, are so prevalent that a slight reduction in efficiency should be easily overcome. "This is a great example of collaboration," said Longaker. "This discovery represents research from four different departments: paediatrics, surgery, cardiology and radiology. We were all doing our own things, and it wasn't until we focused on cross-applications of our research that we realized the potential." "We knew mini-circles worked better than plasmids for gene therapy," agreed Kay, "but it wasn't until I started talking to stem cell people like Joe and Mike that we started thinking of using mini-circles for this purpose. Now it's kind of like 'why didn't we think of this sooner?'" Reference: A non-viral mini-circle vector for deriving human iPS cells Fangjun Jia, Kitchener D Wilson, Ning Sun, Deepak M Gupta, Mei Huang, Zongjin Li, Nicholas J Panetta, Zhi Ying Chen, Robert C Robbins, Mark A Kay, Michael T Longaker & Joseph C Wu Nature Methods Published online: 07 February 2010, doi:10.1038/nmeth.1426 ......... ZenMaster

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Gene Improves Quality of Reprogrammed Stem Cells

Provides 'a better inkling of what we might aim for before differentiating iPS cells to clinically useful cell types' Sunday, 07 February 2010 In the 7 Feb. 2010 issue of the journal Nature, scientists at the Genome Institute of Singapore (GIS), report that a genetic molecule, called Tbx3, which is crucial for many aspects of early developmental processes in mammals, significantly improves the quality of stem cells that have been reprogrammed from differentiated cells. Stem cells reprogrammed from differentiated cells are known as induced pluripotent stem cells or iPS cells. By adding Tbx3 to the existing reprogramming cocktail, GIS scientists successfully produced iPS cells that were much more efficient in recapitulating the entire developmental process. The capability of iPS cells for germ-line transmission represents one of the most stringent tests of their ESC-like quality. This test requires that iPS cells contribute to the formation of germ cells that are responsible for propagating the next generation of offspring. "This represents a significant milestone in raising the current standards of iPS cell research. With this new knowledge, we are now able to generate iPS cells which are, or approach, the true equivalent of ESCs," said Lim Bing, M.D., Ph.D., lead author of the Nature paper and Senior Group Leader at GIS, one of the research institutes of Singapore's A*STAR (Agency for Science, Technology and Research). "When applied to the area of cell therapy-based medicine, we have a better inkling of what we might aim for before differentiating iPS cells to clinically useful cell types. The finding also adds to our insight into the fascinatingly, unchartered but rapidly moving field of reprogramming," Lim added. George Q. Daley, M.D., Ph.D., Director, Stem Cell Transplantation Program, HHMI/Children's Hospital Boston, Harvard Medical School, added: "This paper highlights the rapid progress towards optimized reprogramming strategies. The Singapore group has made an important advance in the production of high quality iPS cells. I would like to congratulate them on this important contribution." Embryonic stem cells (ESCs) are undifferentiated master stem cells that are developmentally important because they give rise to all other differentiated cell types in the human body. It has been shown that with the introduction of a few genetic factors into differentiated cells, these master stem (undifferentiated) cells can be re-created through a process known as reprogramming into iPS cells. Converting adult cells to embryonic cells such as iPS cells represents one of the most astounding breakthrough technologies in biological research. These cells look and behave like normal embryonic stem cells (ESCs) that can generate all other tissue types. Hence the great excitement over iPS potential impact on tissue regeneration and development of therapeutics. Previous studies have demonstrated how scientists can make iPS cells by using different cocktails of genetic factors, as well as improve this efficiency via the addition of chemical supplements. However, not all iPS cells generated with different cocktails resemble true ESCs; that is, the quality of the iPS cells is highly varied. "The ability to produce iPS cells has the potential to accelerate advances in human medicine. To achieve this objective, it is important to establish iPS cells that most closely resemble authentic embryo-derived pluripotent stem cells," said Azim Surani, Ph.D., Professor of Physiology and Reproduction at the Wellcome Trust /Cancer Research UK Gurdon Institute, University of Cambridge. "The new study by Bing Lim and colleagues shows that the inclusion of Tbx3 as one of the reprogramming factors significantly improves the quality of iPS cells. These iPS cells were superior since viable adults composed entirely of these iPS cells could be generated," said Surani. "These iPS cells also showed superior ability for contribution and transmission through the germ line, which is one of the critical criteria for assessing the quality of iPS cells." Reference: Tbx3 improves the germ-line competency of induced pluripotent stem cells Jianyong Han, Ping Yuan, Henry Yang, Jinqiu Zhang, Junliang Tay, Boon Seng Soh, Pin Li, Siew Lan Lim, Suying Cao, Yuriy L. Orlov, Thomas Lufkin, Huck-Hui Ng, Wai-Leong Tam, Bing Lim Nature advance online publication 7 February 2010, doi:10.1038/nature08735 ......... ZenMaster

