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Handle with Care: Telomeres Resemble DNA Fragile Sites

2009-07-10T16:31:00.003+02:00

A protein at the ends of chromosomes, helps prevent DNA replication from stalling at telomeres
Friday, 10 July 2009

Telomeres, the repetitive sequences of DNA at the ends of linear chromosomes, have an important function: They protect vulnerable chromosome ends from molecular attack. Researchers at Rockefeller University now show that telomeres have their own weakness. They resemble unstable parts of the genome called fragile sites where DNA replication can stall and go awry. But what keeps our fragile telomeres from falling apart is a protein that ensures the smooth progression of DNA replication to the end of a chromosome.

The research, led by Titia de Lange, head of the Laboratory of Cell Biology and Genetics, and first author Agnel Sfeir, a postdoctoral associate in the lab, suggests a striking similarity between telomeres and common fragile sites, parts of the genome where breaks tend to occur, albeit infrequently. (Humans have 80 common fragile sites, many of which have been linked to cancer.) De Lange and Sfeir found that these newly discovered fragile sites make it difficult for DNA replication to proceed, a discovery that unveils a new replication problem posed by telomeres.

At the centre of the discovery is a protein known as TRF1, which de Lange, in an effort to understand how telomeres protect chromosome ends, discovered in 1995. Using a conditional mouse knockout, de Lange and Sfeir have now revealed that TRF1, which is part of a six-protein complex called shelterin, enables DNA replication to drive smoothly through telomeres with the aid of two other proteins.

Fragile telomeres. This is a series of images showing chromosomes with fragile telomeres (green). Without the protein TRF1, telomeres resemble common fragile sites, unstable regions on chromosomes that break into segments or stretch due to faulty DNA replication. Credit: Cell.

“Telomeric DNA has a repetitive sequence that can form unusual DNA structures when the DNA is unwound during DNA replication,” says de Lange.

“Our data suggest that TRF1 brings in two proteins that can take out these structures in the telomeric DNA. In other words, TRF1 and its helpers remove the bumps in the road so that the replication fork can drive through.”

The work, published in the July 10 issue of Cell, began when Sfeir deleted TRF1 and saw that the telomeres resembled common fragile sites, suggesting that TRF1 protects telomeres from becoming fragile. Instead of a continuous string of DNA, the telomeres were broken into fragments of twos and threes. To see if the replication fork stalls at telomeres, de Lange and Sfeir joined forces with

Carl L. Schildkraut, a researcher at Albert Einstein College of Medicine in New York City. Using a technique called SMARD, the researchers observed the dynamics of replication across individual DNA molecules — the first time this technique has been used to study telomeres. In the absence of TRF1, the fork often stalled for a considerable amount of time.

The only other known replication problem posed by telomeres was solved in 1985 when it was shown that the enzyme telomerase elongates telomeres, which shorten during every cell division. The second problem posed by telomeres, the so-called end-protection problem, was solved by de Lange and her colleagues when they found that shelterin protects the ends of linear chromosomes, which look like damaged DNA, from unnecessary repair. Working with TRF1, the very first shelterin protein ever to be identified, de Lange and Sfeir have not only unveiled a completely unanticipated replication problem at telomeres, they have also shown how it is solved.

The research lays new groundwork for the study of common fragile sites throughout the genome, explains de Lange.

“Fragile sites have always been hard to study because no specific DNA sequence precedes or follows them,” she says.

“In contrast, telomeres represent fragile sites with a known sequence, which may help us understand how common fragile sites break throughout the genome — and why.”

Reference:
Mammalian Telomeres Resemble Fragile Sites and Require TRF1 for Efficient Replication
Agnel Sfeir, Settapong T. Kosiyatrakul, Dirk Hockemeyer, Sheila L. MacRae, Jan Karlseder, Carl L. Schildkraut and Titia de Lange
Cell, July 10, 2009, 138(1): 90-103
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Key to Maintaining Embryonic Stem Cells in Lab

2009-07-10T04:45:00.003+02:00

Key to Maintaining Embryonic Stem Cells in Lab
Friday, 10 July 2009

In a new study that could transform embryonic stem cell (ES cell) research, scientists at UT Southwestern Medical Center have discovered why mouse ES cells can be easily grown in a laboratory while other mammalian ES cells are difficult, if not impossible, to maintain.

If the findings in mice can be applied to other animals, scientists could have an entirely new palette of research tools to work with, said Dr. Steven McKnight, chairman of biochemistry at UT Southwestern and senior author of the study appearing in the July 9 issue of Science Express.

"This might change the way medical research is done. But it's still a big 'if,'" he said.

According to the research, the activation of a gene called TDH in mouse ES cells results in the cells entering a unique metabolic state that is similar to that of rapidly growing bacterial cells. The gene controls the production of the threonine dehydrogenase (TDH) enzyme in mouse ES cells. This enzyme breaks down an amino acid called threonine into two products. One of the two products goes on to control a cellular process called one carbon metabolism; the other provides ES cells with an essential metabolic fuel.

Both of the threonine breakdown products are necessary to keep the ES cells growing and dividing rapidly in a Petri dish without differentiating into specific tissues.

The various substances currently used by scientists to keep mouse ES cells alive in the laboratory were found by trying many different combinations until something worked, Dr. McKnight said. But until now, it wasn't known that these culture conditions keyed into keeping the TDH gene actively expressed.

"Scientists added this and that until they got the right 'soup,' one that works in the mouse ES cells to somehow activate the TDH gene," he said, adding that exactly how that gene is regulated is still unknown.

Other mammalian species have a functional version of the TDH gene, suggesting the possibility that the process could also be activated in them.

"You would think that the 'mouse soup' would then work for all species, but it doesn't. Researchers have been trying for 20 years to get the right formula for maintaining ES cells from other species. With few exceptions, however, they still haven't gotten it right," Dr. McKnight said.

The research was funded by a National Institutes of Health Director's Pioneer Award, which Dr. McKnight received in 2004. The program encourages investigators to take on creative, unexplored avenues of research that carry a relatively high potential for failure but that also possess a greater chance for truly groundbreaking discoveries.

"By applying a highly innovative technique to manipulate the TDH gene, McKnight's work could be an important breakthrough with a profound impact on future research," said Dr. Raynard S. Kington, acting director of the NIH.

"This research, which was partially funded by our Pioneer Award program, shows the value of supporting exceptionally creative approaches to major challenges in biomedical and behavioral research."

Embryonic stem cells are "blank slate" cells – derived from embryos – that go on to develop into any of the more than 200 types of cells in the adult body.

Because mouse ES cells are easily maintained in the lab, they can be manipulated genetically to produce adult mice in which various genes are either modified or eliminated. So-called "knockout mice" allow scientists to study the genetic aspects of many diseases and conditions, including cancer, Alzheimer's, Parkinson's and paralysis.

In the living mouse, and in other species, ES cells exist for only a short time. In that time, they need to grow rapidly in order to accumulate enough cells to begin the process of differentiating into all the body's cell types. Dr. McKnight hypothesizes that the TDH gene tightly controls this process in the animal, allowing the ES cells to grow, but then it shuts off when it's time to differentiate.

"If we can tweak conditions and determine how to keep the gene turned on in other animals, we might be able to grow and maintain ES cells for study in many species. It's still speculative at this point whether it will work, but if it does, then this may prove to represent a transformational discovery," Dr. McKnight said.

Interestingly, although humans carry a form of the TDH gene, it contains three inactivating mutations. As such, human ES cells do not produce the TDH enzyme.

"In the human embryo, something else is taking the place of this TDH-mediated form of rapid cell growth," Dr. McKnight said.

"Human ES cells may exist in a unique metabolic state, but it would not appear to involve threonine breakdown."

Human ES cells grow slowly and are difficult to maintain in the laboratory, which is a huge impediment to this field of study, Dr. McKnight said.

"If scientists could repair the mutated human TDH gene and replace it into human ES cells, could they make those cells grow faster in culture? I don't know whether this will work or not – it's highly speculative. But if so, it would be profound," he said.

Reference:
Dependence of Mouse Embryonic Stem Cells on Threonine Catabolism
Jian Wang, Peter Alexander, Leeju Wu, Robert Hammer, Ondine Cleaver, and Steven L. McKnight
Science Published online July 9 2009; DOI: 10.1126/science.1173288
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Newborn Brain Cells Show the Way

2009-07-10T04:18:00.004+02:00

Newborn Brain Cells Show the Way
Friday, 10 July 2009

Although the fact that we generate new brain cells throughout life is no longer disputed, their purpose has been the topic of much debate. Now, an international collaboration of researchers made a big leap forward in understanding what all these newborn neurons might actually do. Their study, published in the July 10, 2009, issue of the journal Science, illustrates how these young cells improve our ability to navigate our environment.

"We believe that new brain cells help us to distinguish between memories that are closely related in space," says senior author Fred H. Gage, Ph.D., a professor in the Laboratory for Genetics at the Salk Institute. He is also the Vi and John Adler Chair for Research on Age-Related Neurodegenerative Diseases, who co-directed the study with Timothy J. Bussey, Ph.D., a senior lecturer in the Department of Experimental Psychology at the University of Cambridge, UK, and Roger A. Barker, PhD., honorary consultant in Neurology at Addenbrookes Hospital and Lecturer at the University of Cambridge.

When the first clues emerged that adult human brains continually sprout new neurons, one of the central tenets of neuroscience — we are born with all the brain cells we'll ever have — was about to be overturned. Although it is never easy to shift a paradigm, a decade later the question is no longer whether neurogenesis exists but rather what all these new cells are actually good for.

"Adding new neurons could be a very problematic process if they don't integrate properly into the existing neural circuitry," says Gage.

"There must be a clear benefit to outweigh the potential risk."

The most active area of neurogenesis lies within the hippocampus, a small seahorse-shaped area located deep within the brain. It processes and distributes memory to appropriate storage sections in the brain after readying the information for efficient recall.

"Every day, we have countless experiences that involve time, emotion, intent, olfaction and many other dimensions," says Gage.

"All the information comes from the cortex and is channelled through the hippocampus. There, they are packaged together before they are passed back out to the cortex where they are stored."

Previous studies by a number of laboratories including Gage's had shown that new neurons somehow contribute to hippocampus-dependent learning and memory but the exact function remained unclear.

The dentate gyrus is the first relay station in the hippocampus for information coming from the cortex. While passing through, incoming signals are split up and distributed among 10 times as many cells. This process, called pattern separation, is thought to help the brain separate individual events that are part of incoming memories.

"Since the dentate gyrus also happens to be the place where neurogenesis is occurring, we originally thought that adding new neurons could help with the pattern separation," says Gage.

This hypothesis allowed graduate student Claire Clelland, who divided her time between the La Jolla and the Cambridge labs, to design experiments that would specifically challenge this function of the dentate gyrus using different behavioural tasks and two distinct strategies to selectively shut down neurogenesis in the dentate gyrus.

This image depicts a Paired Associates Learning (PAL) task, in which mice have to choose a specific object in its correct location on a touch screen to obtain a reward. Adult mice deficient in adult neurogenesis showed a specific impairment in pattern separation, identifying a dentate gyrus-specific function for newborn neurons in the adult brain. Adult born neurons are shown in red the background. Credit: Courtesy of Jamie Simon, Salk Institute for Biological Studies.

In the first set of experiments, mice had to learn the location of a food reward that was presented relative to the location of an earlier reward within an eight-armed radial maze.

"Mice without neurogenesis had no trouble finding the new location as long as it was far enough from the original location," says Clelland, "but couldn't differentiate between the two when they were close to each other."

A touch screen experiment confirmed the inability of neurogenesis-deficient mice to discriminate between locations in close proximity to each other but also revealed that these mice had no problem recalling spatial information in general.

"Neurogenesis helps us to make finer distinctions and appears to play a very specific role in forming spatial memories," says Clelland.

