Scientists Generate "Mini-kidney" Structures from Human Stem Cells

Findings may lead to much-needed therapies for kidney disease

Sunday, 17 November 2013

Diseases affecting the kidneys represent a major and unsolved health issue worldwide. The kidneys rarely recover function once they are damaged by disease, highlighting the urgent need for better knowledge of kidney development and physiology.

From left: Salk researchers Ilir Dubova, Ignacio

Sancho Martinez, Yun Xia, Juan Carlos Izpisua

Belmonte and Emmanuel Nivet. Credit: Salk

Institute for Biological Studies. 

Now, a team of researchers led by scientists at the Salk Institute for Biological Studies has developed a novel platform to study kidney diseases, opening new avenues for the future application of regenerative medicine strategies to help restore kidney function.

For the first time, the Salk researchers have generated three-dimensional kidney structures from human stem cells, opening new avenues for studying the development and diseases of the kidneys and to the discovery of new drugs that target human kidney cells. The findings were reported November 17 in Nature Cell Biology.

Scientists had created precursors of kidney cells using stem cells as recently as this past summer, but the Salk team was the first to coax human stem cells into forming three-dimensional cellular structures similar to those found in our kidneys.

"Attempts to differentiate human stem cells into renal cells have had limited success," says senior study author Juan Carlos Izpisua Belmonte, a professor in Salk's Gene Expression Laboratory and holder of the Roger Guillemin Chair.

"We have developed a simple and efficient method that allows for the differentiation of human stem cells into well-organized 3D structures of the ureteric bud (UB), which later develops into the collecting duct system."

For the first time, Salk scientists have grown human

stem cells into early-stage ureteric buds, kidney

structures responsible for reabsorbing water after

toxins have been filtered out. In the laboratory, they

used mouse embryonic kidney cells (seen here in red)

to coax the human stem cells to grow into the nascent

mushroom-shaped buds (blue and green). Their

discovery is a major step in developing regenerative

techniques for growing replacement human kidneys.

Credit: Salk Institute for Biological Studies. 

The Salk findings demonstrate for the first time that pluripotent stem cells (PSCs)-cells capable of differentiating into the many cells and tissue types that make up the body-can made to develop into cells similar to those found in the ureteric bud, an early developmental structure of the kidneys, and then be further differentiated into three-dimensional structures in organ cultures. UB cells form the early stages of the human urinary and reproductive organs during development and later develop into a conduit for urine drainage from the kidneys. The scientists accomplished this with both human embryonic stem cells and induced pluripotent stem cells (iPSCs), human cells from the skin that have been reprogrammed into their pluripotent state.

After generating iPSCs that demonstrated pluripotent properties and were able to differentiate into mesoderm, a germ cell layer from which the kidneys develop, the researchers made use of growth factors known to be essential during the natural development of our kidneys for the culturing of both iPSCs and embryonic stem cells. The combination of signals from these growth factors, molecules that guide the differentiation of stem cells into specific tissues, was sufficient to commit the cells toward progenitors that exhibit clear characteristics of renal cells in only four days.

The researchers then guided these cells to further differentiate into organ structures similar to those found in the ureteric bud by culturing them with kidney cells from mice. This demonstrated that the mouse cells were able to provide the appropriate developmental cues to allow human stem cells to form three-dimensional structures of the kidney.

In addition, Izpisua Belmonte's team tested their protocol on iPSCs from a patient clinically diagnosed with polycystic kidney disease (PKD), a genetic disorder characterized by multiple, fluid-filled cysts that can lead to decreased kidney function and kidney failure. They found that their methodology could produce kidney structures from patient-derived iPSCs.

Because of the many clinical manifestations of the disease, neither gene- nor antibody-based therapies are realistic approaches for treating PKD. The Salk team's technique might help circumvent this obstacle and provide a reliable platform for pharmaceutical companies and other investigators studying drug-based therapeutics for PKD and other kidney diseases.

"Our differentiation strategies represent the cornerstone of disease modelling and drug discovery studies," says lead study author Ignacio Sancho-Martinez, a research associate in Izpisua Belmonte's laboratory.

"Our observations will help guide future studies on the precise cellular implications that PKD might play in the context of kidney development."