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Wednesday, 3 February 2010

Scientists Map Epigenome of Human Stem Cells during Development

Billions of data points provide big picture of 'human epigenome' during critical developmental window Wednesday, 03 February 2010 Scientists at the Genome Institute of Singapore (GIS) and the Scripps Research Institute (TSRI) led an international effort to build a map that shows in detail how the human genome is modified during embryonic development. This detailed mapping is a significant move towards the success of targeted differentiation of stem cells into specific organs, which is a crucial consideration for stem cell therapy. The study was published in the journal Genome Research on Feb. 4, 2010. Chia-Lin Wei, Ph.D., senior author and Senior Group Leader at the GIS, a biomedical research institute of Singapore's Agency for Science, Technology and Research (A*STAR), said: "In this study, we mapped a major component of the epigenome, DNA methylation, for the entire sequence of human DNA, and went further by comparing three types of cells that represented three stages of human development: human embryonic stem cells, human embryonic stem cells that were differentiated into skin-like cells, and cells derived from skin. With these comprehensive DNA methylome maps, scientists now have a blueprint of key epigenetic signatures associated with differentiation." "The cells in our bodies have the same DNA sequence," said TSRI Professor Jeanne Loring, Ph.D., who is a co-senior author of the paper along with Isidore Rigoutsos of IBM Thomas J. Watson Research Center and Chia-Lin Wei of GIS. "Epigenetics is the process that determines what parts of the genome are active in different cell types, making a nerve cell, for example, different from a muscle cell." DNA methylation causes specific subunits of DNA to be chemically modified, which controls which areas of the genome are active and which ones are dormant. DNA methylation is critical to the process in which embryonic cells change from "pluripotent stem cells," which have the ability to turn into hundreds of cell types, to "differentiated cells," distinct types of cells that make up different parts of the body, such as the skin, hair, nerves, etc.. In reviewing the data produced by the study – information on the methylation of three billion base pairs of DNA – the scientists were able to identify previously unknown patterns of DNA methylation. They identified cases in which DNA methylation appeared to enhance, rather than repress, the activity of the surrounding DNA, and found evidence to suggest a role for DNA methylation in the regulation of mRNA splicing. "We produced a very large amount of data," said Loring. "But it actually simplifies the picture. We identified patterns of many genes that are methylated or de-methylated during differentiation. This will allow us to better understand the exquisitely choreographed changes that cells undergo as they develop into different cell types." Louise Laurent of TSRI and the University of California, San Diego, one of the first authors of the study, added: "The data are publicly available, and we are looking forward to learning what other scientists discover from using this information for their own studies on individual genes, embryonic development, and stem cells." "This is definitely an exciting finding in the field of stem cell research," added co-first author Eleanor Wong, who is a graduate student from the GIS in Dr Wei's lab. "Using this knowledge, scientists can now survey different cell types and developmental pathways, identify the genes affected, and characterize the functions of these genes in the process of differentiation. It's all very exciting!" Reference: Dynamic changes in the human methylome during differentiation Louise Laurent, Eleanor Wong, Guoliang Li, Tien Huynh, Aristotelis Tsirigos, Chin Thing Ong, Hwee Meng Low, Ken Wing Kin Sung, Isidore Rigoutsos, Jeanne Loring and Chia-Lin Wei Genome Research, Feb. 4, 2010 online issue. ......... ZenMaster

