Tuesday, 20 December 2011

Bone Marrow-derived Cells Differentiate in the Brain through Mechanisms of Plasticity

Bone Marrow-derived Cells Differentiate in the Brain through Mechanisms of Plasticity
Monday, 19 December 2011

Bone marrow-derived stem cells (BMDCs) have been recognized as a source for transplantation because they can contribute to different cell populations in a variety of organs under both normal and pathological conditions. Many BMDC studies have been aimed at repairing damaged brain tissue or helping to restore lost neural function, with much research focused on BMDC transplants to the cerebellum at the back of the brain. In a recent study, a research team from Spain has found that BMDCs, can contribute to a variety of neural cell types in other areas of the brain as well, including the olfactory bulb, because of a mechanism of "plasticity".

Their results are published in the current issue of Cell Transplantation (20:8).

"To our knowledge, ours is the first work reporting the BMDC's contribution to the olfactory neurons," said study corresponding author Dr. Eduardo Weruaga of the University of Salamanca, Spain.

"We have shown for the first time how BMDCs contribute to the central nervous system in different ways in the same animal depending on the region and cell-specific factors."

In this study, researchers grafted bone marrow cells into mutant mice suffering from the degeneration of specific neuronal populations at different ages, then compared them to similarly transplanted healthy controls. An increase in the number of BMDCs was found along the lifespan in both experimental groups. Six weeks after transplantation, however, more bone marrow-derived microglial cells were observed in the olfactory bulbs of the test animals where the degeneration of mitral cells was still in progress. The difference was not observed in the cerebellum where cell degeneration had been completed.

"Our findings demonstrate that the degree of neurodegenerative environment can foster the recruitment of neural elements derived from bone marrow," explained Dr. Weruaga.

"But we also have provided the first evidence that BMDCs can contribute simultaneously to different encephalic areas through different mechanisms of plasticity – cell fusion for Purkinje cells - among the largest and most elaborately dendritic neurons in the human brain – and differentiation for olfactory bulb interneurons."

Dr. Weruaga noted that they confirmed that BMDCs fuse with Purkinje cells but, unexpectedly, they found that the neurodegenerative environment had no effect on the behavior of the BMDCs.

"Interestingly, the contribution of BMDCs occurred through these two different plasticity mechanisms, which strongly suggests that plasticity mechanisms may be modulated by region and cell type-specific factors," he said.

"This study shows a potential new contribution of bone marrow derived cells following transplantation into the brain, making these cells highly versatile, in their ability to both differentiate into and fuse with endogenous neurons" said Dr. Paul R. Sanberg , coeditor-in-chief of Cell Transplantation and distinguished professor of Neuroscience at the Center of Excellence for Aging and Brain Repair, University of South Florida.

Source: Cell Transplantation Center of Excellence for Aging and Brain Repair
Contact: David Eve

Bone Marrow Contributes Simultaneously to Different Neural Types in the Central Nervous System Through Different Mechanisms of Plasticity
Recio, J. S.; Álvarez-Dolado, M.; Díaz, D.; Baltanás, F. C.; Piquer-Gil, M.; Alonso, J. R.; Werunga, E.
Cell Transplant. 20(8):1179-1192; 2011

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Grafting of Human Spinal Stem Cells into ALS Rats Best with Immunosuppressant Combination

Grafting of Human Spinal Stem Cells into ALS Rats Best with Immunosuppressant Combination  
Monday, 19 December 2011

A team of researchers grafting human spinal stem cells into rats modeled with amyotrophic lateral sclerosis (ALS), also known as "Lou Gehrig's Disease," a degenerative, lethal, neuromuscular disease, have tested four different immunosuppressive protocols aimed at determining which regimen improved long-term therapeutic effects. Their study demonstrated that a combined, systematically delivered immunosuppression regimen of two drugs significantly improved the survival of the human spinal stem cells. Their results are published in the current issue of Cell Transplantation (20:8).

"There are no therapeutic strategies that successfully modify ALS progression or outcome," said study corresponding author Dr. Michael P. Hefferan of the University of California at San Diego Neurodegeneration Laboratory.

"Cell-based transplantation therapies have emerged as potential treatments for several neurological disorders, including ALS. However, cell graft survival seems to greatly depend on an accompanying immunosuppression regimen, yet there are differential responses to identical immunosuppressive therapies."