Adds Gage, "There is value in knowing something about the relationship between separate events and the closer they get the more important this information becomes."

But pattern separation might not be the only role that new neurons have in adult brain function: a computer model simulating the neuronal circuits in the dentate gyrus based on all available biological information suggested an additional function.

"To our surprise, it turned out that newborn neurons actually form a link between individual elements of episodes occurring closely in time," says Gage.

Given this, he and his team are now planning experiments to see whether new neurons are also critical for coding temporal or contextual relationships.

About the Salk Institute for Biological Studies:
The Salk Institute for Biological Studies is one of the world's preeminent basic research institutions, where internationally renowned faculty probe fundamental life science questions in a unique, collaborative, and creative environment. Focused on both discovery and mentoring future generations of researchers, Salk scientists make groundbreaking contributions to our understanding of cancer, aging, Alzheimer's, diabetes, and cardiovascular disorders by studying neuroscience, genetics, cell and plant biology, and related disciplines.

Faculty achievements have been recognized with numerous honours, including Nobel Prizes and memberships in the National Academy of Sciences. Founded in 1960 by polio vaccine pioneer Jonas Salk, M.D., the Institute is an independent non-profit organization and architectural landmark.

Reference:
A Functional Role for Adult Hippocampal Neurogenesis in Spatial Pattern Separation
C. D. Clelland, M. Choi, C. Romberg, G. D. Clemenson, Jr., A. Fragniere, P. Tyers, S. Jessberger, L. M. Saksida, R. A. Barker, F. H. Gage, T. J. Bussey
Science 10 July 2009: Vol. 325. no. 5937, pp. 210 – 213, DOI: 10.1126/science.1173215
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Stem Cells' 'Suspended' State Preserved by Key Step in Chromatin Remodelling

2009-07-09T02:51:00.001+02:00

Stem Cells' 'Suspended' State Preserved by Key Step in Chromatin Remodelling
Thursday, 09 July 2009

Scientists have identified a gene that is essential for embryonic stem cells to maintain their all-purpose, pluripotent state. Exploiting the finding may lead to a greater understanding of how cells acquire their specialized states and provide a strategy to efficiently reprogram mature cells back into the pluripotent state, an elusive step in stem cell research but one crucial to a range of potential clinical treatments.

The research was led by University of California, San Francisco scientists. It is being reported Wednesday, July 8, 2009, in the advanced online edition of the journal Nature, and will be published in the journal's print edition at the end of July.

Embryonic stem cells are suspended in an "open" state, uniquely poised to become any one of many types of specialized cells, as genetic instructions dictate. Directing the specialization of embryonic stem cells to cells needed by patients is an area of enormous promise in stem cell research. Reversing the natural process – converting specialized cells back into the all-purpose stem cell stage – is another great promise of stem cell research.

Reprogramming specialized cells from Parkinson's patients, for example, would allow scientists to study the mechanisms that cause neurons in the brain to develop the disease. It also could lead to treatments by directing the restored stem cells to produce healthy neurons to introduce into patients.

The new research, conducted on mouse embryo cells, revealed that a gene known as Chd1 loosens the packaging that normally protects DNA in the cell nucleus. This step, known as chromatin remodelling, allows the cell's protein-making machinery to gain access to the DNA and transform progenitor cells into specialized cells and tissue, such as neurons, muscle and bone.

A number of genes are known to trigger chromatin remodelling, allowing small sections of DNA to become accessible in order to make specific proteins. Chd1 is the first gene found to regulate a "global" loosening of the DNA in embryonic stem cells, the scientists report. The global condition sets the stage for turning on many different genes to make a broad range of specialized cells.

"Embryonic stem cells are characterized by this open state, but, up to now, we didn't know the mechanisms that maintain this state, or even if it is necessary for the full stem cell potential," said Alexandre Gaspar-Maia, lead author of the paper.

"We found that Chd1 is critical for both, and for allowing an efficient reprogramming. Chd1 is important for allowing the normal differentiation process, and it is essential for playing the 'differentiation tape' backwards – bringing differentiated cells back to pluripotency."

Gaspar-Maia is a graduate student (from the PhD Program in Experimental Biology and Biomedicine, at the University of Coimbra, Portugal) in the lab of senior author Miguel Ramalho-Santos, PhD, of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF.

The scientists discovered the pivotal role of Chd1 by using the powerful technique of RNA interference, or RNAi, to screen this gene and 40 other candidate genes. (RNAi is a naturally occurring process in which small RNAs bind to other RNAs to increase or decrease their activity.) In this case, the scientists used the technique to silence Chd1. When they did so, embryonic stem cells could not make the full range of specialized cells.

In a laboratory test used to simulate normal cell specialization, the scientists detected no differentiation of cardiac muscle, and also no formation of a tissue known as primitive endoderm, which is essential for the embryo to survive and develop.

Chd1 also was shown by the research team to be necessary for the reprogramming of specialized cells back to the pluripotent stem cell state. The team plans to study chromatin remodelling in still more detail to clarify what other molecules work in concert with the Chd1 gene to direct the process. This would aid efforts to increase the efficiency and safety of reprogramming cells. This research may also shed light on how cells transition from one type to another, a process that happens normally during embryonic development and goes astray in cancer.

"We now know that Chd1 is essential, and, so far, appears unique in its global effect, but we expect that there are major players yet to be discovered," said senior author Ramalho-Santos, UCSF assistant professor of obstetrics, gynaecology and reproductive sciences, and pathology.

"If we can understand how Chd1 works, that will also tell us more about how the cells regulate their precise specialization during development, and turn on their pluripotency program during reprogramming."

The scientists conclude that embryonic stem cells exist in a dynamic state, poised between the open condition that may assure the cell's full potential, and the more constrained state that allows only certain kinds of cells to progress. Chd1, they say, is central to maintaining the open, pluripotent stem cell state.

Reference:
Chd1 regulates open chromatin and pluripotency of embryonic stem cells
Alexandre Gaspar-Maia, Adi Alajem, Fanny Polesso, Rupa Sridharan, Mike J. Mason, Amy Heidersbach, João Ramalho-Santos, Michael T. McManus, Kathrin Plath, Eran Meshorer & Miguel Ramalho-Santos
Nature advance online publication 8 July 2009 doi:10.1038/nature08212
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Reprogramming Adult Testis Cells to Pluripotency

2009-07-08T12:44:00.005+02:00

Playing it safe without the use of genes, viruses or reprogramming proteins
Wednesday, 08 July 2009

Kinarm Ko and Hans Schöler's team at the Max Planck Institute for Molecular Biomedicine in Münster have succeeded for the first time in culturing a clearly defined cell type from the testis of adult mice and converting these cells into pluripotent stem cells without introduced genes, viruses or reprogramming proteins. These stem cells have the capacity to generate all types of body tissue. The culture conditions alone were the crucial factor behind the success of the reprogramming process. (Cell Stem Cell, July 2, 2009)

The testis is a sensitive organ and an astonishing one at that. Even at the age of 70, 80 or 85, men have cells that constantly produce new sperm. Therefore, they can conceive embryos and become fathers at almost any age - assuming they can find a sufficiently young female partner. Based on this, researchers have long assumed that cells from the testis have a similar potential as in embryonic stem cells: that is, a pluripotency that enables them to form over 200 of the body's cell types.

In fact, a number of researchers have recently stumbled on the multiple talents in the male gonads of humans and mice. It all began with the work of Takashi Shinohara's team in 2004. The Japanese scientists discovered that, like embryonic stem cells, certain cells in the testis of newborn mice are able to develop into different kinds of tissue. In 2006, scientists working with Gerd Hasenfuß and Wolfgang Engel in Gottingen reported that such adaptable cells can also be found in adult male mice. Additionally, Thomas Skutella and his colleagues at the University of Tübingen recently made headlines when they cultured comparable cells from human testis tissue.

A bewildering variety of cells
"At first glance, it would appear that it has long been established that pluripotent cells exist in the testis of adult humans and mice," says Schöler.

"However, it is often unclear as to exactly which cells are being referred to in the literature and what these cells can actually do." (See Background article)

This is not only due to the fact that the testis contains a multitude of different cells. Scientists who dismantle tissue in the laboratory must carefully separate and analyse the cells to establish which cell type they have under the microscope. The question of potency is a controversial one among stem cell researchers, as binding benchmarks have yet to be defined. What some scientists would define as "pluripotent" is just about deemed "multi-potent", that is, as having a limited capacity for differentiation, by others. Greater certainty can be provided by carrying out the relevant tests. These include, among other things, a test to establish whether, after injection into early embryos, the cells are able to contribute to the development of the new organism and gamete formation, and to pass on their genes to further generations. However, not every team carries out all of these tests and important questions are left unanswered, even in articles published in renowned journals.

Stable original cell line
With their work, Ko and his colleagues wanted to establish clarity from the outset. To this end, they started by culturing a precisely defined type of cell, so-called germline stem cells (GSCs), from the testis of adult mice. In their natural environment, these cells can only do one thing: constantly generate new sperm. Moreover, their own reproduction is an extremely rare occurrence. Only two or three of them will be found among the 10,000 cells in the testis tissue of a mouse. However, they can be isolated individually and reproduced as cell lines with stable characteristics. Under the usual cell culturing conditions, they retain their unipotency for weeks and years. Consequently, all they can do is reproduce or form sperm.

What nobody had guessed until now, however, was that a simple trick is enough to incite these cells to reprogram. If the cells are distributed on new Petri dishes, some of them revert to an embryonic state once they are given sufficient space and time.

"Each time we filled around 8000 cells into the individual wells of the cell culture plates, some of the cells reprogrammed themselves after two weeks," reports Ko. And when the switch in these germline-derived pluripotent stem cells (gPS) has been reversed, they start to reproduce rapidly.

The researchers have proven that the "reignition" of the cells has actually taken place with the aid of numerous tests. Not only can the reprogrammed cells be used to generate heart, nerve or endothelial cells, as is the case with embryonic stem cells, the scientists can also use them to produce mice with mixed genotypes, known as chimeras, from the new gPs, and thus demonstrate that cells obtained from the testis can pass their genes on to the next generation.

Whether this process can also be applied to humans remains an open question. There is much to suggest, however, that gPS cells exceed all previously artificially reprogrammed cells in terms of the simplicity of their production and their safety.

Reference:
Induction of pluripotency in adult unipotent germline stem cells
Kinarm Ko, Natalia Tapia, Guangming Wu, Jeong Beom Kim, Marcos J Araúzo-Bravo, Philipp Sasse, Tamara Glaser, David Ruau, Dong Wook Han, Boris Greber, Kirsten Hausdörfer, Vittorio Sebastiano, Martin Stehling, Bernd K. Fleischmann, Oliver Brüstle, Martin Zenke and Hans R. Schöler
Cell Stem Cell, July 2, 2009, doi:10.1016/j.stem.2009.05.025

Background article:
The germ of pluripotency
Mito Kanatsu-Shinohara und Takashi Shinohara
Nature Biotechnology 24(6), June 2006, doi: 10.1038/nbt0606-663
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How to Make Human Sperm in the Lab

2009-07-08T12:37:00.003+02:00

Newcastle University researchers make human sperm from embryonic stem cells
Wednesday, 08 July 2009

Human sperm have been created using embryonic stem cells for the first time in a scientific development which will lead researchers to a better understanding of the causes of infertility.

Researchers led by Professor Karim Nayernia at Newcastle University and the NorthEast England Stem Cell Institute (NESCI) have developed a new technique which has made the creation of human sperm possible in the laboratory.

The work is published today (8th July 2009) in the academic journal Stem Cells and Development.

The NorthEast England Stem Cell Institute (NESCI) is a collaboration between Newcastle and Durham Universities, Newcastle NHS Foundation Trust and other partners.