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Human Embryonic Stem Cells Arrests Acute Lung Injury in Mice

Human Embryonic Stem Cells Arrests Acute Lung Injury in Mice Wednesday, 03 February 2010 A new study by Rick Wetsel, Ph.D., left and Dachun Wang, M.D., of the University of Texas Health Science Center at Houston, explores the potential use of transplantable lung cells derived from human embryonic stem cells to treat respiratory disease. Credit: The University of Texas Health Science Center at Houston.Stem cell researchers exploring a new approach for the care of respiratory diseases report that an experimental treatment involving transplantable lung cells was associated with improved outcomes in tests on mice with acute lung injury. The lung cells were derived from human embryonic stem cells (hESCs). Findings by investigators at the University of Texas Health Science Center at Houston are scheduled to appear in the March issue of Molecular Therapy. Mice receiving the transplantable lung cells lived longer, sustained less scarring in their lungs and had normal amounts of oxygen in their blood, said Rick Wetsel, Ph.D., the study's senior author and a professor in the university's Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases (IMM). "Respiratory diseases are a major cause of mortality and morbidity worldwide," wrote Wetsel and his colleagues in the paper. "Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease." Giuseppe N. Colasurdo, M.D., dean of The University of Texas Medical School at Houston and physician-in-chief at Children's Memorial Hermann Hospital, said: "This research work will provide a useful model for studying the pathogenesis and treatment of a variety of lung disorders. I am confident future studies will advance our knowledge on the cellular mechanisms responsible for the improvement observed in the study." Colasurdo, who specializes in lung disorders in children and infants, said a better understanding of the basic mechanisms involved in the healing phase of lung diseases is critical to the development of treatments. "These are diseases involving a variety of cells and cell products," he said. Much human embryonic stem cell research is focused on conditions like lung injury in which the body has difficulty healing itself. Because these early stage cells can mature into many different cell types, they are being explored as a way to replace or repair missing or damaged tissue. These cells also divide rapidly providing researchers with a large supply of cells. Scientists compared the outcomes of mice with damaged lungs receiving the treatment to those not receiving the treatment. Researchers reported that the experimental stem cell treatment "not only prevented or reversed visual hallmarks of pulmonary injury, but also restored near normal lung function to mice." Lung cells can be damaged by exposure to pollution and disease agents. Wetsel and his colleague Dachun Wang, M.D., an IMM instructor, used a genetic selection procedure they created to generate a type of lung cell known as alveolar epithelial type II. These cells secrete surfactant, a substance that keeps the lung inflated, and can also turn into another important lung cell that regulates the transfer of oxygen into the blood and the removal of carbon dioxide. The human embryonic stem cells used in this research were approved by the National Institutes of Health (NIH) for study. Wetsel called the results "promising" but added that additional tests in other animal models and eventually humans will be needed before these cell transplants can be used to treat respiratory diseases. The scientists used mice with weakened immune systems to reduce the possibility that the human cells would be rejected. Should research proceed to the clinical trial stage, there are at least two ways to address rejection issues, Wetsel said. Patients could be treated with immunosuppressive drugs. Scientists may also be able to take one of the patient's own skin cells and convert it into "induced pluripotent stem cells" or iPS cells, which are believed to have many of the same capabilities as human embryonic stem cells. Reference: Transplantation of Human Embryonic Stem Cell-Derived Alveolar Epithelial Type II Cells Abrogates Acute Lung Injury in Mice Dachun Wang, John E Morales, Daniel G Calame, Joseph L Alcorn and Rick A Wetsel Molecular Therapy (2010); doi:10.1038/mt.2009.317 ......... ZenMaster

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3-D Scaffold Provides Clean, Biodegradable Structure for Stem Cell Growth