While the reason for this differential response is unclear, the study authors suggest that several mechanisms, including distinct types of acute and inflammatory responses, may be to blame.

Their study aimed at optimizing an immunosuppressive protocol for transplanting human spinal cord cells into pre-symptomatic ALS G93A rats with the G93A superoxide dismutase (SOD1) mutation. Two drugs, tacrolimus (FK506) and mycophenolate, were used alone and in combination.

"Although FK506 has been used successfully as monotherapy in our previous studies of spinal ischemia, it failed in the present study on ALS," explained Dr. Hefferan, who speculated that inflammation played a role in the failure.

"In contrast to ALS, where spinal inflammation continues and likely worsens until end stage, the traumatically-injured spinal cord is typically characterized by an acute inflammatory phase followed by a progressive loss of most inflammatory markers."

According to the researchers, the animals receiving combined immunosuppression of both FK506 and mycophenolate likely benefited from the longer half-life of mycophenolate rather than from its action.

"The addition of mycophenolate seemed to supplement inhibition of T-cell formation and led to a robust graft survival when analyzed three weeks after grafting," concluded Dr. Hefferan.

Source: Cell Transplantation Center of Excellence for Aging and Brain Repair
Contact: David Eve

Optimization of Immunosuppressive Therapy for Spinal Grafting of Human Spinal Stem Cells in a Rat Model of ALS
Hefferan, M. P.; Johe, K.; Hazel, T.; Feldman, E. L.; Lunn, J. S.; Marsala, M.
Cell Transplant. 20(8):1153-1161; 2011


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iPS Cells Match Embryonic Stem Cells in Modeling Human Disease

iPS Cells Match Embryonic Stem Cells in Modeling Human Disease 
Monday, 19 December 2011

Stanford University School of Medicine investigators have shown that iPS cells, viewed as a possible alternative to human embryonic stem cells, can mirror the defining defects of a genetic condition — in this instance, Marfan syndrome — as well as embryonic stem cells can. An immediate implication is that iPS cells could be used to examine the molecular aspects of Marfan on a personalized basis. Embryonic stem cells, on the other hand, can't do this because their genetic contents are those of the donated embryo, not the patient's.

This proof-of-principle regarding the utility of induced pluripotent stem cells also has more universal significance, as it advances the credibility of an exciting approach that's been wildly acclaimed by some and viewed through gimlet eyes by others: the prospect of using iPS cells in modeling a broad range of human diseases. These cells, unlike ESCs, are easily obtained from virtually anyone and harbor a genetic background identical to the patient from which they were derived. Moreover, they carry none of the ethical controversy associated with the necessity of destroying embryos.

"Our in vitro findings strongly point to the underlying mechanisms that may explain the clinical manifestations of Marfan syndrome," said Michael Longaker, MD, professor of surgery and senior author of the study, which will be published online Dec. 12 in Proceedings of the National Academy of Sciences. Longaker is the Dean P. and Louise Mitchell Professor in the School of Medicine and co-director of the school's Institute for Stem Cell Biology and Regenerative Medicine. The study's first author is Natalina Quarto, PhD, a senior research scientist in Longaker's laboratory.

Marfan syndrome is an inherited connective-tissue disorder that occurs in one in 10,000 to one in 20,000 individuals. It is caused by any of a large number of defects in one gene. People with this condition tend to be very tall and thin and to suffer from osteopenia, or poor bone mineralization. Medical experts speculate that Abraham Lincoln, for example, suffered from this disorder. Marfan can also profoundly affect the eyes and cardiovascular system.

In this study, both iPS cells and embryonic stem cells carrying a mutation that causes Marfan syndrome showed impaired ability to form bone, and all too readily formed cartilage. These aberrations mirror the most prominent clinical manifestation of the disease.

Discovered in 2006, induced pluripotent stem cells, or iPS cells, are derived from fully differentiated tissues such as the skin. Yet they harbor the same capacity of embryonic stem cells to differentiate into all the tissues of the body as well as to replicate for indefinite periods in a dish. Because they offer an ethically uncomplicated alternative to embryonic stem cells, iPS cells have fueled the hope that they can replace ESCs in scientists' efforts to analyze, in a dish, the cellular defects ultimately responsible for diseases ranging from diabetes to Parkinson's and even such complex conditions as cardiovascular disease and autism.