Professor Nayernia says:

"This is an important development as it will allow researchers to study in detail how sperm forms and lead to a better understanding of infertility in men – why it happens and what is causing it. This understanding could help us develop new ways to help couples suffering infertility so they can have a child which is genetically their own."

"It will also allow scientists to study how cells involved in reproduction are affected by toxins, for example, why young boys with leukaemia who undergo chemotherapy can become infertile for life – and possibly lead us to a solution."

The team also believe that studying the process of forming sperm could lead to a better understanding of how genetic diseases are passed on.

In the technique developed at Newcastle, stem cells with XY chromosomes (male) were developed into germline stem cells which were then prompted to complete meiosis - cell division with halving of the chromosome set. These were shown to produce fully mature, sperm called scientifically, In Vitro Derived sperm (IVD sperm).

In contrast, stem cells with XX chromosomes (female) were prompted to form early stage sperm, spermatagonia, but did not progress further. This demonstrates to researchers that the genes on a Y chromosome are essential for meiosis and for sperm maturation.

IVD sperm
The IVD sperm will not and cannot be used for fertility treatment. As well as being prohibited by UK law, the research team say fertilization of human eggs and implantation of embryos would hold no scientific merit for them as they want to study the process as a model for research.

"While we can understand that some people may have concerns, this does not mean that humans can be produced 'in a dish' and we have no intention of doing this. This work is a way of investigating why some people are infertile and the reasons behind it. If we have a better understanding of what's going on it could lead to new ways of treating infertility," adds Professor Nayernia.

Technique
The Newcastle University team have developed a method for establishing early stage sperm from human embryonic stem cells in the laboratory.

The embryonic stem cells were cultured in a new medium containing vitamin A derivative (retinoic acid), in a new technique established by the team. Based on this technique, the cells differentiated into germline stem cells.

These expressed a protein which was stained with a green fluorescent marker and they were separated out by FACSTM (Fluorescence-activated cell sorting) using a laser.

After further differentiation, these in vitro derived germline stem cells expressed markers which are specific to primordial germ cells, spermatogonial stem cells, meiotic (spermatocytes) and post meiotic germ cells (spermatids and sperm).

These results indicated maturation of the primordial germ cells to haploid male gametes – called IVD sperm – characterised by containing half a chromosome set (23 chromosomes).
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NIH Issues New Guidelines for Human Stem Cell Research

2009-07-07T05:39:00.001+02:00

NIH Issues New Guidelines for Human Stem Cell Research
Tuesday, 07 July 2009

On March 9, 2009, President Barack H. Obama issued Executive Order (EO)13505 Removing Barriers to Responsible Scientific Research Involving Human Stem Cells. The EO states that the Secretary of Health and Human Services, through the Director of NIH, may support and conduct responsible, scientifically worthy human stem cell research, including human embryonic stem cell (hESC) research, to the extent permitted by law. NIH published draft Guidelines for research involving hESCs in the Federal Register for public comment, 74 Fed. Reg. 18578 on April 23, 2009. The comment period ended on May 26, 2009. Approximately 49,000 comments on the draft Guidelines were submitted to NIH by patient advocacy groups, scientists and scientific societies, academic institutions, medical organizations, religious organizations, private citizens, and members of Congress.

The final NIH Guidelines for Human Stem Cell Research implementing the EO and establishing policy and procedures under which the NIH will fund such research, were released today. They will be effective on July 7, 2009. Public comments on the draft Guidelines were also released today and are available at.

The Guidelines will ensure that NIH-funded research in this area is ethically responsible, scientifically worthy, and conducted in accordance with applicable law. Internal NIH policies and procedures, consistent with the EO and these Guidelines, will govern the conduct of intramural NIH stem cell research.

The Guidelines prescribe the assurances and supporting documentation that must accompany requests for NIH funding for research using hESCs, and describe research that is not eligible for NIH funding. NIH will provide additional guidance concerning the implementation of the Guidelines and the status of pending applications in future Guide Notices.

Ongoing NIH-supported research involving previously approved hESC lines may continue. No new uses of hESC may be initiated in ongoing funded studies unless reviewed and approved by the NIH.

Reference:
National Institutes of Health Guidelines on Human Stem Cell Research
Stem Cell Information, Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services, 2009 [cited Monday, July 06, 2009]
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Genetic Factors That Hold Promise for Treatment of Vascular Diseases

2009-07-05T23:42:00.001+02:00

microRNAs turns stem cells into vascular smooth muscle cells
Sunday, 05 July 2009

Researchers at the Gladstone Institute of Cardiovascular Disease (GICD) have discovered a key switch that makes stem cells turn into the type of muscle cells that reside in the wall of blood vessels. The same switch might be used in the future to limit growth of vascular muscle cells that cause narrowing of arteries leading to heart attacks and strokes, limit formation of blood vessels that feed cancers, or make new blood vessels for organs that are not getting enough blood flow.

In a study published in the current issue of the journal Nature, the researchers found that a tiny RNA molecule, called microRNA-145 (miR-145), not only had all the information necessary to turn a stem cell into a vascular smooth muscle cell (VSMC), but could also affect VSMCs in the adult artery. VSMCs have the unique property that they can start dividing when an artery is injured or during atherosclerosis, ultimately causing narrowing of the vessel leading to occlusion. miR-145 and its sister microRNA, miR-143, work together to stop the pathologic division of VSMCs. In the setting of vessel disease, their activity was turned down, allowing the VSMCs to divide and clog up the artery.

microRNAs are small RNA molecules that do not make protein, but instead affect that amount of protein synthesized by the cell from their target mRNAs — the blueprints for translating the genetic code into proteins. miR-145 and miR-143 together controlled the synthesis of a network of "master regulators" that control VSMCs, and thereby were able to function as a central "switch" for the behaviour of these important cells.

"The ability of miR-145 to efficiently direct the cell fate of vascular smooth muscle cells from stem cells represents the power of these tiny microRNAs to exert major effects on cells," said Deepak Srivastava, MD, GICD director and senior author of the study.

"We hope that we can use this knowledge to control when the body makes or does not make new blood vessels," he added.

Previously, GICD researchers had shown that miR-143 is highly enriched when embryonic stem cells turned into cardiac stem cells. Here they found that miR-143 and miR-145 were both present as the heart was forming in mice, but became localized to the smooth muscle of blood vessels and of the gut after birth.

Further analysis revealed that miR-143 and miR-145 are directly controlled by a protein called myocardin, which itself is sufficient to "reprogram" an adult non-muscle cell into a VSMC. Furthermore, the activation of these microRNAs by myocardin was a necessary event for myocardin to induce the VSMC fate. In one type of stem cell, miR-145 by itself was enough to completely push the stem cell into a functioning VSMC.

These findings suggested that miR-143 and miR-145 are involved in the switch between the differentiation and proliferation of VSMCs — and thus contribute to vessel narrowing in heart disease. In a mouse model of this switch generated by collaborator Joseph Miano, PhD, a professor at the Cardiovascular Research Institute of the University of Rochester, expression of miR-143 and miR-145 was markedly reduced in injured arteries containing proliferating, less differentiated smooth muscle cells. Interestingly, miR-145 mRNA was also reduced to almost undetectable levels in atherosclerotic blood vessels with thickened walls.

"miR-145 was necessary and sufficient for differentiation of VSMCs, so it is possible that restoring its activity could prevent the vessel narrowing in atherosclerosis," said Kimberly Cordes, PhD, a postdoctoral fellow in the Srivastava lab and lead author of the study.

Since the effects of miRNAs depend on their mRNA targets, the researchers looked for mRNA targets of miR-143 and miR-145. They found that miR-143 and miR-145 cooperate in targeting a network of transcription factors, including Klf4, myocardin, and Elk-1, to promote the differentiation and repress proliferation of smooth muscle cells.

"The multiple targets we identified for miR-143 and miR-145 reveal an elegant mechanism by which these miRNAs promote differentiation and simultaneously repress proliferation of VSMCs" said Dr. Srivastava.

The targets miR-145 and miR-143 regulate are not only major regulators of VSMCs, but also control whether cells divide excessively in conditions such as cancer.

According to Dr. Cordes, "the down regulation of miR-145 in numerous cancers and our findings in this study raise the possibility that miR-145 could function as a pro-differentiation factor in cancers also and could be a new therapeutic target."

"Our findings in this study offer insights into regulatory mechanisms that govern the differentiation and proliferation of smooth muscle," said Dr. Srivastava.

"They have fundamental implications for the treatment of vessel diseases like atherosclerosis and also may be important for cancer."

About the Gladstone Institutes
The J. David Gladstone Institutes, an independent, non-profit biomedical research organization, affiliated with the University of California, San Francisco, is dedicated to the health and welfare of humankind through research into the causes and prevention of some of the world's most devastating diseases. Gladstone is comprised of the Gladstone Institute of Cardiovascular Disease, the Gladstone Institute of Virology and Immunology and the Gladstone Institute of Neurological Disease.

Reference:
miR-145 and miR-143 regulate smooth muscle cell fate and plasticity
Kimberly R. Cordes, Neil T. Sheehy, Mark P. White, Emily C. Berry, Sarah U. Morton, Alecia N. Muth, Ting-Hein Lee, Joseph M. Miano, Kathryn N. Ivey & Deepak Srivastava
Nature advance online publication 5 July 2009, doi:10.1038/nature08195
.........

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Call for Public Debates on Future Uses of Stem Cells

2009-07-03T00:09:00.001+02:00

Science is running ahead of public debate and guidelines to grapple with use of stem cell-derived eggs and sperm
Thursday, 02 July 2009

More than 40 scientists, bioethicists, lawyers and science journal editors are calling on their colleagues, policy makers and the public to begin developing guidelines for the research and reproductive use of stem cell-derived eggs and sperm, even though such use may be a decade or more away.

"Science has always moved faster than social debate or society's ability to grapple with these issues," says Debra Mathews, Ph.D., lead author of a paper published in the July issue of Cell Stem Cell and assistant director of science programs at the Johns Hopkins Berman Institute of Bioethics. The paper calls for all parties to begin engaging in open discussion and debates, and describes the need for informed social policy well in advance of the eventual use of eggs and sperm derived from pluripotent stem cells.

Mathews said stem cell researchers need to be better prepared to address public questions about uses of so-called pluripotent stem cell-derived gametes – regardless of how realistic or soon those uses may be. Such uses would potentially include reproductive uses such as the creation of sperm and eggs for in vitro fertilization, embryo selection based on genetic profile, and the creation of embryos from the tissues of foetuses, children and the deceased.

The issues are too complex, and the stakes are too high, the authors suggest, for the public to be caught unaware by some new capability for using stem cell-derived gametes, and the research already is moving rapidly toward generation of sperm and eggs capable of making human embryos and potentially children.

"Because derived-gamete research will require the creation and destruction of human embryos, this line of research will be morally objectionable to those who imbue human embryos with full moral status, and those objections must be addressed," the authors state.

In their paper, the Johns Hopkins-led team described an analysis of the current state of pluripotent stem cell science and suggested a framework for the debates that need to take place.

There was consensus by the authors that policymakers should not restrict scientific inquiry solely because ethical or moral disagreement exists about the use of these cells. Instead, they offered recommendations for guidelines that would be the focus of social debate. Among them were that restrictions should be specific to those aspects of the technology that are deemed morally unacceptable in a given nation or state, and that specific consent should be required of tissue donors whose cells will be used to derive gametes for use in reproduction. This approach would rule out using for reproduction any tissue from foetuses, minors and the deceased. Consent, they said, need not be required in situations involving laboratory studies that produce no embryos.

The authors emphasized that significant oversight rules must be in place before any reproductive uses of gametes even begins, and early attempts to use gametes for these purposes should take place only as part of clinical research that follows the highest ethical standards.

Assuming that reproductive use of stem cell-derived gametes does occur, the health of women carrying the resulting foetuses, and of children born to them, should be monitored rigorously and tracked in long-term studies.