3-D Scaffold Provides Clean, Biodegradable Structure for Stem Cell Growth Wednesday, 03 February 2010 The biodegradable scaffold was first built as a cylinder (right) and then cut into dime-sized slices. Credit: Miqin Zhang, University of Washington.Medical researchers were shocked to discover that virtually all human embryonic stem cell lines being used in 2005 were contaminated. Animal by-products used to line Petri dishes had left traces on the human cells. If those cells had been implanted in a human body they likely would have been rejected by the patient's immune system. Even today, with new stem cell lines approved for use in medical research, there remains a risk that these cells will be contaminated in the same way. Most research labs still use animal-based "feeder layers" because it remains the cheapest and most reliable way to get stem cells to multiply. Materials scientists at the University of Washington have now created an alternative. They built a three-dimensional scaffold out of a natural material that mimics the binding sites for stem cells, allowing the cells to reproduce on a clean, biodegradable structure. Results published in the journal Biomaterials show that human embryonic stem cells grow and multiply readily on the structure. "The major challenge for stem cell therapy today is it's very difficult to make a lot of them with high purity," said lead author Miqin Zhang, a UW professor of materials science and engineering. "So far it seems like this material is very good for stem cell renewal." Medical researchers hope to someday use stem cells to grow new tissues and organs. Key to the research is the fact that new cells maintain the property that holds medical promise — the ability to differentiate into any of the more than 220 cell types in the adult human body. Growing the cells in three dimensions better resembles conditions in the human body. It also allows mass production, which will be needed for any clinical applications. This is a magnified view of the scaffold shows the pores, each about a tenth of a millimeter wide, where stem cells can grow. Credit: Miqin Zhang, University of Washington."Three-dimensional scaffolds are an active area of research," said Carol Ware, a UW professor of comparative medicine and expert on stem cells. "They are not commonly used yet, but will be important to transition embryonic stem cells to the clinic. To date, nobody has found a perfect matrix." Zhang's cylindrical scaffold is made of chitosan, found in the shells of crustaceans, and alginate, a gelatinous substance found in algae. Chitosan and alginate have a structure similar to the matrix that surrounds cells in the body, to which cells can attach. Different processing techniques can make the scaffold out of interconnected pores of almost any size, Zhang said. Researchers first seeded the scaffold with 500,000 embryonic stem cells, and after 21 days the scaffold was completely saturated. The cells infiltrated the structure, Zhang added, unlike other materials where cells often grow only on the surface. "This scaffold mimics the extracellular matrix at the atomic level, and so the cells are able to grow in this environment," Zhang said. To retrieve the cells, researchers immersed the scaffold in a mild solution. The structure is biodegradable and so dissolved to release the stem cells. One also could implant the stem cell-covered scaffold directly into the body. Analysis of gene activity and testing in the lab and in mice showed that the new stem cells retained the same properties as their predecessors. Other researcher groups are also looking for alternatives to feeder layers. The leading contenders are scaffolds coated with custom proteins designed to mimic the key properties of the animal cells in the feeder layer. Such products are expensive and difficult to produce in a consistent manner, Zhang said. The proteins also get used up in a few days and have to be replaced, making them costly and time-consuming for everyday use. "Our scaffold is made of natural materials that are already FDA-approved for food and biomedical applications. Also, these materials are unlimited, and the cost is cheap," she said. Zhang's group is now working to build a scaffold larger than the current dime-sized prototype, and is collaborating with the UW's Institute for Stem Cells and Regenerative Medicine and UW School of Medicine to try growing different types of stem cells, including those from umbilical cord blood and bone marrow, in the material. They will try to get the resulting cells to differentiate into bone, neuron, muscle and liver cells. ......... ZenMaster