One hope for iPS cells is to be able to differentiate them in a dish into tissues of interest — say, nerve cells of a patient with Parkinson's or autism — and study these resulting cells' characteristics with an eye to understanding the disease in a patient-specific way. This would be impossible to do with embryonic stem cells, unless ESCs from donated human eggs could be modified through the so-far insurmountable feat of substituting a patient's own genetic material into these eggs to reflect the patient's own genetic background.

While scientists have set the goal of using these cells for more than research purposes — developing therapeutic applications in regenerative medicine — that prospect is more distant. Scientists will have to develop the capacity first to repair within such cells, whether iPS or ESC, the genetic defects determined to be responsible for a patient's condition, and then differentiate the cells in bulk into the affected tissue, which could be used for regenerative medicine. Again, iPS cells in theory might be a better bet because, being initially derived from a particular patient, they could differentiate into tissues that are less likely to provoke graft rejection than similar tissues produced using a donor embryo's ESCs.

However, a number of studies have reported subtle differences between iPS cells and ESCs, implying that the two may not be equivalent. Experts have wondered whether these differences may render iPS cells inadequate substitutes for ESCs in modeling disease states. This study suggests otherwise, Longaker said.

The opportunity for a head-to-head comparison of ESCs and iPS cells arose serendipitously when Barry Behr, PhD, professor of obstetrics and gynecology and director of the Stanford Fertility & Reproductive Medicine Center, performed pre-implantation genetic diagnosis to select embryos for in vitro fertilization. Behr and Renee Reijo-Pera, PhD, professor of obstetrics and gynecology and director of the Stanford Center for Reproductive and Stem Cell Biology, discovered that one candidate embryo carried a genetic mutation that causes Marfan syndrome. This embryo was thus not deemed fit for implantation. But it was a potential source of embryonic stem cells, each of which would carry the Marfan-causing mutation. So, rather than discarding or storing it, the researchers received permission to derive the embryonic stem cells Longaker's team studied. (Both Behr and Reijo-Pera are co-authors of the study.)

What followed was a collaboration featuring an all-star cast that included senior faculty members from several departments in the medical school as well as researchers at the University of Naples Federico II in Italy. The researchers generated ESCs from the Marfan-carrying embryo. They also obtained skin biopsies from Marfan patients from another Stanford co-author, Uta Francke, MD, professor of genetics and of pediatrics, and used cells called fibroblasts from these samples to derive iPS cells by means of what have now become routine procedures.

"Here we had both iPS cells and embryonic stem cells side by side in culture dishes, both containing the defective gene responsible for Marfan. This was a perfect opportunity to compare them head to head," Longaker said.

When they did that, Longaker, Quarto and their associates found that both the iPS cells derived from the skin of Marfan patients and the ESCs from the embryonic Marfan carrier exhibited aberrations identical to those that characterize the disorder's observed skeletal symptoms — a diminished capacity to form bone and a heightened propensity for forming cartilage instead.

The scientists began the study with the knowledge that mutations causing Marfan syndrome are found in a gene that codes for a protein called FIBRILLIN-1. Importantly, FIBRILLIN-1 is known, in turn, to inhibit the activity of an intercellular signaling molecule named TGF-beta. Mouse studies have indicated that the absence or mutation of FIBRILLIN-1 results in a failure of this inhibition. This study showed for the first time in humans that the reason for stem cells' failure to form bone and overzealous conversion to cartilage directly resulted from their consequent exposure to more, and more-activated, TGF-beta than normal people's cells are.

The success of iPS cells in faithfully reproducing Marfan's cellular and molecular defects every bit as well as ESCs do may allow the disease to be studied (and, in the long run, even treated) in a case-by-case manner. While Marfan is a single-gene disorder, it can and does result from any of a large number of mutations to that one gene — upward of 600 have been identified so far —which manifest as a spectrum of subtle differences in symptoms from one patient to the next.