Pluripotent stem cell-derived gamete research brings together several of today's most contentious ethical issues, including the use of embryonic stem cells, the increasing ability to identify and understand risks associated with particular parts of the human genome, advanced reproductive technologies to treat infertility and interest in "human enhancement."

Mathews noted that pluripotent stem cell-derived gamete research already is producing significant advances in basic understanding of how eggs and sperm develop from germ cells, infertility, genetic diseases and some cancers.

Mathews said the most difficult scientific issue the study team faced was predicting how long it would take to get from a human stem cell to a set of gametes capable of successful test-tube fertilization, and how long, if ever, it would be until such gametes are used in clinical care. The group believes it will take at least a decade to develop derived human gametes and that clinical applications likely will not be available for several years beyond that.

Whatever the time frame, she said determining whether pluripotent stem cell-derived gametes can function reliably and normally is critical for both non-reproductive and reproductive purposes.

Scientists and the public also must prepare, Mathews noted, for the potential production of large numbers of human gametes that facilitate multigenerational laboratory studies of human genetics and disease.

"Although many welcome the prospects for disease prevention and health promotion that such research should facilitate, many others will find the treatment of human embryos in such blatantly manipulative ways to be ethically unacceptable," the authors said in their paper.

Reference:
Pluripotent Stem Cell-Derived Gametes: Truth and (Potential) Consequences
Debra J.H. Mathews, Peter J. Donovan, John Harris, Robin Lovell-Badge, Julian Savulescu and Ruth Faden
Cell Stem Cell, Volume 5, Issue 1, 11-14, 2 July 2009, doi:10.1016/j.stem.2009.06.005
.........

ZenMaster

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Embryonic Stem Cells and iPS Cells are Different

2009-07-02T23:49:00.003+02:00

UCLA scientists find molecular differences between ESCs and reprogrammed skin cells
Thursday, 02 July 2009

UCLA researchers have found that embryonic stem cells and skin cells reprogrammed into embryonic-like cells have inherent molecular differences, demonstrating for the first time that the two cell types are clearly distinguishable from one another.

The data from the study suggest that embryonic stem cells and the reprogrammed cells, known as induced pluripotent stem (iPS) cells, have overlapping but still distinct gene expression signatures. The differing signatures were evident regardless of where the cell lines were generated, the methods by which they were derived or the species from which they were isolated, said Bill Lowry, a researcher with the Broad Stem Cell Research Center and a study author.

"We need to keep in mind that iPS cells are not perfectly similar to embryonic stem cells," said Lowry, an assistant professor of molecular, cell and developmental biology.

"We're not sure what this means with regard to the biology of pluripotent stem cells. At this point our analyses comprise just an observation. It could be biologically irrelevant, or it could be manifested as an advantage or a disadvantage."

The study appears in the July 2, 2009 issue of the journal Cell Stem Cell.

The iPS cells, like embryonic stem cells, have the potential to become all of the tissues in the body. However, iPS cells do not require the destruction of an embryo.

The study was a collaboration between the labs of Lowry and UCLA researcher Kathrin Plath, who were among the first scientists and the first in California to reprogram human skin cells into iPS cells. The researchers performed microarray gene expression profiles on embryonic stem cells and iPS cells to measure the expression of thousands of genes at once, creating a global picture of cellular function.

Lowry and Plath noted that, when the molecular signatures were compared, it was clear that certain genes were expressed differently in embryonic stem cells than they were in iPS cells. They then compared their data to that stored on a National Institutes of Health data base, submitted by laboratories worldwide. They analyzed that data to see if the genetic profiling conducted in other labs validated their findings, and again they found overlapping but distinct differences in gene expression, Lowry said.

"This suggested to us that there could be something biologically relevant causing the distinct differences to arise in multiple labs in different experiments," Lowry said.

"That answered our first question: Would the same observation be made with cell lines created and maintained in other laboratories?"

Next, UCLA researchers wanted to confirm their findings in iPS cell lines created using the latest derivation methods. The cells from the UCLA labs were derived using an older method that used integrative viruses to insert four genes into the genome of the skin cells, including some genes known to cause cancer. They analyzed cell lines derived with newer methods that do not require integration of the reprogramming factors. Their analysis again showed different molecular signatures between iPS cells and their embryo-derived counterparts, and these signatures showed a significant degree of overlap with those generated with integrative methods.

To determine if this was a phenomenon limited to human embryonic stem cells, Lowry and Plath analyzed mouse embryonic stem cells and iPS lines derived from mouse skin cells and again validated their findings. They also analyzed iPS cell lines made from mouse blood cells with the same result.

"We can't explain this, but it appears something is different about iPS cells and embryonic stem cells," Lowry said.

"And the differences are there, no matter whose lab the cells come from, whether they're human or mouse cells or the method used to derive the iPS cells. Perhaps most importantly, many of these differences are shared amongst lines made in various ways."

Going forward, UCLA researchers will conduct more sophisticated analyses on the genes being expressed differently in the two cell types and try to understand what is causing that differential expression. They also plan to differentiate the iPS cells into various lineages to determine if the molecular signature is carried through to the mature cells. In their current study, Lowry and Plath did not look at differentiated cells, only the iPS and embryonic stem cells themselves.

Further study is crucial, said Mark Chin, a postdoctoral fellow and first author of the study.

"It will be important to further examine these cells lines in a careful and systematic manner, as has been done with other stem cell lines, if we are to understand the role they can play in clinical therapies and what effect the observed differences have on these cells," he said.

Reference:
Induced Pluripotent Stem Cells and Embryonic Stem Cells Are Distinguished by Gene Expression Signatures
Mark H. Chin, Mike J. Mason, Wei Xie, Stefano Volinia, Mike Singer, Cory Peterson, Gayane Ambartsumyan, Otaren Aimiuwu, Laura Richter, Jin Zhang, Ivan Khvorostov, Vanessa Ott, Michael Grunstein, Neta Lavon, Nissim Benvenisty, Carlo M. Croce, Amander T. Clark, Tim Baxter, April D. Pyle, Mike A. Teitell, Matteo Pelegrini, Kathrin Plath, andWilliam E. Lowry
Cell Stem Cell, Volume 5, Issue 1, 111-123, 2 July 2009, doi:10.1016/j.stem.2009.06.008
.........

ZenMaster

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Blood Stem Cell Growth Factor Reverses Memory Decline in Mice

2009-07-02T01:39:00.002+02:00

The new study shows GCSF impacts both bone marrow and brain to improve cognition
Thursday, 02 July 2009

A human growth factor that stimulates blood stem cells to proliferate in the bone marrow reverses memory impairment in mice genetically altered to develop Alzheimer's disease, researchers at the University of South Florida and James A. Haley Hospital found. The granulocyte-colony stimulating factor (GCSF) significantly reduced levels of the brain-clogging protein beta amyloid deposited in excess in the brains of the Alzheimer's mice, increased the production of new neurons and promoted nerve cell connections.

The findings are reported online in Neuroscience and are scheduled to appear in the journal's print edition in August.

GCSF is a blood stem cell growth factor or hormone routinely administered to cancer patients whose blood stem cells and white blood cells have been depleted following chemotherapy or radiation. GCSF stimulates the bone marrow to produce more white blood cells needed to fight infection. It is also used to boost the numbers of stem cells circulating in the blood of donors before the cells are harvested for bone marrow transplants. Advanced clinical trials are now investigating the effectiveness of GCSF to treat stroke, and the compound was safe and well tolerated in early clinical studies of ischemic stroke patients.

"GCSF has been used and studied clinically for a long time, but we're the first group to apply it to Alzheimer's disease," said USF neuroscientist Juan Sanchez-Ramos, MD, PhD, the study's lead author.

"This growth factor could potentially provide a powerful new therapy for Alzheimer's disease – one that may actually reverse disease, not just alleviate symptoms like currently available drugs."

The researchers showed that injections under the skin of filgrastim (Neupogen®) – one of three commercially available GCSF compounds – mobilized blood stem cells in the bone marrow and neural stem cells within the brain and both of these actions led to improved memory and learning behaviour in the Alzheimer's mice.

"The beauty in this less invasive approach is that it obviates the need for neurosurgery to transplant stem cells into the brain," Dr. Sanchez-Ramos said.

Based on the promising findings in mice, the Alzheimer's Drug Discovery Foundation is funding a pilot clinical trial at USF's Byrd Alzheimer's Center. The randomized, controlled trial, led by Dr. Sanchez-Ramos and Dr. Ashok Raj, will test the safety and effectiveness of filgrastim in 12 patients with mild to moderate Alzheimer's disease.

The researchers worked with 52 elderly mice, equivalent to the human ages of 60 to 80 years. About half (24) were mice genetically altered to develop symptoms mimicking Alzheimer's disease by the time they reach 5-months old. The others (28 normal, or non-Alzheimer's, mice) were not. The researchers confirmed through a series of tests that the Alzheimer's mice were memory impaired before beginning the experiments.

Some mice were treated for three weeks with injections of the GCSF compound filgrastim. At the end of study, the Alzheimer's mice treated with GCSF demonstrated clearly improved memory, performing as well on behavioural tests as their non-Alzheimer's counterparts. The Alzheimer's mice administered saline injections instead of GCSF continued to perform poorly. GCSF treatment did not boost the already excellent memory performance demonstrated by the non-Alzheimer's mice tested before the study began.

Further experiments showed that the size and extent of beta amyloid deposited in the brains of the Alzheimer's mice was significantly less in those treated with GCSF. Depending on their ages, mice treated with GCSF had a 36 to 42-percent reduction in beta amyloid, the protein considered a major culprit in the development of Alzheimer's disease.

GCSF reduced the burden of beta amyloid deposited in the brains of the Alzheimer's mice by several means, the researchers found. One was by recruiting reinforcements to clear beta amyloid accumulating abnormally in the brain. The growth factor prodded bone-marrow derived microglia outside the brain to join forces with the brain's already-activated microglia in eliminating the Alzheimer's protein from the brain. Microglia are brain cells that act as the central nervous system's main form of immune defence. Like molecular "Pac-men," they rush to the defence of damaged or inflamed areas to gobble up toxic substances.

The growth factor also appeared to increase the production of new neurons in the area of the brain (hippocampus) associated with memory decline in Alzheimer's disease and to form new neural connections.

"The concept of using GCSF to harness bone marrow-derived cells for Alzheimer's therapy is exciting and the findings in mice are promising, but we still need to prove that this works in humans," said Dr. Raj, a physician researcher at the Byrd Alzheimer's Center at USF Health.
.........

ZenMaster

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Cell Transplantation and Cardiac Repair

2009-07-02T01:25:00.001+02:00

Cell Transplantation and Cardiac Repair
Thursday, 02 July 2009

The frontiers of cell transplantation for cardiac repair are discussed in the current issue of Cell Transplantation (Vol. 18 No.3).

Two studies are highlighted, one by a Brazil-based research team who looked at two different bone marrow cell delivery methods following myocardial infarction, and a second study from a team of researchers in Germany who used bone marrow stem cell transplants to repair limb ischemia with a goal of preventing amputations.

Two delivery techniques for stem cells:
With evidence mounting that cell-based therapies can repair the injured myocardium following acute infarction, a Brazil-based research team addressed questions of the best way to safely deliver bone-marrow mononuclear cells (BMMNC) derived from the same patient (autologous cells) to the heart, following a heart attack caused by a prolonged interruption of blood flow leading to changes in the electrocardiogram (ST elevation myocardial infarction). They compared two different delivery techniques – through the anterograde intra-coronary (ICA) or via the retrograde intra-coronary artery vein (ICV). Researchers used radiolabelled cells to evaluate cell distribution patterns in the heart and their relationship with left ventricle function improvement.

"BMMNC retention by damaged heart tissue was apparently higher when the anterograde approach was used, although further studies are required to confirm this data," said corresponding author Dr. Hans Dohman of the Hospital Pro-Cardiaco in Rio de Janeiro, Brazil.