Source: Salk Institute for Biological Studies

Contact: Kat Kearney 

Reference:

Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells

Yun Xia, Emmanuel Nivet, Ignacio Sancho-Martinez, Thomas Gallegos, Keiichiro Suzuki, Daiji Okamura, Min-Zu Wu, Ilir Dubova, Concepion Rodriguez Estban, Nuria Montserrat, Josep Maria Campistol, and Juan Carlos Ispisua Belmonte

Nature Cell Biology (2013), doi:10.1038/ncb2872

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Un-junking Junk DNA

Un-junking Junk DNA

Wednesday, 13 November 2013

A study led by researchers at the University of California, San Diego School of Medicine shines a new light on molecular tools our cells use to govern regulated gene expression. The study was published on line in advance of print November 10 in the journal Nature Structural and Molecular Biology.

"We uncovered a novel mechanism that allows proteins that direct pre-mRNA splicing – RNA-binding proteins – to induce a regulatory effect from greater distances than was thought possible," said first author Michael T. Lovci, of the Department of Cellular and Molecular Medicine, the Stem Cell Research Program and Institute for Genomic Medicine at UC San Diego.

Researchers from California, Oregon, Singapore and Brazil made this finding while working toward an understanding of the most basic signals that direct cell function. According to Lovci, the work broadens the scope that future studies on the topic must consider. More importantly, it expands potential targets of rationally designed therapies which could correct molecular defects through genetic material called antisense RNA oligonucleotides (ASOs).

"This study provides answers for a decade-old question in biology," explained principal investigator Gene Yeo, PhD, assistant professor of Cellular and Molecular Medicine, member of the Stem Cell Research Program and Institute for Genomic Medicine at UC San Diego, as well as with National University of Singapore.

"When the sequence of the human genome was fully assembled, under a decade ago, we learned that less than 3 percent of the entire genome contains information that encodes for proteins. This posed a difficult problem for genome scientists – what is the other 97 percent doing?"

The role of the rest of the genome was largely a mystery and was thus referred to as "junk DNA." Since then sequencing of other, non-human, genomes has allowed scientists to delineate the sequences in the genome that are remarkably preserved across hundreds of millions of years of evolution. It is widely accepted that this evidence of evolutionary constraint implies that, even without coding for protein, certain segments of the genome are vital for life and development.

Using this evolutionary conservation as a benchmark, scientists have described varied ways cells use these non-protein-coding regions. For instance, some exist to serve as DNA docking sites for proteins which activate or repress RNA transcription. Others, which were the focus of this study, regulate alternative mRNA splicing.

Eukaryotic cells use alternative pre-mRNA splicing to generate protein diversity in development and in response to the environment. By selectively including or excluding regions of pre-mRNAs, cells make on average ten versions of each of the more than 20,000 genes in the genome. RNA-binding proteins are the class of proteins most closely linked to these decisions, but very little is known about how they actually perform their roles in cells.

"For most genes, protein-coding space is distributed in segments on the scale of islands in an ocean," Lovci said.

"RNA processing machinery, including RNA-binding proteins, must pick out these small portions and accurately splice them together to make functional proteins. Our work shows that not only is the sequence space nearby these 'islands' important for gene regulation, but that evolutionarily conserved sequences very far away from these islands are important for coordinating splicing decisions."

Since this premise defies existing models for alternative splicing regulation, whereby regulation is enacted very close to protein-coding segments, the authors sought to define the mechanism by which long-range splicing regulation can occur. They identified RNA structures – RNA that is folded and base-paired upon itself – that exist between regulatory sites and far-away protein-coding "islands." Dubbing these types of interactions "RNA-bridges" for their capacity to link distant regulators to their targets, the authors show that this is likely a common and under-appreciated mechanism for regulation of alternative splicing.

These findings have foreseeable implications in the study of biomedicine, the researchers said, as the RNA-binding proteins on which they focused – RBFOX1 and RBFOX2 – show strong associations with neurodevelopmental disorders, such as autism and also certain cancers. Since these two proteins act upstream of a cascade of effects, understanding how they guide alternative splicing decisions may lead to advancements in targeted therapies which correct the inappropriate splicing decisions that underlie many diseases.