Contact: Bruce Goldman


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Saturday, 3 December 2011

Adult Stem Cells Use Special Pathways to Repair Damaged Muscle

Discovery could help future treatments for muscle repairs, disorders  
Saturday, 03 December 2011

When a muscle is damaged, dormant adult stem cells called satellite cells are signaled to "wake up" and contribute to repairing the muscle. University of Missouri researchers recently found how even distant satellite cells could help with the repair, and are now learning how the stem cells travel within the tissue. This knowledge could ultimately help doctors more effectively treat muscle disorders such as muscular dystrophy, in which the muscle is easily damaged and the patient's satellite cells have lost the ability to repair.
D Cornelison, an associate professor of biological
sciences in the College of Arts and Science and
a researcher in the Bond Life Sciences Center,
says knowing how adult stem cells travel could
help treatments for muscle disorders such as
muscular dystrophy. Credit: MU News Bureau.
"When your muscles are injured, they send out a 'mayday' for satellite cells to come and fix them, and those cells know where to go to make more muscle cells, and eventually new muscle tissue," said D Cornelison, an associate professor of biological sciences in the College of Arts and Science and a researcher in the Bond Life Sciences Center.

"There is currently no effective satellite cell-based therapy for muscular dystrophy in humans. One problem with current treatments is that it requires 100 stem cell injections per square centimeter, and up to 4,000 injections in a single muscle for the patient, because the stem cells don't seem to be able to spread out very far. If we can learn how normal, healthy satellite cells are able to travel around in the muscles, clinical researchers might use that information to change how injected cells act and improve the efficiency of the treatment."
Video: When a muscle is damaged, dormant
adult stem cells called satellite cells are signaled
to “wake up” and contribute to repairing the
muscle. Credit: MU News Bureau.
In a new study, researchers in Cornelison's lab used time-lapse microscopy to follow the movement of the satellite cells over narrow "stripes" of different proteins painted onto the glass slide. The researchers found that several versions of a protein called ephrin had the same effect on satellite cells: the cells that touch stripes made of ephrin immediately turn around and travel in a new direction.

"The stem cell movement is similar to the way a person would act if asked to walk blindfolded down a hallway. They would feel for the walls," Cornelison said.

"Because the long, parallel muscle fibers carry these ephrin proteins on their surface, ephrin might be helping satellite cells move in a straighter line towards a distant 'mayday' signal."

Contact: Steven Adams

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Not All Cellular Reprogramming is Created Equal

Not All Cellular Reprogramming is Created Equal
Friday, 02 December 2011 

Like embryonic stem cells, iPS cells can become any cell type in the body, a characteristic that could make them well-suited for therapeutic cell transplantation or for creating cell lines to study such diseases as Parkinson's and Alzheimer's. Inconsistencies in iPS cell quality reported in a number of recent studies have tarnished their promise, dampened enthusiasm, and fueled speculation that they may never be used therapeutically.

Tweaking the levels of factors used during the reprogramming of adult cells into induced pluripotent stem (iPS) cells greatly affects the quality of the resulting iPS cells, according to Whitehead Institute researchers.

"This conclusion is something that I think is very surprising or unexpected — that the levels of these reprogramming factors determine the quality of the iPS cells," says Whitehead Founding Member Rudolf Jaenisch.

"We never thought they'd make a difference, but they do."

An article describing this work is published in the December 2 issue of Cell Stem Cell.

iPS cells are made by introducing specific reprogramming genes into adult cells. These factors push the cells into a pluripotent state similar to that of embryonic stem (ES) cells. Like ES cells, iPS cells can become any cell type in the body, a characteristic that could make them well-suited for therapeutic cell transplantation or for creating cell lines to study such diseases as Parkinson's and Alzheimer's.

Since the creation of the first iPS cells in 2006, researchers using various reprogramming techniques have reported a broad spectrum of efficiency rates and quality of resulting iPS cells. Although researchers have shown iPS cells can fulfill all developmental tests applied to ES cells, recent reports have identified molecular differences that can influence their developmental potential and render them less-than-equivalent partners to ES cells. These inconsistencies have tarnished the promise of iPS cells, dampened enthusiasm, and fueled speculation that they may never be used therapeutically.