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Monday, 1 February 2010

Growing Cartilage – No Easy Task

New nanoscopic material enables cartilage to do what it doesn't do naturally Monday, 01 February 2010 Northwestern University researchers are the first to design a bioactive nanomaterial that promotes the growth of new cartilage in vivo and without the use of expensive growth factors. Minimally invasive, the therapy activates the bone marrow stem cells and produces natural cartilage. No conventional therapy can do this. The results will be published online the week of Feb. 1 by the Proceedings of the National Academy of Sciences (PNAS). "Unlike bone, cartilage does not grow back, and therefore clinical strategies to regenerate this tissue are of great interest," said Samuel I. Stupp, senior author, Board of Trustees Professor of Chemistry, Materials Science and Engineering, and Medicine, and director of the Institute for BioNanotechnology in Medicine. Countless people – amateur athletes, professional athletes and people whose joints have just worn out – learn this all too well when they bring their bad knees, shoulders and elbows to an orthopaedic surgeon. Damaged cartilage can lead to joint pain and loss of physical function and eventually to osteoarthritis, a disorder with an estimated economic impact approaching $65 billion in the United States. With an aging and increasingly active population, this figure is expected to grow. "Cartilage does not regenerate in adults. Once you are fully grown you have all the cartilage you'll ever have," said first author Ramille N. Shah, assistant professor of materials science and engineering at the McCormick School of Engineering and Applied Science and assistant professor of orthopaedic surgery at the Feinberg School of Medicine. Shah is also a resident faculty member at the Institute for BioNanotechnology in Medicine. Type II collagen is the major protein in articular cartilage, the smooth, white connective tissue that covers the ends of bones where they come together to form joints. "Our material of nanoscopic fibres stimulates stem cells present in bone marrow to produce cartilage containing type II collagen and repair the damaged joint," Shah said. "A procedure called microfracture is the most common technique currently used by doctors, but it tends to produce a cartilage having predominantly type I collagen which is more like scar tissue." The Northwestern gel is injected as a liquid to the area of the damaged joint, where it then self-assembles and forms a solid. This extracellular matrix, which mimics what cells usually see, binds by molecular design one of the most important growth factors for the repair and regeneration of cartilage. By keeping the growth factor concentrated and localized, the cartilage cells have the opportunity to regenerate. Together with Nirav A. Shah, a sports medicine orthopaedic surgeon and former orthopaedic resident at Northwestern, the researchers implanted their nanofibre gel in an animal model with cartilage defects. The animals were treated with microfracture, where tiny holes are made in the bone beneath the damaged cartilage to create a new blood supply to stimulate the growth of new cartilage. The researchers tested various combinations: microfracture alone; microfracture and the nanofibre gel with growth factor added; and microfracture and the nanofibre gel without growth factor added. They found their technique produced much better results than the microfracture procedure alone and, more importantly, found that addition of the expensive growth factor was not required to get the best results. Instead, because of the molecular design of the gel material, growth factor already present in the body is enough to regenerate cartilage. The matrix only needed to be present for a month to produce cartilage growth. The matrix, based on self-assembling molecules known as peptide amphiphiles, biodegrades into nutrients and is replaced by natural cartilage. ......... ZenMaster

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Stem Cells Rescue Nerve Cells by Direct Contact

Stem Cells Rescue Nerve Cells by Direct Contact Monday, 01 February 2010 Two exogenous murine neural stem cells (NSCs) filled with the gap junction-permeable dye Calcein (green) and the gap junction-impermeable dye DiI (red) following engraftment to an organotypic stritatal tissue culture. Cell nuclei are displayed by DAPI nuclear counterstain (blue). Copyright: Johan Jäderstad and Eric Herlenius.Scientists at the Swedish medical university Karolinska Institute have shown how transplanted stem cells can connect with and rescue threatened neurons and brain tissue. The results point the way to new possible treatments for brain damage and neurodegenerative diseases. A possible strategy for treating neurodegenerative diseases is to transplant stem cells into the brain that prevent existing nerve cells from dying. The method has proved successful in different models, but the mechanisms behind it are still unknown. According to one hypothesis, the stem cells mature into fully-mature neurons that communicate with the threatened brain tissue; according to another, the stem cells secrete various growth factors that affect the host neurons. The new report, co-authored by several international research groups and lead by Karolinska Institute, shows that stem cells transplanted into damaged or threatened nerve tissue quickly establish direct channels, called gap junctions, to the nerve cells. Stem cells actively bring diseased neurons back from the brink via cross talk through gap junctions, the connections between cells that allow molecular signals to pass back and forth. The study found that the nerve cells were prevented from dying only when these gap junctions were formed. The results were obtained from mice and human stem cells in cultivated brain tissue, and from a series of rodent models for human neurodegenerative diseases and acute brain injuries. "Many different molecules can be transported through gap junctions," says Eric Herlenius, who led the study. "This means that a new door to the possible future treatment of neuronal damage has been opened, both figuratively and literally." Reference: Communication via gap junctions underlies early functional and beneficial interactions between grafted neural stem cells and the host Johan Jäderstad, Linda M. Jäderstad, Jianxue Li, Satyan Chintawar, Carmen Salto, Massimo Pandolfo, Vaclav Ourednik, Yang D. Teng, Richard L. Sidman, Ernest Arenas, Evan Y. Snyder and Eric Herlenius PNAS Online Early Edition, 1 Feb 2010 ......... ZenMaster