Source: University of California - San Diego 

Contact: Debra Kain

Reference:

Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges

Michael T Lovci, Dana Ghanem, Henry Marr, Justin Arnold, Sherry Gee, Marilyn Parra, Tiffany Y Liang, Thomas J Stark, Lauren T Gehman, Shawn Hoon, Katlin B Massirer, Gabriel A Pratt, Douglas L Black, Joe W Gray, John G Conboy & Gene W Yeo

Nature Structural & Molecular Biology, 10 November 2013 | doi:10.1038/nsmb.2699

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Human Stem Cells Used to Elucidate Mechanisms of Beta-cell Failure in Diabetes

Mechanisms that impair insulin production in diabetes identified using a human stem cell model of Wolfram syndrome, a rare form of diabetes

Wednesday, 13 November 2013

Insulin-producing beta-cells. 

Scientists from the New York Stem Cell Foundation (NYSCF) Research Institute and Columbia University Medical Center (CUMC) have used stem cells created from the skin of patients with a rare form of diabetes — Wolfram syndrome — to elucidate an important biochemical pathway for beta-cell failure in diabetes. The findings by Linshan Shang and colleagues were published today in Diabetes.

Scientists from NYSCF produced induced pluripotent stem (iPS) cells from skin samples from individuals with a rare form of diabetes, Wolfram syndrome. They then derived insulin-producing cells (beta cells) from these iPS cells, creating a human diabetes model in vitro. Next, they showed that the beta cells failed to normally secrete insulin because of protein-folding — or endoplasmic reticulum (ER) — stress. They found that a chemical, 4-phenyl butyric acid, that relieves this stress, prevents the cells from failing, suggesting a potential target for clinical intervention.

"These cells represent an important mechanism that causes beta-cell failure in diabetes. This human iPS cell model represents a significant step forward in enabling the study of this debilitating disease and the development of new treatments," said Dieter Egli, PhD, principal investigator of the study, and Senior Research Fellow at NYSCF and NYSCF–Robertson Stem Cell Investigator.

Wolfram syndrome is a rare, often fatal genetic disorder characterized by the development of insulin-dependent diabetes, vision loss, and deafness. Since all forms of diabetes are ultimately the result of an inability of pancreatic beta cells to provide sufficient insulin in response to blood sugar concentrations, this Wolfram patient stem cell model enables analysis of a specific pathway leading to beta-cell failure in more prevalent forms of diabetes. It also enables the testing of strategies to restore beta-cell function that may be applicable to all types of diabetes.

"Utilizing stem cell technology, we were able to study a devastating condition to better understand what causes the diabetes symptoms as well as discover possible new drug targets," said Susan L. Solomon, Co-Founder and Chief Executive Officer of The New York Stem Cell Foundation.

"This report highlights again the utility of close examination of rare human disorders as a path to elucidating more common ones," said co-author Rudolph L. Leibel, MD, the Christopher J. Murphy Professor of Diabetes Research and co-director of the Naomi Berrie Diabetes Center at CUMC.

"Our ability to create functional insulin-producing cells using stem cell techniques on skin cells from patients with Wolfram's syndrome has helped to uncover the role of ER stress in the pathogenesis of diabetes. The use of drugs that reduce such stress may prove useful in the prevention and treatment of diabetes."

Clinicians from the Naomi Berrie Diabetes Center recruited Wolfram syndrome patients to donate a skin sample. All Wolfram patients had childhood-onset diabetes requiring treatment with injected insulin, and all had vision loss. Additional cell lines were obtained from Coriell Institute for Medical Research. The researchers at NYSCF "reprogrammed," or reverted, the skin cells to an embryonic-like state to become iPS cells. An iPS cell line generated from a healthy individual was used as a normal control.

The researchers differentiated the iPS cells from the Wolfram subjects and the controls into beta cells, an intricate process that took several weeks. They implanted both Wolfram and control iPS cell-derived beta cells under the kidney capsule of immuno-compromised mice. Beta cells from the Wolfram subjects produced less insulin in the culture dish and secreted less insulin into the bloodstream of the mice when they were challenged with high blood-sugar levels.

A key finding was that these beta cells showed elevated markers of ER stress. Treatment with 4-phenyl butyric acid reduced the ER stress and increased the amount of insulin produced by the beta cells, thereby increasing the ability to secrete insulin in response to glucose.