In one example reported last year, a lab created iPS cells using a cutting-edge technique in which a piece of DNA containing four reprogramming genes is safely integrated in the genome of adult mouse cells. In this highly publicized study, the resulting iPS cells performed poorly in tests of pluripotency and failed to produce adult mice, which is the most stringent test of pluripotency. Yet again this called into question the fidelity by which reprogramming factors could consistently generate fully reprogrammed cells equivalent to ES cells. Many in the field saw this as another nail in the coffin of iPS cells.

To Bryce Carey, first author of the Cell Stem Cell paper and a graduate student in Jaenisch's lab at the time, this death knell seemed premature. He repeated the experiment, changing a few details, including the order in which the reprogramming factors were placed on the inserted piece of DNA. Surprisingly, such small alterations had a profound effect — more adult cells were converted to high-quality iPS cells than in the earlier, nearly identical study.

"We are trying to show that the reprogramming process is not as flawed as some have thought, and that you can isolate these fully pluripotent iPS cells that have all of the developmental potential as embryonic stem cells at a pretty high frequency," says Carey, who is now a postdoctoral associate at Rockefeller University.

"A lot of times these parameters are very difficult to control, so while the approach first described by [Shinya] Yamanaka back in 2006 is still the most reliable method for research purposes, we should be cautious in concluding there are inherent limitations. We show recovery of high-quality cells doesn't have to be the exception."

Contact: Nicole Giese

Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells
Bryce W. Carey, Styliani Markoulaki, Jacob Hanna, Dina A. Faddah, Yosef Buganim, Jongpil Kim, Kibibi Ganz, Eveline J. Steine, John P. Cassady, Menno P. Creyghton, G. Grant Welstead, Qing Gao, and Rudolf Jaenisch
Cell Stem Cell, December 2, 2011, 10.1016/j.stem.2011.11.003


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Japanese Researchers Repairing Spinal Cord Injury with Human Dental Pulp Stem Cells

Japanese Researchers Repairing Spinal Cord Injury with Human Dental Pulp Stem Cells 
Saturday, 03 December 2011

One of the most common causes of disability in young adults is spinal cord injury. Currently, there is no proven reparative treatment. Hope that a stem cell population, specifically dental pulp stem cells, might be of benefit to individuals with severe spinal cord injury has now been provided by the work of Akihito Yamamoto and colleagues, at Nagoya University Graduate School of Medicine, Japan, in a rat model of this devastating condition.

In the study, when rats with severe spinal cord injury were transplanted with human dental pulp stem cells they showed marked recovery of hind limb function. Detailed analysis revealed that the human dental pulp stem cells mediated their effects in three ways: they inhibited the death of nerve cells and their support cells; they promoted the regeneration of severed nerves; and they replaced lost support cells by generating new ones. Yamamoto and colleagues therefore hope that this approach can be translated into an effective treatment for severe spinal cord injury.

Contact: Karen Honey

Human dental pulp–derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms
Kiyoshi Sakai, Akihito Yamamoto, Kohki Matsubara, Shoko Nakamura, Mami Naruse, Mari Yamagata, Kazuma Sakamoto, Ryoji Tauchi, Norimitsu Wakao, Shiro Imagama, Hideharu Hibi, Kenji Kadomatsu, Naoki Ishiguro and Minoru Ueda
J Clin Invest. 2011, doi:10.1172/JCI59251


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Bush Embryonic Stem Cell Lines Different from Newly Derived Cell Lines

Bush Embryonic Stem Cell Lines Different from Newly Derived Cell Lines
Friday, 02 December 2011

Established human embryonic cell lines, including those approved for federal research funding under former President George W. Bush, are different than newly derived human embryonic stem cell lines, according to a study by UCLA stem cell researchers.

The finding, by scientists with the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, points to the importance of continuing to derive new stem cell lines so researchers can better understand pluripotency, the ability of these cells to make every cell in the human body, said study senior author Amander Clark, an assistant professor of molecular, cell and developmental biology in Life Sciences.

"It is critical to find out the characteristics that result in the highest quality pluripotent stem cell lines that we can make," Clark said.

"It is possible that we have not set the bar high enough yet for embryonic stem cells or induced pluripotent stem cells. We now know that established lines are different from newly derived lines and now we have to find out how important that is."

The study appears Nov. 30, 2011 in the early online edition of the peer-reviewed journal Human Molecular Genetics.