While previous reports observed that micro-vascular obstruction impairing cell uptake by the heart could be an issue, the team hypothesized that an intravenous approach may overcome that potential.

"We hypothesized that an intravenous approach might overcome this issue since the passage (diapedesis) of circulating cells into the adjacent cardiac tissue occurs on the venous side of microcirculation," added Dr. Dohman.

"In addition, we found that the grade of obstruction of microcirculation does not correlate with the efficiency of cell delivery to the infarcted tissue."

The research team also found that higher cell retention correlated with better changes in observed ejection fraction from baseline to a six-month follow-up.

"Our data point toward a causal relationship between the total number of cells that participate in infarct repair and the final enhancement of cardiac function," concluded Dr. Dohman.

Bone marrow cell transplants for limb ischemia:
Bone marrow cell transplantation has been shown to induce new blood vessel growth (angiogenesis) and foster improvements in patients with ischemic artery disease. This study, conducted by a Berlin-based group, evaluated the long-term effects of intermuscular, autologous bone marrow cell (aBMC) transplants in patients at major risk for amputation due to the loss of blood flow to a limb (limb ischemia) and who have failed or who are not candidates for surgical techniques to remove or bypass blockages. Patients who have failed surgical steps have high rates of amputation. In this study, 90 percent of patients participating in the study had been scheduled for major amputations; their long-term limb salvage rate was 53 percent.

"Our results suggest that aBMC transplants have the potential to treat severely ischemic limbs," said corresponding author Dr. Berthold Amann of the Franziskuskrankenhaus Berlin-Vascular Center.

"Among the patients in whom limb salvage was successful and amputation avoided, the leg was also pain-free and usable."

Dr. Amann noted that the patients with limb salvage had better baseline perfusion than the eventually amputated patients. Among the limb-salvaged group, analgesic consumption was reduced by 62 percent and their total walking distance improved from zero to 40 meters.

"This study found that after aBMC, in addition to being spared amputation, a critically ischemic leg can have increased blood flow and support wound healing while patients have reduced pain," concluded Dr. Amann.

Researchers suggested that their results are yet to be confirmed, but a double-blind, placebo controlled study is currently underway.

"Should ours and other studies prove this therapy to be effective, adoption by hospitals other than large academic centres will require a simple method for processing the bone marrow," added Dr. Amann.

"The use of bone marrow derived cells for improvement in ischemic muscle in the heart or leg is very promising," said Amit N. Patel, associate professor of surgery at the University of Utah School of Medicine and the cardiovascular, skin, other tissue section editor of Cell Transplantation.

"Both articles demonstrate that the route of delivery is one of the key determinants in having positive outcomes in the early clinical trials."
.........

ZenMaster

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How Brain Stem Cells Develop Into Cells Which Repair Damaged Tissue

2009-07-01T21:10:00.002+02:00

Insight could lead to new therapies to repair damage caused by MS
Wednesday, 01 July 2009

The joint research, funded by the National Multiple Sclerosis Society and the UK MS Society as well as the National Institutes of Health and Howard Hughes Medical Institute, was conducted by scientists at the University of California San Francisco (UCSF) and University of Cambridge and was published today (01 July) in the journal Genes and Development.

Multiple sclerosis is an autoimmune disease, which is caused by the body's immune system attacking nerve fibres and their protective insulation, the myelin sheath, in the central nervous system. This damage prevents the nerves from 'firing' properly, and then leads to their destruction, resulting in physical and intellectual disabilities.

It is currently thought that two components determine clinical outcomes in MS. First, it is important to stop ongoing damage (mainly achieved by controlling inflammation in the central nervous system). The second is to repair the damage that has occurred to the protective myelin sheaths surrounding the nerve fibres (this involves a regenerative process called remyelination in which new myelin sheaths are restored to nerve fibres).

While there exist several effective treatments to reduce inflammatory damage, no treatments are available to augment remyelination to repair the damage to nerve fibres. Critical to the development of such repair therapies is to understand how the brain's own stem cells can replace the myelin forming cells (oligodendrocytes) lost in the disease. During early stages of the disease, the brains own stem cells are surprisingly good at repairing damage in MS. However, for reasons that until now have not been well explained, they become less efficient as the disease progresses.

In this study the researchers have identified the Wnt pathway, which plays an active role in the maintenance and proliferation of stem cells, as a crucial determinant of whether oligodendrocytes can efficiently make myelin. Their studies demonstrate that if the Wnt pathway is abnormally active, then the process is inhibited. This opens up the exciting possibility that the repair can be enhanced in MS patients by drugs that block the Wnt pathway.

Professor Robin Franklin from the University of Cambridge, a co-senior author of the study, explained the significance of their findings:

"The pathway we identified plays a critical role in whether repair to the damaged cells will or will not occur. Interestingly, mutations in this particular pathway are also involved in several cancers. In this regard, drugs that inhibit this pathway from signalling have been sought which might suppress tumour growth. These same drugs may also find a role in promoting repair in MS."

Lead author of the study, Stephen Fancy, PhD, a postdoctoral fellow in the lab of co-senior author David Rowitch, MD, PhD, a Howard Hughes Medical Institute Investigator at the University of California, San Francisco, said:

"We believe we have made a significant step forward in understanding why repair might fail in neurological diseases such as MS by identifying a pathway which inhibits the myelin repair process."

MS Society Director of Research, Jayne Spink, said:

"We are delighted with the outcome of this outstanding research, which gives us greater knowledge of the mechanics of MS. This works opens up new avenues of research and lends itself to more study. Being able to uncover the secrets behind the damage caused in MS will take us forward in our understanding of this debilitating condition."

"Our studies work have implications for other diseases," said UCSF's Rowitch.

"In a condition called periventricular leukomalacia (PVL), which can lead to cerebral palsy in extremely premature infants, recent studies show a similar inability of oligodendrocytes to perform their important repair function. In respect to failed myelin repair, we see a parallel between the chronic demyelinated plaques of multiple sclerosis and the lesions of PVL."

Reference:
Dysregulation of the Wnt pathway inhibits timely myelination and remyelination in the mammalian CNS
Stephen P.J. Fancy, Sergio E. Baranzini, Chao Zhao, Dong-In Yuk, Karen-Amanda Irvine, Sovann Kaing, Nader Sanai, Robin J.M. Franklin, and David H. Rowitch
Genes Dev. July 1, 2009 23: 1571-1585, doi:10.1101/gad.1806309

See also:
Multiple Sclerosis: A New Theory for Why Repair of the Brain’s Wiring Fails
HHMI News – July 1, 2009
.........

ZenMaster

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Chromosomal Problems Affect Nearly All Human Embryos

2009-07-01T20:43:00.004+02:00

Discovery may explain low fertility rates in humans
Wednesday, 01 July 2009

Scientists have shown that chromosomal abnormalities are present in more than 90% of IVF embryos, even those produced by young, fertile couples. Ms Evelyne Vanneste, a PhD student in the Centre for Human Genetics and the University Fertility Center, Leuven University, Belgium, told the 25th annual conference of the European Society for Human Reproduction and Embryology today, that the surprising finding meant that current techniques used in preimplantation genetic screening (PGS), where embryos are screened genetically in order to select the best embryo for transfer, do nothing to improve pregnancy and live birth rates. Indeed, it can lead to potentially viable embryos being discarded, she said.

Ms Vanneste and her team studied each cell from 23 three or four day-old IVF embryos from young (less than 35 years old), fertile couples who had asked for preimplantation genetic diagnosis (PGD). PGD is carried out where one or both parents have a known genetic abnormality, in this case an X-linked disorder or the microdeletions (loss of a tiny piece of a chromosome) that can cause such disorders as the cancer predisposition syndrome neurofibromatosis type 1. The embryos are screened to avoid the implantation of one carrying that abnormality. Such embryos are the most representative of normal human embryogenesis, the process that begins once an egg has been fertilised.

Using new technologies that can detect chromosomal aberrations in the whole genome (all human chromosomes) of a single cell, the team was able to screen embryonic cells at a much higher resolution than previously. Therefore, they could identify more chromosomal abnormalities than has been possible using the current technique, fluorescent in situ hybridisation (FISH), which can only analyse ten of the approximately 32,000 genetic regions at the same time.

"Until now, the majority of studies analysing the genetic composition of human embryos used low resolution techniques on embryos derived from couples with fertility problems who are at risk for embryonic aneuploidy, an aberrant number of chromosomes, such as three copies of chromosome 21 that results in Down's syndrome. Therefore, little was known about the frequency and type of chromosomal imbalances in embryos from normal, fertile women," said Ms Vanneste.

"Our new technique has enabled us to show that chromosomal abnormalities are far more common and complex than previously anticipated, even in embryos from young, normal fertile couples. This leads us to believe that such abnormalities must be present in all human IVF-ICSI embryos.”

"Although in vitro culture conditions are known to have a limited influence on the rate of chromosomal imbalances in IVF/ICSI embryos, it is probable that the chromosome instability observed in vitro also occurs in spontaneous pregnancies since, at most, 30% of human conceptions result in a live birth and more than 50% of spontaneous abortions carry chromosomal aberrations. The high rate of chromosomal abnormalities is almost certainly responsible for the low fecundity of humans compared with other mammals," she added.

The scientists say that their work has important implications for preimplantation genetic screening (PGS) in fertility treatment. PGS is routinely used in many fertility centres for couples who encounter problems with conception, particularly for advanced maternal age, repeated failure of implantation, repeated miscarriages, or severe male fertility problems. In PGS, a single cell is removed from the early embryo for genetic testing, since it is hypothesised that the selection of chromosomally normal embryos for uterine transfer would increase the live birth rate and decrease the spontaneous abortion rate per embryo transferred.

"Although PGS is promoted as a way of increasing the chances of a successful pregnancy," said Ms Vanneste, "there has never been any significant evidence that it does, in fact, increase live birth rates after IVF. Our findings have shown that almost every cell of a human embryo carries a different genetic composition; consequently, the one cell that is analysed genetically is not representative of the rest of the embryo. If the tested cell is genetically abnormal, the embryo will not be transferred. But the rest of the embryo might be normal and develop into a healthy person. Therefore, the use of PGS means that potentially viable embryos will be discarded. The prevalent chromosomal instability in all early human IVF embryos explains the failure of PGS to improve the live birth rate per embryo transferred.”

"I think that we have made a crucial breakthrough that will change the way we do preimplantation genetic diagnosis and PGS and help to advance our ability to improve human fertility," said Ms Vanneste.
.........

ZenMaster

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Neural Stem Cell Differentiation Factor Discovered

2009-07-01T05:58:00.001+02:00

Why neural stem cells divide and differentiate
Wednesday, 01 July 2009

Neural stem cells represent the cellular backup of our brain. These cells are capable of self-renewal to form new stem cells or differentiate into neurons, astrocytes or oligodendrocytes. Astrocytes have supportive functions in the environment of neurons, while oligodendrocytes form the myelin layer around axons in order to accelerate neuronal signal transmission. But how does a neural stem cell “know” which way it is supposed to develop? On the molecular level receptors of the Notch family play a significant role in this process. So far, only stimulating extracellular ligands of Notch receptors had been described. Biochemists of Goethe University Medical School now describe a long time assumed but not yet identified soluble Notch inhibitor.

Frankfurt scientists led by Mirko Schmidt and Ivan Dikic reported in the renowned journal “Nature Cell Biology” that the secreted protein EGFL7 (Epidermal Growth Factor-like domain 7) is such an inhibitory factor. EGFL7 had already been known from its involvement in the development of blood vessels.

“It was a surprise when we discovered that EGFL7 bound the extracellular domains of Notch receptors and competed with known Notch ligands" explains Ivan Dikic from the Institute of Biochemistry and CEF Institute in Frankfurt. Researchers analyzed the antagonistic effects of EGFL7 in adult neural stem cells. The self-renewal potential of these cells depends on an intact interaction of the ligand Jagged1 and its receptor Notch1. Addition of EGFL7 blocked the essential interaction and reduced the division of neural stem cells. At the same time, EGFL7 stimulated the differentiation of neural stem cells into neurons.