Direct evidence in mice, as well as circumstantial evidence in humans with both type 1 and type 2 diabetes, highlights the role of the ER stress response mechanism in the survival of insulin-producing beta cells. The ER stress response mechanisms oppose both the stress of immune assault in type 1 diabetes and the metabolic stress of high blood glucose in both types of diabetes. When the ER stress response fails cell death occurs, potentially reducing the number of insulin-producing cells.

Source: New York Stem Cell Foundation 

Contact: David McKeon

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Why Stem Cells Need to Stick With Their Friends

Why Stem Cells Need to Stick With Their Friends

Thursday, 07 November 2013

Embryonic stem cells stick together (green, left

image) while they express Oct4 protein in their

nuclei (magenta, left). When Oct4 is removed

their shape changes and their adhesion to each

other is reduced (right panel, nuclei in blue).

Credit: DanStem. 

Scientists at University of Copenhagen and University of Edinburgh have identified a core set of functionally relevant factors which regulates embryonic stem cells' ability for self-renewal. A key aspect is the protein Oct4 and how it makes stem cells stick together. The identification of these factors will be an important tool in devising better and safer ways of making specialised cells for future regenerative cell therapies for treatment of diseases like diabetes and Parkinson's disease. The results have just been published in the scientific journal Current Biology.

Scientists have known that the protein Oct4 plays a key role in maintaining the embryonic stem cells in pure form by turning on stem cell genes, however up until now it has not been know which of the 8.000 or more possible genes that Oct4 can choose from actually support self-renewal.

By comparing the evolution of stem cells in frogs, mice and humans, scientists at the Danish Stem Cell Center (DanStem) and the MRC Centre for Regenerative Medicine in Edinburgh have now been able to link the protein Oct4 with the ability of cells to stick together. They found that for embryonic stem cells to thrive they need to stick together and Oct4's role is to make sure they stay that way.

"Embryonic stem cells can stay forever young unless they become grown-up cells with a specialised job in a process called differentiation. Our study shows that Oct4 prevents this process by pushing stem cells to stick to each other," says Dr Alessandra Livigni, Research Fellow at the University of Edinburgh.

Identification of specific genes

The research teams in Edinburgh and Copenhagen successfully identified 53 genes, out of more than 8.000 possible candidates that together with Oct4, functionally regulate cell adhesion. Almost like finding needles in a haystack the scientists have paved the way for a more efficient way of maintaining stem cells as stem cells.

"Embryonic stem cells are characterized, among other things, by their ability to perpetuate themselves indefinitely and differentiate into all the cell types in the body – a trait called pluripotency. Though to be able to use them medically, we need to be able to maintain them as stem cells, until they're needed. When we want to turn a stem cell into a specific cell for example; an insulin producing beta cell, or a nerve cell like those in the brain, we'd like this process to occur accurately and efficiently. We cannot do this if we don't understand how to maintain stem cells as stem cells," says Professor Joshua Brickman from DanStem, University of Copenhagen.

Future potential

As well as maintaining embryonic stem cells in their pure state more effectively, this new insight will also enable scientists to more efficiently manipulate adult cells to revert to a stem cell like stage known as induced pluripotent stem cells (iPS cells). These cells have many of the same traits and characteristics as embryonic stem cells but can be derived from the patients to both help study degenerative disease and eventually treat them.

"This research knowledge has the potential for us to change the way we grow stem cells, enabling us to use them in a less costly and more efficient way. It will help us devise better and safer ways to create specialised cells for future regenerative medicine therapies," concludes Professor Joshua

Source: University of Copenhagen 

Contact: Joshua Brickman

Reference:

A Conserved Oct4/POUV-Dependent Network Links Adhesion and Migration to Progenitor Maintenance

Alessandra Livigni, Hanna Peradziryi, Alexei A. Sharov, Gloryn Chia, Fella Hammachi, Rosa Portero Migueles, Woranop Sukparangsi, Salvatore Pernagallo, Mark Bradley, Jennifer Nichols, Minoru S.H. Ko, Joshua M. Brickman

Current Biology, 07 November 2013, 10.1016/j.cub.2013.09.048

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Human Muscle Stem Cell Therapy Gets Help from Zebrafish

HSCI researchers improve therapy prospects

Thursday, 07 November 2013

Zebrafish in Research Lab for Animal Testing.