The study looked at the first six human embryonic stem cell lines developed by Clark's research team at UCLA from 2009 to 2011, which have since been accepted by the National Institute of Health's embryonic stem cell registry, founded by executive order in March 2009. Acceptance into the registry allows the UCLA lines to be used in federally funded research projects.

In her study, Clark decided to examine X chromosome inactivation and the mechanisms by which female stem cells turn off one X chromosome during development because it is a large physical marker that is easy to visualize in individual cells. Clark wanted to compare this specific molecular signature in established embryonic stem cell lines versus what occurs during the derivation of new embryonic stem cell lines from human blastocysts.

The established lines examined in the study were from a group of stem cell lines derived prior to 2001. The field has known for many years that the majority of established lines, Clark said, had already undergone X chromosome inactivation, and her work confirmed this finding. However, with the progression of time, Clark found that the molecular signature no longer reflected the normal process of X chromosome inactivation.

The X chromosome normally is inactivated by non-coding RNA and a special form of chromatin in female cells. In abnormal states, such as those found in the older, established human embryonic stem cells, the X chromosome is inactive, but this process is not regulated by the non-coding RNA and the chromatin is different.

"The classic signature is gone, so something else is regulating X chromosome inactivation in the established cell lines," Clark said.

"It will be important not only to find out what that is, but also to discover what else is changing in the nucleus that we cannot see regardless of whether the cell line is male or female."

The new cell lines generated by Clark's research team were derived from human embryos that were donated to the Broad Stem Cell Research Center by couples who had previously undergone in vitro fertilization to overcome infertility. The couples no longer planned to store or use their frozen embryos for reproductive purposes and had declined to donate the embryos to others for reproductive use.

The human embryos were transferred from the fertility clinic to the derivation lab at UCLA in frozen vials. They were then thawed by Clark's research team, and at six to seven days of development the embryos, or blastocysts, contained a cluster of cells called the inner cell mass. The inner cell mass is the source of new embryonic stem cell lines.

Clark's lab examined the human embryonic stem cell lines three to four weeks after growth from the inner cell mass and found that both X chromosomes were still active in many cells, making them more like the cells from the original inner cell mass.

Slowly, with time in culture and cryopreservation – how the lines are ultimately stored – one X chromosome is inactivated and the cell lines become identical to the older, established lines, including abnormal X chromosome inactivation, Clark said.

The question, Clark said, is whether the first cells to grow out from the inner cell mass are of a higher quality, and therefore the ones researchers should be aspiring to use for research and potentially therapeutically.

"It may prove to be important to stabilize these cells at that very young state, one that's closest in identity to the inner cell mass," Clark said.

"And then we can ask whether these cells give the best quality when differentiated into clinical cell types."

Keeping both X chromosomes active will also be important in modeling diseases such as Rett syndrome.

Going forward, Clark will study human embryonic stem cells in three states, lines in which the X chromosome is inactivated by normal means, lines in which the chromosome is inactivated abnormally and lines in which both X chromosomes remain active. Clark will seek to understand the differentiation potential of each of the three states.

"Our data highlights the importance of maintaining human embryonic stem cell derivation efforts. Gold standard human embryonic stem cell lines should be the benchmark for all human pluripotent stem cell research," the study states.

"Developing new experimental approaches aimed at sustaining human pluripotent nuclei in an epigenetic state closer in identity to the day six or seven day human blastocyst is to work towards a more robust gold standard."

Contact: Kim Irwin


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Friday, 2 December 2011

Singapore Scientists Lead Human Embryonic Stem Cell Study

Singapore Scientists Lead Human Embryonic Stem Cell Study
Friday, 02 December 2011

Researchers from A*STAR Singapore took lead roles in a study that identified a portion of the genome mutated during long-term culture of human embryonic stem cells (hESCs). The study was a worldwide collaboration, led by Drs Peter Andrews of the University of Sheffield (UK), Paul Robson of the Genome Institute of Singapore (GIS), Steve Oh of Singapore's Bioprocessing Technology Institute (BTI), and Barbara Knowles and others in the international stem cell community. The GIS, IMB and BTI are research institutes under the umbrella of the Agency for Science, Technology and Research, (A*STAR), Singapore.