“It has been well defined that Notch signalling drives the formation of astrocytes from neural stem cells while it suppresses the formation of neurons and the maturation of oligodendrocytes" explains Mirko Schmidt at the Institute of Neurology. Inhibition of Notch signalling reverses the situation and more neural stem cells differentiate into neurons. This is exactly what happened upon the addition of EGFL7. In order to verify their findings in vivo, the researchers analyzed mouse brains and identified mature neurons as a source of EGFL7 in the adult brain. The distribution of these cells in the brain was biologically significant, as EGFL7 was absent from regions with high amounts of neural stem cells, e.g. the sub-ventricular zone.

“This way EGFL7 may promote the formation of new neurons" suggests Schmidt.

The findings of Schmidt and Dikic offer a plethora of medical applications. Maturation of adult stem or precursor cells is significant for the development of multiple tissues, e.g. in the central nerve system or in the heart. Moreover, cancer stem cells have been described, which are important for the formation of tumours, especially in the human brain. EGFL7 might also be applied as a neuronal differentiation factor in ischemic insults or neurodegenerative diseases such as Alzheimer or Parkinson predict both researchers. Future work will unravel in which diseases EGFL7 can unfold its therapeutic potential.

Reference:
Epidermal growth factor-like domain 7 (EGFL7) modulates Notch signalling and affects neural stem cell renewal
Mirko H.H. Schmidt, Frank Bicker, Iva Nikolic, Jeannette Meister, Tanja Babuke, Srdjan Picuric, Werner Müller-Esterl, Karl H. Plate & Ivan Dikic
Nature Cell Biology, 7 June 2009, doi:10.1038/ncb1896
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ZenMaster

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First Human Receives Cardiac Stem Cells

2009-07-01T05:39:00.001+02:00

Clinical trial to heal damage caused by heart attacks
Wednesday, 01 July 2009

Doctors at the Cedars-Sinai Heart Institute announced today the completion of the first procedure in which a patient’s own heart tissue was used to grow specialized heart stem cells that were then injected back into the patient’s heart in an effort to repair and re-grow healthy muscle in a heart that had been injured by a heart attack.

The minimally-invasive procedure was completed on the first patient on Friday, June 26.

The procedure is part of a Phase I investigative study approved by the U.S. Food and Drug Administration and supported by the Specialized Centers for Cell-based Therapies at the National Heart, Lung, and Blood Institute and the Donald W. Reynolds Foundation. It is the first to use adult cells from a patient’s own heart to attempt to heal injured heart muscle.

“This procedure signals a new and exciting era in the understanding and treatment of heart disease,” said Eduardo Marbán, MD PhD, director of the Cedars-Sinai Heart Institute, who developed the technique and is leading the clinical trial.

“Five years ago, we didn’t even know the heart had its own distinct type of stem cells. Now we are exploring how to harness such stem cells to help patients heal their own damaged hearts.”

The study is directed by the Cedars-Sinai Heart Institute, with the collaboration of the Johns Hopkins University, where Dr. Marbán worked prior to joining Cedars-Sinai in 2007. The 24 patients participating in the study have hearts that were damaged and scarred by heart attacks. Once enrolled in the study, patients go through a three-step procedure.

After undergoing extensive imaging so doctors can pinpoint the exact location and severity of the scars wrought by the heart attack, the patient undergoes a minimally-invasive biopsy, with local anaesthesia. Using a catheter inserted through a vein in the patient’s neck, doctors remove a small piece of heart tissue, about half the size of a raisin.

The heart tissue is then taken to a specialized lab at Cedars-Sinai, where heart stem cells are cultured using methods invented by Marbán and his team. It takes about four weeks for the cells to multiply to numbers sufficient for therapeutic use, approximately 10 to 25 million.

In the third and final step, the now-multiplied stem cells are re-introduced into the patient’s coronary arteries during a second catheter procedure.

All patients in the study had to have experienced heart attacks within four weeks prior to enrolling in the research project. Four patients will receive 12.5 million stem cells and two patients will serve as controls. Later this summer, it is anticipated that 12 more patients will undergo procedures to receive 25 million stem cells, while six additional patients will be monitored as controls.

The first patient, Kenneth Milles, a 39-year-old controller for a small construction company in the San Fernando Valley, experienced a heart attack on May 10 due to a 99 percent blockage in the left anterior descending artery, a major artery of the heart. Milles’ heart attack left 21 percent of his heart muscle infarcted, or scarred. He underwent his biopsy May 24 and received his infusion of stem cells on June 29.

The patients will be monitored for six months. Complete results are scheduled to be available in late-2010.

Marbán, who holds the Mark Siegel Family Foundation Chair at the Cedars-Sinai Heart Institute and directs Cedars-Sinai’s Board of Governors Heart Stem Cell Center, also said the cardiac stem cell procedure is a logical step forward from recent studies in which cardiac patients have been treated with stem cells derived from bone marrow. Studies over the past eight years have shown that more than 500 cardiac patients have experienced modest improvement when treated with bone marrow stem cells.

However, bone marrow stem cells are not predestined to regenerate heart muscle. When cardiac stem cells were discovered five years ago by various teams worldwide, Marbán began developing a method for isolating heart stem cells from minimally-invasive biopsies and then multiplying the cells. Unlike bone marrow cells, heart stem cells are naturally programmed to regrow heart tissue, so they could prove more effective in healing the injury caused by heart attacks.

“If successful, we hope the procedure could be widely available in a few years and could be more broadly applied to cardiac patients,” Marbán said. For example, if patients are able to re-grow damaged heart muscle via stem cell therapy, there could be lesser demand for expensive and risky treatments such as heart transplants.

The process to grow the cardiac-derived stem cells involved in the study was developed by Marbán when he was on the faculty of Johns Hopkins University. The university has filed for a patent on that intellectual property, and has licensed it to a company in which Dr. Marbán has a financial interest. No funds from that company were used to support the clinical study. All funding was derived from the National Institutes of Health, the Donald W. Reynolds Foundation and Cedars-Sinai Medical Center.

About the Cedars-Sinai Heart Institute:
The Cedars-Sinai Heart Institute is internationally recognized for outstanding heart care built on decades of innovation and leading-edge research. From cardiac imaging and advanced diagnostics to surgical repair of complex heart problems to the training of the heart specialists of tomorrow and research that is deepening medical knowledge and practice, the Cedars-Sinai Heart Institute is known around the world for excellence and innovations.
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ZenMaster

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Early Heart Attack Therapy

2009-07-01T05:23:00.002+02:00

Bone marrow extract improves cardiac function
Wednesday, 01 July 2009

A UCSF study for the treatment of heart failure after heart attack found that the extract derived from bone marrow cells is as effective as therapy using bone marrow stem cells for improving cardiac function, decreasing the formation of scar tissue and improving cardiac pumping capacity after heart attack.

Findings were published online and in the July 2009 issue of the Journal of Molecular Therapy. The cover of the journal features a microscope image of cells from the UCSF study.

The studies were done in mice using a novel stem cell delivery method developed by UCSF researchers to show that the extract from bone marrow cells is as beneficial to cardiac function as are intact, whole cells. Both the cell and cell extract therapies resulted in the presence of more blood vessels and less cardiac cell death, or apoptosis, than no therapy. The study also showed that heart function benefitted despite the finding that few of the injected cells remained in the heart at one month after therapy.

"Peer-reviewed medical literature is controversial as to whether bone marrow cells differentiate into cardiomyocytes, or cardiac muscle cells, but there is general agreement that stem cell therapy with these cells results in some level of functional improvement after a heart attack. The exact mechanism for this is not yet clear. Our results confirm that whole cells are not necessarily required in order to see the beneficial effects of bone marrow cell therapy," said Yerem Yeghiazarians, MD, study author, cardiologist and director of UCSF's Translational Cardiac Stem Cell Development Program.

UCSF researchers are investigating these new therapies to improve cardiac function after heart attack in an effort to prevent heart failure. Heart failure occurs when cardiac muscle is damaged and scar tissue replaces beating cardiomyocytes. As scar replaces healthy tissue, it causes the heart to enlarge and lose its pumping capacity. When the pumping capacity decreases, the heart fills with fluid, which moves to the lungs and can lead to organ failure and death.

"Current therapies improve symptoms but do not replace scar tissue. Our hope is to use stem cells to decrease the scar, minimize the loss of cardiac muscle and maintain or even improve the cardiac function after a heart attack," Yeghiazarians said.

Using a novel, closed-chest, ultrasound-guided injection technique developed by Yeghiazarians and his colleagues, the team administered three different groups with bone marrow cells, bone marrow cell extract, or saline (for the control group). The injections were administered at day three after heart attack – a timeframe somewhat similar to human biology on days six-to-seven after heart attack.

The team found at day 28 that both the bone marrow cell group and the extract group had significantly smaller heart damage than the control group.

Left-ventricular ejection fraction (LVEF), or the measurement of blood pumped out of the ventricles per heart beat, fell uniformly in each group after heart attack from a level of about 57.2 percent to 38.4 percent. At day 28 (and after the therapies had been administered on day three), LVEF improved in both the bone marrow cell and extract groups to approximately 40.6 and 39.1 percent as compared to approximately 33.2 percent for the control group.

"We hope our findings can help in the development of new therapies for improving heart function after the deleterious effects of a heart attack," says Yeghiazarians.

The team is continuing to evaluate bone marrow cell and extract therapies in order to identify the proteins and factors within the extract and gain insight into the possible mechanisms of cardiac functional improvement.

"The best acute therapy for a heart attack remains early recognition and revascularization of the blocked artery to minimize the damage to the heart muscle," said Yeghiazarians.

"Although the prognosis depends on multiple factors, what we know for sure is that the sooner a heart attack gets diagnosed and cardiologists open the blocked artery, the better the long-term outcome. There are a number of ongoing stem cell-based clinical trials, and depending on further research and the outcome of these studies, we might have new therapies for the treatment of patients who suffer from a heart attack in the not-too-distant future."

Reference:
Injection of Bone Marrow Cell Extract Into Infarcted Hearts Results in Functional Improvement Comparable to Intact Cell Therapy
Yerem Yeghiazarians, Yan Zhang, Megha Prasad, Henry Shih, Shereen A Saini, Junya Takagawa, Richard E Sievers, Maelene L Wong, Neel K Kapasi, Rachel Mirsky, Juha Koskenvuo, Petros Minasi, Jianqin Ye, Mohan N Viswanathan, Franca S Angeli, Andrew J Boyle, Matthew L Springer and William Grossman
Mol Ther 17: 1250-1256; doi:10.1038/mt.2009.85
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ZenMaster

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Pigs' Connective Tissue Cells Converted into Stem Cells

2009-06-25T22:01:00.004+02:00

New finding could result in better tests for stem cell therapy, more accurate model
Thursday, 25 June 2009

For years, proponents have touted the benefits of embryonic stem cell research, but the potential therapies still face hurdles. Side effects such as tumour development, a lack of an effective and long-term animal model to test new therapies, and genetic incompatibility between the host and donor cells are some of the problems faced by researchers. Now, scientists at the University of Missouri-Columbia have developed the ability to take regular cells from a pig's connective tissues, known as fibroblasts, and transform them into stem cells, eliminating several of these hurdles. The new study appeared in a recent issue of the Proceedings of the National Academy of Sciences (PNAS).

"It's important to develop a good, accurate animal model to test these new therapies," said R. Michael Roberts, Curator's Professor of Animal Science and Biochemistry and a researcher in the Bond Life Sciences Center.

"Cures with stem cells are not right around the corner, but the pig could be an excellent model for testing new therapies because it is so similar to humans in many ways."