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Cells Send Dirty Laundry Home to Mom

How mother cells manage to make young daughter’s Monday, 01 February 2010 Bright green protein aggregates are transported from the young daughter cell into the larger mother cell using conveyor-like structures called actin cables. Credit: University of Gothenburg.Understanding how aged and damaged mother cells manage to form new and undamaged daughter cells is one of the toughest riddles of ageing, but scientists now know how yeast cells do it. In a groundbreaking study researchers from the University of Gothenburg, Sweden, show how the daughter cell uses a mechanical "conveyor belt" to dump damaged proteins in the mother cell. "This ensures that the daughter cell is born without age-related damage," says Professor Thomas Nyström from the Department of Cell and Molecular Biology. Thomas Nyström is a professor of microbiology at the University of Gothenburg and one of Sweden's leading researchers in the field of cellular and molecular biology. His research group has published countless scientific discoveries about cell ageing which have provided a new understanding of aging and age-related diseases. Now he and his colleagues have identified a key piece in the ageing puzzle. Mechanic transport In a study published as a feature article in the scientific journal Cell, two collaborating research groups at the Department of Cell and Molecular Biology have been able to show how newly formed yeast cells transport damaged and aged proteins back to the mother cell, guaranteeing that the new cell is born young and healthy. Mother dustbin "Previously it was believed that these structures allowed only one-way traffic of proteins and organelles from mother cell to daughter cell," says Nyström. "We can now show that damaged proteins are transported in the opposite direction. In principle, this means that the daughter cell uses the mother cell as a dustbin for all the rubbish resulting from the ageing process, ensuring that the newly formed cell is born without age-related damage." Conveyor belt In the study, the researchers show that this transportation is mechanical, using conveyor-like structures called actin cables. A special gene, which controls the rate of ageing, called SIR2, is needed for these cables to form properly. Previous research has shown that changing the SIR2 gene can markedly extend the life span of an organism. Longer life "Increased SIR2 activity means a longer life, whereas a damaged SIR2 gene accelerates ageing," says Nyström. "This has been demonstrated in studies of yeast, worms, flies and fish, and may also be the case in mammals." Future treatment This knowledge of how age-damaged proteins are transported from daughter cell to mother cell could eventually be used in the treatment of age-related diseases caused by protein toxicity in humans, but Nyström says that it is too early to say how. The first step "The first step is to study whether this transportation of damaged proteins also occurs in the cells of mammals, including humans, for example in the formation of sex sells and stem cells." Reference: The Polarisome Is Required for Segregation and Retrograde Transport of Protein Aggregates Beidong Liu, Lisa Larsson, Antonio Caballero, Xinxin Hao, David Öling, Julie Grantham, Thomas Nyström Cell, Volume 140, Issue 2, 257-267, 22 January 2010, 10.1016/j.cell.2009.12.031 ......... ZenMaster

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Dogs May Provide an Excellent Model for Understanding Human Complex Diseases