Involving 125 ethnically diverse hESC lines originating from 38 laboratories globally, and now identified to represent multiple ethnic groups from different parts of the globe, the study is the largest to be conducted on the genetic stability of cultured hESCs. The findings are published today in the journal Nature Biotechnology.

Research into the variability of hESCs is very important as these cells may lead to future cell therapy and regenerative medicine. During long-term culture, however, these cells can acquire genetic changes (mutations), some of which could compromise the cells' utility for regenerative medicine. It is believed that mutations that arise and endure over long-term culture provide a selective advantage for the cells, such as a greater propensity for self-renewal.

The study re-emphasized that many chromosome changes occur repeatedly, resulting in increased copies in specific areas of the genome. Interestingly, through molecular karyotyping performed in Dr Robson's laboratory at the GIS, about 20% of the karyotypically normal cell lines exhibited subkaryotypic amplifications of a specific region in chromosome 20. This is also one of the karyotypically defined areas of change. The minimal region common to these cells contains three ES-cell expressed genes, and one of them, BCL2L1, is a strong candidate for driving hESC culture adaptation. The data generated in this study will be useful for understanding the frequency and types of genetic changes affecting cultured hESCs, an important issue in evaluating the cells for potential therapeutic applications.

Dr Paul Robson, Senior Group Leader of the Developmental Cellomics Laboratory, GIS, said:
"Not only does this work provide important information for evaluating human embryonic stem cell genetic integrity, it also highlights the general utility of these cells in understanding human biology and disease. This same region has recently been identified to repeatedly occur in numerous human cancer cell types, this likely indicative of similar selection pressures at play in stem cells and cancer cells. Interestingly, we found the propensity for mutation at this location is associated with a relatively recent chromosomal rearrangement that occurred in the last common ancestor of the human, chimp, and gorilla thus pointing to the value of having a comparative perspective for understanding human biology."

Dr Barbara Knowles, Principle Investigator at IMB added:
"This is a prodigious piece of community work comparing the genome of cell lines from around the world that were sampled after they had been grown in cell culture for a short period of time to samples from the same cell lines taken after they had been in culture for a longer period of time. Scientists at GIS used these globally obtained samples to pinpoint an area of the genome that contains a gene(s) that affects the cell's ability to control its own growth."

Dr Steve Oh, Principal Scientist at BTI said:
"This study took over three years to complete and is a great testimony of the international stem cell community working persistently together as a force for good. A special thanks goes to Prof Peter Andrews for his leadership! The fact that of the 125 cell lines tested, over 65% of them exhibited normal karyotypes in long term culture bodes well for the use of human embryonic stem cells for cell therapy in the future."

Contact: Winnie Serah Lim

Screening a large, ethnically diverse population of human embryonic stem cells identifies a chromosome 20 minimal amplicon that confers a growth advantage
The International Stem Cell Initiative
Nature Biotechnology, 27 November, 2011, doi:10.1038/nbt.2051


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Sunday, 27 November 2011

Neurons Grown from Skin Cells May Hold Clues to Autism

Rare syndrome's workings could help explain how brain wiring goes awry
Sunday, 27 November 2011

Potential clues to how autism miswires the brain are emerging from a study of a rare, purely genetic form of the disorders that affects fewer than 20 people worldwide. Using cutting-edge "disease-in a-dish" technology, researchers funded by the National Institutes of Health have grown patients' skin cells into neurons to discover what goes wrong in the brain in Timothy Syndrome. Affected children often show symptoms of autism spectrum disorders along with a constellation of physical problems.

Representative iPSC-derived neurons from
Timothy syndrome patient (bottom) shows
increased numbers of neurons that produce
the chemical messengers norepinephrine
and dopamine, compared to those from a
control subject (top). Credit: Ricardo
Dolmetsch, Ph.D., Stanford University.
Abnormalities included changes in the composition of cells in the cortex, the largest brain structure in humans, and of neurons that secrete two key chemical messengers. Neurons that make long-distance connections between the brain's hemispheres tended to be in short supply.
Most patients with Timothy Syndrome meet diagnostic criteria for an autism spectrum disorder. Yet, unlike most cases of autism, Timothy syndrome is known to be caused by a single genetic mutation.