In their research, Roberts; Toshihiko Ezashi, a research assistant professor of animal sciences in the College of Agriculture, Food and Natural Resources and lead author on the study; and Bhanu Telugu, a post-doctoral fellow in animal sciences; cultured fibroblasts from a foetal pig. The scientists then inserted four specific genes into the cells. These genes have the ability to "re-program" the differentiated fibroblasts so that they "believe" they are stem cells, take on many of the properties of stem cells that would normally be derived from embryos, and, like embryonic stem cells, differentiate into many, possibly all, of the more than 250 cell types found in the body of an adult pig.

Since these "induced pluripotent stem cells" were not derived from embryos and no cloning technique was used to obtain them, the approach eliminates some of the controversy that has accompanied stem cell research in the past. The next step is for Roberts and his team to remove the four genes that reprogrammed the original cells. Then the researchers will determine what needs to be done to direct the new stem cells to develop into specific cell types.

"Right now, we researchers have not answered questions concerning how to make stem cells develop into just one type of cell, such as those of liver, kidney or blood cells, rather than a mixture," Roberts said.

"Now that we have been able to turn regular cells into stem cells, we need to learn how to make the right type of tissue and then test putting that new tissue back into the animal."

Roberts also noted that using the same animal for both the beginning and end of the research would eliminate any host rejection of the transplanted cells once scientists reach the point where they are putting the new tissue back into the animal. Using pigs rather than mice allows researchers to observe any long-term effects of the therapies. Because mice typically have a short life span and differ from humans more than pigs, it is less difficult to predict and/or study long-term effects using pigs, Telugu said.

Reference:
Derivation of induced pluripotent stem cells from pig somatic cells
Toshihiko Ezashi, Bhanu Prakash V. L. Telugu, Andrei P. Alexenko, Shrikesh Sachdev, Sunilima Sinha and R. Michael Roberts
PNAS June 18, 2009, doi: 10.1073/pnas.0905284106
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ZenMaster

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Artificial Liver for Drug Tests

2009-06-25T17:55:00.003+02:00

Artificial Liver for Drug Tests
Thursday, 25 June 2009

If you have hay fever, headaches or a cold, it's only a short way to the nearest chemist. The drugs, on the other hand, can take eight to ten years to develop. Until now animal experiments have been an essential step, yet they continue to raise ethical issues.

"Our artificial organ systems are aimed at offering an alternative to animal experiments," says Professor Heike Mertsching of the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB in Stuttgart.

"Particularly as humans and animals have different metabolisms. 30 per cent of all side effects come to light in clinical trials."

The test system, which Professor Mertsching has developed jointly with Dr. Johanna Schanz, should in future give pharmaceutical companies greater security and shorten the path to new drugs. Both researchers received the "Human-centered Technology" prize for their work.

"The special feature, in our liver model for example, is a functioning system of blood vessels," says Dr. Schanz.

"This creates a natural environment for cells." Traditional models do not have this, and the cells become inactive.

"We don't build artificial blood vessels for this, but use existing ones – from a piece of pig's intestine."

All of the pig cells are removed, but the blood vessels are preserved. Human cells are then seeded onto this structure – hepatocytes, which, as in the body, are responsible for transforming and breaking down drugs, and endothelial cells, which act as a barrier between blood and tissue cells. In order to simulate blood and circulation, the researchers put the model into a computer-controlled bioreactor with flexible tube pump, developed by the IGB. This enables the nutrient solution to be fed in and carried away in the same way as in veins and arteries in humans.

"The cells were active for up to three weeks," says Dr. Schanz.

"This time was sufficient to analyze and evaluate the functions. A longer period of activity is possible, however."

The researchers established that the cells work in a similar way to those in the body. They detoxify, break down drugs and build up proteins. These are important pre-conditions for drug tests or transplants, as the effect of a substance can change when transformed or broken down – many drugs are only metabolized into their therapeutic active form in the liver, while others can develop poisonous substances. The researchers have demonstrated the basic possibilities for use of the tissue models – liver, skin, intestine and windpipe. At the moment, the test system is being examined. Within two years it could provide a safer alternative to animal experiments.
.........

ZenMaster

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Age Affects Function of Embryonic Muscle Stem Cell Genes

2009-06-25T17:44:00.005+02:00

Adult satellite cells have distinct genetic requirements
Thursday, 25 June 2009

Scientists working at the Carnegie Institution's Department of Embryology, with colleagues, have overturned previous research that identified critical genes for making muscle stem cells. It turns out that the genes that make muscle stem cells in the embryo are surprisingly not needed in adult muscle stem cells to regenerate muscles after injury. The finding challenges the current course of research into muscular dystrophy, muscle injury, and regenerative medicine, which uses stem cells for healing tissues, and it favours using age-matched stem cells for therapy. The study is published in the June 25 advance on-line edition of Nature.

Previous studies have shown that two genes Pax3 and Pax7, are essential for making the embryonic and neonatal muscle stem cells in the mouse. Lead researcher Christoph Lepper, a predoctoral fellow in Carnegie's Chen-Ming Fan's lab and a Johns Hopkins student, for the first time looked at these two genes in promoting stem cells at varying stages of muscle growth in live mice after birth.

As Christoph explained:

"The paired-box genes, Pax3 and Pax7 are involved in the development of the skeletal muscles. It is well established that both genes are needed to produce muscle stem cells in the embryo. A previous student, Alice Chen, studied how these genes are turned on in embryonic muscle stem cells (also published in Nature). I thought that if they are so important in the embryo, they must be important for adult muscle stem cells. Using genetic tricks, I was able to suppress both genes in the adult muscle stem cells. I was totally surprised to find that the muscle stem cells are normal without them."

This cross section of hind limb muscle tissue is from a mouse five days after injury. The uninjured cells are at top and stained red. The blue cells below are regenerating muscles cells. They were labelled with a blue stain and formed from muscle stem cells. Credit: Christoph Lepper.

The researchers then looked at whether the same was true upon injury, after which the repair process requires muscle stem cells to make new muscles. For this, they injured the leg muscles between the knee and ankle. They were again surprised that these muscle stem cells, without the two key embryonic muscle stem cell genes, could generate muscles as well as normal muscle stem cells. They even performed a second round of injury and found that the stem cells were still active.

The scientists then wondered when these genes become unnecessary for muscle stem cells to regenerate muscles. It turned out that these embryonic genes are important to muscle stem cell creation up to the first three weeks after birth. What makes the muscle stem cells different after three weeks?

The scientist believe that these two embryonic muscle stem cell genes also tell the stem cells to become quiet as the organism matures. After that time is reached, they "hand over" their jobs to a different set of genes. The researchers suggest that since the adult muscle stem cells are only activated when injury occurs (by trauma or exercise), they use a new set of genes from those used during embryonic development, which proceeds without injury. The scientists are eager to find these adult muscle stem cell genes.

"We are just beginning to learn the basics of stem cell biology, and there are many surprises," remarked Allan Spradling, director of Carnegie's Department of Embryology.

"This work illustrates the importance of carrying out basic research using animal models before rushing into the clinic with half-baked therapies."

Reference:
Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements
Christoph Lepper, Simon J. Conway & Chen-Ming Fan
Nature advance online publication 25 June 2009, doi:10.1038/nature08209
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Placenta: New Source for Harvesting Stem Cells

2009-06-23T17:14:00.002+02:00

Children's Hospital Oakland scientists first to discover new source for harvesting stem cells
Tuesday, 23 June 2009

A groundbreaking study conducted by Children's Hospital & Research Center at Oakland, California, is the first to reveal a new avenue for harvesting stem cells from a woman's placenta, or more specifically the discarded placentas of healthy newborns. The study also finds there are far more stem cells in placentas than in umbilical cord blood, and they can be safely extracted for transplantation. Furthermore, it is highly likely that placental stem cells, like umbilical cord blood and bone marrow stem cells, can be used to cure chronic blood-related disorders such as sickle cell disease, thalassaemia, and leukaemia.

The study, led by Children's Hospital & Research Center Oakland scientists Frans Kuypers, PhD, and Vladimir Serikov, PhD, will be the feature story in the July 2009 issue of Experimental Biology and Medicine. The doctors and their team made the discoveries by harvesting term placentas from healthy women undergoing elective Caesarean sections.

"Yes, the stem cells are there; yes, they are viable; and yes, we can get them out," declared Dr. Kuypers.

Stem cells are essentially blank cells that can be transformed into any type of cell such as a muscle cell, a brain cell, or a red blood cell. Using stem cells from umbilical cord blood, Children's Hospital Oakland physicians have cured more than 100 kids with chronic blood-related diseases through their sibling donor cord blood transplantation program, which began in 1997. However, according to the American Cancer Society, each year at least 16,000 people with serious blood- related disorders are not able to receive the bone marrow or cord blood transplant they need because they cannot find a match.

Dr. Kuypers explained that even when a patient receives a cord blood transplant, there may not be enough stem cells in the umbilical cord to successfully treat their disorder. Placentas, however, contain several times more stem cells than umbilical cord blood.

"The greater supply of stem cells in placentas will likely increase the chance that an HLA (human leukocyte antigen) matched unit of stem cells engrafts, making stem cell transplants available to more people. The more stem cells, the bigger the chance of success," said Dr. Kuypers.

This is a microphotograph of chorionic villus of human term placenta immunostained for CD34 (Marker of endothelial and haematopoietic stem cells, red), CD31 (marker of endothelial cell, green) and nuclei (DAPI, blue). Non-endothelial CD34-positive cell is clearly observed in tissue of placenta. Credit: Society for Experimental Biology and Medicine.

In this report, said Dr. Serikov, we demonstrate for the first time that human placentas could provide abundant amounts of CD34+ CD133+ colony-forming cells, as well as other primitive hematopoietic progenitors, suitable for transplantation in humans. The total amount of live haematopoietic stem cells, or colony-forming units in culture that could be obtained from placentas was an order of magnitude larger than the number of hematopoietic stem cells obtained from cord blood from the same source.

Haematopoietic stem cells which maintain their differentiation capacity, as well as stromal stem cells that support long-term culture of haematopoietic cells, can be harvested from perfusate of placenta following CXCR4 receptor blockade, said Dr. F. Kuypers. Importantly, live HPCs can similarly be obtained from whole cryopreserved placentas. Cells derived from placental tissue differentiated into all blood lineages in vitro. Animal experiments further demonstrated successful engraftment of placenta-derived HSC, which reconstituted haematopoiesis in immunodeficient mice.

In summary, said Dr. F. Kuypers, our results indicate for the first time that human term placenta is a high capacity source of live and functional hematopoietic stem cells. By using placental circulation and stem cell receptor blockade an abundant amounts of hematopoietic stem cell could be easily obtained in sterile conditions by non-destructive methods.

Dr. Steven R. Goodman, Editor-in-Chief of Experimental Biology and Medicine said "the outstanding importance of these results for practical haematology is determined by the fact that total number of stem cells that can be harvested from cord blood limits the efficacy of this stem cell source for transplants only to small children. These novel findings demonstrate that placenta may provide a source of autologous stem cells sufficient for reconstitution of haematopoiesis in adult patients. Use of methods to obtain haematopoietic cells from placenta, developed by Dr. Serikov and Dr. Kuypers as augmentation of cord blood-based therapy or replacement of bone marrow for transplantation will dramatically change whole field of transplantology."

Drs. Kuypers and Serikov have also developed a patent-pending method that will allow placental stem cells to be safely harvested and made accessible for transplantation. The process involves freezing placentas in a way that allows them to later be defrosted and suffused with a compound that enables the extraction of viable stem cells. The method will make it possible for companies to gather, ship and store placentas in a central location.

"We're looking for a partnership with industry to get placenta-derived stem cells in large quantities to the clinic," said Dr. Kuypers. He adds that much more research and grant funding are needed to explore the maximum potential of this latest discovery. He remains encouraged.