Dogs May Provide an Excellent Model for Understanding Human Complex Diseases Monday, 01 February 2010 In a new Swedish-Finnish study, published in Nature Genetics, researchers at Uppsala University and the Swedish University of Agricultural Sciences (SLU), in collaboration with scientists at University of Helsinki, Finland and USA, identified five loci that predispose to an SLE-related disease in Nova Scotia duck tolling retrievers. The study indicates that the homogeneity of strong genetic risk factors within dog breeds make dogs an excellent model in which to identify pathways involved in human complex diseases. The results of the study also open the door for further studies of specific T-cell activation pathways in human populations. To find genes for human common diseases, thousands of blood samples are needed from both patients and healthy controls. The structure provided by dog breeding, and the refinement of various properties within the breeds, make it much easier to find pathogenic genes with a smaller number of samples. The unique canine breed structure makes dogs an excellent model for studying genetic diseases. Incidences of specific diseases are elevated in different breeds, indicating that a few genetic risk factors might have accumulated through drift or selective breeding. In the new Swedish-Finnish study with 81 affected dogs and 57 controls from the Nova Scotia duck tolling retriever breed the researchers identified five loci associated with a canine systemic lupus erythematosus (SLE) -related disease complex. Fine mapping with twice as many dogs validated these loci. "It's extremely interesting and feels fantastic that we can so readily find genes even for complex diseases in dogs. The study also provides entirely new avenues for studying SLE in humans," says Professor Kerstin Lindblad-Toh, who directed the study. "Our results indicate that the homogeneity of strong genetic risk factors within dog breeds allows multigenic disorders to be mapped with fewer than 100 cases and 100 controls, making dogs an excellent model in which to identify pathways involved in human complex diseases," says Professor Hannes Lohi, University of Helsinki and Folkhälsan Research Center, Finland. Nova Scotia duck tolling retrievers (NSDTRs) are strongly predisposed to many immune-mediated diseases, including a systemic lupus erythematosus (SLE) -related disease complex comprising an immune-mediated rheumatic disease (IMRD) and steroid-responsive meningitis-arthritis (SRMA). The NSDTR breed was developed in the Yarmouth region of Nova Scotia in the early 1800s as a hunting and retrieving dog. The breed descended from a very small population of dogs that survived two devastating outbreaks of canine distemper virus in 1908 and 1912. One hypothesis for the abnormally high rates of autoimmune diseases in modern NSDTRs world-wide is that dogs with particularly strong or reactive immune systems were much more likely to survive these outbreaks. Pedigree analysis of the SLE disease complex in NSDTRs has indicated that it involves multi-genetic inheritance, like most autoimmune diseases in humans. The IMRD disease complex involves chronic musculoskeletal signs with a clinical picture indicative of immune-mediated non-erosive polyarthritis. Many of the clinical features of the canine IMRD complex are similar to those of human SLE. "It's worth pointing out that the canine risk factors are very strong," says Kerstin Lindblad-Toh. "The risk factors that have been found thus far in humans with SLE may double the risk, but in dogs, each disease gene increases the risk about five times." "In this study, we have identified five loci that predispose to an SLE-related disease in NSDTRs. The study highlights the strength of disease mapping in dogs, where a canine breed may carry a few disease loci, each with a strong effect, that together are sufficient to predispose to a complex disease," Professor Lohi states. Some types of genetic risk factor will be more easily traced in dogs than in humans, and the dog studies might be a valuable complement to human study for identifying new genes and pathways that are important in disease pathogenesis. "The genes that have thus far been found in humans with SLE do not primarily regulate T cells, but a major share of the genetic risk factors are still unknown in humans. It will therefore be interesting to move on and look at various subtypes of SLE and see whether genes that regulate T cells cause any of them," says Kerstin Lindblad-Toh. "Although we plan to identify and characterize the functions of the canine mutations, this study opens the door for further studies of specific T-cell activation pathways in human populations. In the more long term, the development of clinical treatment regimens based on a dog's particular risk genotype might be possible. For instance, the effect of calcineurin inhibitors could be studied in dogs as a complement or alternative to traditional corticosteroid therapy. Such studies might also lead to better treatment options for human rheumatic diseases and SLE," Lohi says. Reference: Genome-wide association mapping identifies multiple loci for a canine SLE-related disease complex Maria Wilbe, Päivi Jokinen, Katarina Truvé, Eija H Seppala, Elinor K Karlsson, Tara Biagi, Angela Hughes, Danika Bannasch, Göran Andersson, Helene Hansson-Hamlin, Hannes Lohi & Kerstin Lindblad-Toh Nature Genetics, Published online: 31 January 2010, doi:10.1038/ng.525 ......... ZenMaster

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