"Studying the consequences of a single mutation, compared to multiple genes with small effects, vastly simplifies the task of pinpointing causal mechanisms," explained Ricardo Dolmetsch, Ph.D., of Stanford University, a National Institute of Mental Health (NIMH) grantee who led the study. His work was partially funded by a NIH Director's Pioneer Award.

Dolmetsch, and colleagues, report on their findings Nov. 27, 2011 in the journal Nature Medicine.

"Unlike animal research, the cutting-edge technology employed in this study makes it possible to pinpoint molecular defects in a patient's own brain cells," said NIMH Director Thomas R. Insel, M.D..

"It also offers a way to screen more rapidly for medications that act on the disordered process."

Prior to the current study, researchers knew that Timothy syndrome is caused by a tiny glitch in the gene that code for a calcium channel protein in cell membranes. The mutation results in too much calcium entering cells, causing a tell-tale set of abnormalities throughout the body. Proper functioning of the calcium channel is known to be particularly critical for proper heart rhythm – many patients die in childhood of arrhythmias – but its role in brain cells was less well understood.

To learn more, Dolmetsch and colleagues used a new technology called induced pluripotent stem cells (iPSCs). They first converted skin cells from Timothy Syndrome patients into stem cells and then coaxed these to differentiate into neurons.

"Remarkable reproducibility" observed across multiple iPSC lines and individuals confirmed that the technique can reveal defects in neuronal differentiation – such as whether cells assume the correct identity as the brain gets wired-up in early development, said the researchers. Compared to those from controls, fewer neurons from Timothy Syndrome patients became neurons of the lower layers of the cortex and more became upper layer neurons. The lower layer cells that remained were more likely to be the kind that project to areas below the cortex. In contrast, there were fewer-than-normal neurons equipped to form a structure, called the corpus callosum, which makes possible communications between the left and right hemispheres.

Forebrain of a mouse genetically engineered
to express the mutated gene that causes
Timothy syndrome (TS) shows fewer neurons
contributing to a brain structure responsible
for long-distance communications between
the left and right hemispheres, called the
corpus callosum, compared to the same
structure in a control animal (Ctrl). Human
iPSCs from TS patients showed a similar
reduction. Credit: Ricardo Dolmetsch, Ph.D.,
Stanford University.
Many of these defects were also seen in parallel studies of mice with the same genetic mutation found in Timothy syndrome patients. This supports the link between the mutation and the developmental abnormalities.

Several genes previously implicated in autism were among hundreds found to be expressed abnormally in Timothy Syndrome neurons. Excess cellular calcium levels also caused an overproduction of neurons that make key chemical messengers. Timothy Syndrome neurons secreted 3.5 times more norepinephrine and 2.3 times more dopamine than control neurons. Addition of a drug that blocks the calcium channel reversed the abnormalities in cultured neurons, reducing the proportion of catecholamine-secreting cells by 68 percent.

The findings in Timothy Syndrome patient iPSCs follow those in Rett Syndrome, another single gene disorder that often includes autism-like symptoms. About a year ago, Alysson Muotri, Ph.D., and colleagues at University of California, San Diego, reported deficits in the protrusions of neurons, called spines, which help form connections, or synapses. The Dolmetsch team's discovery of earlier (neuronal fate) and later (altered connectivity) defects suggest that disorders on the autism spectrum affect multiple stages in early brain development.

"Most of these abnormalities are consistent with other emerging evidence that ASDs arise from defects in connectivity between cortex areas and show decreased size of the corpus callosum," said Dolmetsch.

"Our study reveals how these might be traceable to specific mechanisms set in motion by poor regulation of cellular calcium. It also demonstrates that neurons derived from iPSCs can be used to identify the cellular basis of a neurodevelopmental disorder."

The mechanisms identified in this study may become potential targets for developing new therapies for Timothy Syndrome and may also provide insights into the neural basis of deficits in other forms of autism, said Dolmetsch.

Contact: Jules Asher

Using iPS cell-derived neurons to uncover cellular phenotypes associated with Timothy Syndrome
Pasca SP, Portmann T, Voineagu I, Yazawa M, Shcheglovitov O, Pasca AM, Cord B, Palmer TD, Chikahisa S, Seiji N, Bernstein JA, Hallmayer J, Geschwind DH, Dolmetsch RE.
Nature Medicine, November 27, 2011, doi:10.1038/nm.2576


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