"Someday, we will be able to save a lot more kids and adults from these horrific blood disorders."
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ZenMaster

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Researchers Edit Genes in Human Stem Cells

2009-06-18T22:45:00.001+02:00

Researchers Edit Genes in Human Stem Cells
Thursday, 18 June 2009

Researchers at the Johns Hopkins School of Medicine have successfully edited the genome of human- induced pluripotent stem cells, making possible the future development of patient-specific stem cell therapies. Reporting this week in Cell Stem Cell, the team altered a gene responsible for causing the rare blood disease paroxysmal nocturnal haemoglobinuria, or PNH, establishing for the first time a useful system to learn more about the disease.

"To date, only about six genes have been successfully targeted or edited in human stem cells out of countless people and attempts — that's just not efficient enough if we want to move disease research and therapy forward," says Linzhao Cheng, Ph.D., an associate professor of gynaecology and obstetrics and member of the Johns Hopkins Institute of Cell Engineering.

"We've been able to improve gene targeting and editing in human embryonic stem cells more than 200 fold."

Cheng's lab and collaborators at Johns Hopkins study PNH, a condition where "friendly fire" kills patients' own blood cells and the body can't replenish the lost blood cells due to loss of normal blood stem cells. PNH is an acquired disease that occurs only in adults, according to Cheng.

"It's a tough condition to study because we need to study it in blood stem cells and they're difficult to grow in the lab. So for years we've been trying to develop another cell system to better understand and perhaps fix what's going on in PNH."

To establish a system for research, they used human embryonic stem cells which can be expanded unlimitedly in the laboratory, but they also had to create a mutation as found in a PNH patient.

To target and remove the function of the one specific gene known to cause PNH, the research team improved on the standard approach of gene targeting, which can remove a functional gene or replace a dysfunctional gene. The gene targeting technology, first used successfully for mouse embryonic stem cells, won a Nobel Prize in Physiology or Medicine in 2007.

Gene targeting exploits a cell's own ability to repair broken DNA. When DNA breaks from exposure to mutagens or other agents like DNA-cutting enzymes, DNA-repairing enzymes in the cell find and re-join the two exposed DNA ends. However, if another piece of DNA with exposed ends is floating around, it effectively can be spliced into the broken DNA during repair, and replace the defective copy.

The team's technological improvement includes the use of custom-designed molecular scissors that are made by collaborators at Harvard University and University of Texas Southwestern Medical Center. These engineered DNA cutting enzymes make a precise break at specific locations in a cell's DNA — in this case in the gene that causes PNH. They added the molecular scissors and a fragment of DNA containing a gene that confers selection of rare targeted clones in both human embryonic stem cells and induced pluripotent stem cells. The latter, also known as iPS cells, are very similar to embryonic stem cells in biological properties, but generated by using adult tissues such as skin.

Of all the cells surviving selection, they picked and grew eight iPS cell lines to study further, and five of those contained a targeted insertion at the gene site. Further examination showed that the cells contained the correct number of chromosomes, no longer contained any trace of the molecular scissors and had characteristics as cells from PNH patients that lack a group of cell surface molecules.

"I commend my team, especially Dr. Jizhong Zou who spent three years with the help of many collaborators on this challenging project," says Cheng.

"We're very excited about this accomplishment; it will enable better studies for other blood diseases. But there's still much to do before we can really use human iPS cells in clinical therapies."

Cheng's team will continue to improve on techniques and begin applying these techniques to iPS cells from patients.

Reference:
Gene Targeting of a Disease-Related Gene in Human Induced Pluripotent Stem and Embryonic Stem Cells
Jizhong Zou, Morgan L. Maeder, Prashant Mali, Shondra M. Pruett-Miller, Stacey Thibodeau-Beganny, Bin-Kuan Chou, Guibin Chen, Zhaohui Ye, In-Hyun Park, George Q. Daley, Matthew H. Porteus, J. Keith Joung, and Linzhao Cheng
Cell Stem Cell, 18 June 2009, doi:10.1016/j.stem.2009.05.023
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ZenMaster

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Fallopian Tubes Offer New Stem Cell Source

2009-06-18T02:20:00.001+02:00

Fallopian Tubes Offer New Stem Cell Source
Thursday, 18 June 2009

Human tissues normally discarded after surgical procedures could be a rich additional source of stem cells for regenerative medicine. New research from BioMed Central's open access Journal of Translational Medicine shows for the first time that human fallopian tubes are abundant in mesenchymal stem cells which have the potential of becoming a variety of cell types.

It has previously been shown that mesenchymal stem cells obtained from umbilical cords, dental pulp and adipose tissue, which are all biological discards, are able to differentiate into muscle, fat, bone and cartilage cell lineages; therefore, the search for sources to obtain multipotent stem cells from discarded tissues and without ethical problems is of great interest.

Tatiana Jazedje, and the research team from Human Genome Research Centre at the University of São Paulo, directed by Mayana Zatz, with the collaboration of medical doctors from the reproductive area, set out to isolate and assess the differentiation potential of mesenchymal stem cells from discarded human fallopian tubes. In the study, human fallopian tubes were obtained from hysterectomy and other gynaecological procedures from fertile women in their reproductive years (range 35-53 years) who had not undergone hormonal treatment for at least three months prior to surgery.

The Brazilian team found that human fallopian tube mesenchymal stem cells could be easily isolated and expanded in vitro, and are able to differentiate into muscle, fat, cartilage and bone cell lines. The cells' chromosome complement showed no abnormalities, suggesting chromosomal stability.

"In addition to providing an additional potential source for regenerative medicine, these findings might contribute to reproductive science as a whole," Jazedje commented.

"Moreover, the use of human tissue fragments that are usually discarded in surgical procedures does not pose ethical problems," Jazedje concluded.

Reference:
Human fallopian tube: a new source of multipotent adult mesenchymal stem cells discarded in surgical procedures
Tatiana Jazedje, Paulo M Perin, Carlos E Czeresnia, Mariangela Maluf, Silvio Halpern, Mariane Secco, Daniela F Bueno, Natassia M Vieira, Eder Zucconi and Mayana Zatz
Journal of Translational Medicine (in press)
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ZenMaster

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Gene Vital to Early Embryonic Cells Forming a Normal Heart and Skull

2009-06-16T02:01:00.002+02:00

Gene Vital to Early Embryonic Cells Forming a Normal Heart and Skull
Tuesday, 16 June 2009

New research from Cincinnati Children's Hospital Medical Center highlights the critical role a certain gene and its protein play during early embryonic development on formation of a normal heart and skull.

In a study posted online June 15 by the Proceedings of the National Academy of Sciences, a research team at Cincinnati Children's reports that too little of the gene/protein SHP2 interferes with the normal developmental activity of what are called neural crest cells. These cells, which occur very early in embryonic development, migrate to specific regions of the embryo. While doing so, the cells are supposed to differentiate and give rise to certain nerve tissues, craniofacial bones or smooth muscle tissue of the heart.

"Our findings show that a deficiency of SHP2 in neural crest cells results in a failure of cell differentiation at diverse sites in the developing embryo," said Jeffrey Robbins, Ph.D., co-director of the Heart Institute at Cincinnati Children's and senior investigator of the study.

"This leads to anatomical and functional deficits so severe that it precludes viability of the developing foetus."

SHP2 is a tyrosine phosphatase – an enzyme that helps trigger a cascade of biochemical reactions in cells as they specify to form certain tissues.

Although the study was conducted using mouse embryos, the findings are significant in efforts to understand congenital malformations of the heart and craniofacial region in people. Especially relevant, the researchers said, is the insight gained into early molecular events during embryonic development that might help explain such birth defects.

Dr. Robbins said the findings from this study could be used to develop specific drugs that could target the affected pathway, leading to treatment of heart and craniofacial malformations. About 4 percent of human infants are born with congenital malformations. Abnormal heart development is the most common human birth defect, affecting about 1 percent of newborns. The researcher team also wants to explore the exact alterations in neural crest cell migration, expansion and differentiation that contribute to birth defects of other organ systems.
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ZenMaster

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Huntington's disease Deciphered

2009-06-15T01:21:00.002+02:00

Mutated huntingtin activates a neuron specific kinase called JNK3
Monday, 15 June 2009

Researchers at the University of Illinois at Chicago College of Medicine have discovered how the mutated huntingtin gene acts on the nervous system to create the devastation of Huntington's disease. The report of their findings is available in Nature Neuroscience online.

The researchers were able to show that the mutated huntingtin gene activates a particular enzyme, called JNK3, which is expressed only in neurons and, further, to show what effect activation of that enzyme has on neuron function.

Huntington's disease is an adult onset neurodegenerative disease marked by progressive mental and physical deterioration. It has been known for more than a decade that everyone who develops the disease has mutations in a particular gene, called huntingtin, according to Scott Brady, professor and head of anatomy and cell biology at the UIC College of Medicine.

"There are several puzzling aspects of this disease," said Brady, who is co-principal investigator on the study.

"First, the mutation is there from day one. How is it that people are born with a perfectly functioning nervous system, despite the mutation, but as they grow up into their 30s and 40s they start to develop these debilitating symptoms? We need to understand why the protein is bad at 40 but it wasn't bad at 4."

The second problem, according to Brady, is that the gene is expressed not just in the nervous system but in other parts of the body. However, the only part of the body that is affected is the nervous system. Why are neurons being affected?

Brady, Gerardo Morfini, assistant professor of anatomy and cell biology at UIC and co-principal investigator of the study, and their colleagues began looking for a mechanism that could explain all the pieces of the puzzle. They found that at extremely low concentrations, huntingtin was a potent inhibitor of axonal transport, the system within the neuron that shuttles proteins from the cell body where they are synthesized to the synaptic terminals where they are needed.

A neuron's critical role in making connections may require it to make the cellular trunk, called an axon, between the cell body and the synaptic terminal to be very long. Some cells have axons that reach half the body's length – for a tall person, a meter or more. But even in the brain, axonal projections are very long compared to other cells. In addition to the challenge of distance, neurons are very complex cells with many specialized areas necessary to carry out synaptic connections, requiring a robust transport system.

"Inhibition of neuronal transport is enough to explain what is happening in Huntington's," said Brady. Loss of delivery of materials to the terminals results in loss of transmission of signals from the neuron. Loss of signal transmission causes the neurons to begin to die back, leading to reduced transmissions, more dying back and eventual neuronal cell death.

This mechanism also explains the late onset of the disease, Brady said. Activation of JNK3 reduces transport but does not eliminate it. Young neurons have a robust transport system, but transport gradually declines with age.

"If you take a hit when you're very young, you still are making more and transporting more proteins in each neuron than you need," Brady said.

"But as you get older and older, the neuron produces and transports less. Each hit diminishes the system further. Eventually, the neuron falls below the threshold needed to maintain cell health."

Brady's group has also linked this pattern of progressive neurodegeneration – marked by a loss of signalling between neurons, a slow dying back of neurons, and eventual neuron death – to damage to the transport system in several other hereditary adult-onset neurodegenerative diseases and to Alzheimer's disease.

"There is a common theme and a common Achilles heel of the neuron that underlies all these diseases," Brady said.

"We've invented a word, dysferopathy, (from the Greek 'fero', to carry or transport) for these adult-onset neurodegenerative diseases. All have disruption of the axonal transport system in common."

Reference:
Pathogenic huntingtin inhibits fast axonal transport by activating JNK3 and phosphorylating kinesin
Gerardo A Morfini, Yi-Mei You, Sarah L Pollema, Agnieszka Kaminska, Katherine Liu, Katsuji Yoshioka, Benny Björkblom, Eleanor T Coffey, Carolina Bagnato, David Han, Chun-Fang Huang, Gary Banker, Gustavo Pigino & Scott T Brady
Nature Neuroscience Published online: 14 June 2009, doi:10.1038/nn.2346
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ZenMaster

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