Harvard Stem Cell scientists have discovered that the same chemicals that stimulate muscle development in zebrafish can also be used to differentiate human stem cells into muscle cells in the laboratory, a historically challenging task that, now overcome, makes muscle cell therapy a more realistic clinical possibility.

The work, published this week in the journal Cell, began with a discovery by Boston Children's Hospital researchers, led by Leonard Zon, MD, and graduate student Cong (Tony) Xu, who tested 2,400 different chemicals in cultures of zebrafish embryo cells to determine if any could increase the numbers of muscle cells formed. Using fluorescent reporter fish in which muscle cells were visible during their creation, the researchers found six chemicals that were very effective at promoting muscle formation.

Zon shared his results with Harvard Department of Stem Cell and Regenerative Biology professor Amy Wagers, PhD, and Mohammadsharif Tabebordbar, a graduate student in her laboratory, who tested the six chemicals in mice. One of the six, called forskolin, was found to increase the numbers of muscle stem cells from mice that could be obtained when these cells were grown in laboratory dishes. Moreover, the cultured cells successfully integrated into muscle when transplanted back into mice.

Inspired by the successful application of these chemicals in mice, Salvatore Iovino, PhD, a joint postdoctoral fellow in the Wagers lab and the lab of C. Ronald Kahn, MD, at the Joslin Diabetes Center, investigated whether the chemicals would also affect human cells and found that a combination of three chemicals, including forskolin, could induce differentiation of human induced pluripotent stem (iPS) cells, made by reprogramming skin cells. Exposure of iPS cells to these chemicals converted them into skeletal muscle, an outcome the Wagers and Kahn labs had been striving to achieve for years using conventional methods. When transplanted into a mouse, the human iPS-derived muscle cells also contributed to muscle repair, offering early promise that this protocol could provide a route to muscle stem cell therapy in humans.

The interdisciplinary, cross-laboratory collaboration between Zon, Wagers, and Kahn highlights the advantage of open exchange between researchers.

"If we had done this screen directly on human iPS cells, it would have taken at least 10 times as long and cost 100 times as much," said Wagers.

"The zebrafish gave us a big advantage here because it has a fast generation time, rapid development, and can be easily and relatively cheaply screened in a culture dish."

"This research demonstrates that over 300 million years of evolution, the pathways used in the fish are conserved through vertebrates all the way up to the human," said Wagers' fellow HSCRB professor Leonard Zon, chair of the Harvard Stem Cell Institute Executive Committee and director of the stem cell program at Boston Children's Hospital.

"We can now make enough human muscle progenitors in a dish to allow us to model diseases of the muscle lineage, like Duchenne muscular dystrophy, conduct drug screens to find chemicals that correct that disease, and in the long term, efficiently transplant muscle stem cells into a patient."

In a similar biomedical application, Kahn, who is chief academic officer at the Joslin, plans to apply the new ability to quickly produce muscle stem cells for diabetes research. His lab will generate iPS-derived muscle cells from people who are at risk for diabetes and people who have diabetes to identify alterations that lead to insulin resistance in the muscle.

Going forward, Zon plans to apply this platform of cross-species discovery to other stem cell lines, including those involved in blood and eye development.

"We have a new system to use to study tissue development, and it's not just muscle that can be studied, every single organ can be studied in the zebrafish system," he said.

Source: Harvard University 

Contact: B. D. Colen

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Study Identifies Mechanism That Makes Ordinary Stem Cells Create Tumours

Epigenetic effects on cell signalling leads healthy stem cells to create benign fibromas in the jaw

Thursday, 07 November 2013

This is Professor Songtao Shi of the Ostrow

School of Dentistry at the University of Southern

California. Credit: John Skalicky. 

A new study from the Ostrow School of Dentistry at the USC published in Cell Stem Cell illustrates how changes in cell signalling can cause ordinary stem cells in the jaw to start forming benign but potentially harmful tumours.

Principal investigator Songtao Shi, professor at the Ostrow School of Dentistry Center for Craniofacial Molecular Biology, says ossifying fibromas, the tumours focused on in the study, are benign but can grow aggressively and cause progressive enlargement of the jaw.

"The only treatment option for ossifying fibromas is surgical, which leads to major loss of vital tissues and challenging post-surgical reconstruction," Shi says.

"Quality of life is largely compromised. Thus, there is an urgent need to understand the underlying mechanism by which stem cells may contribute to the pathophysiology of oro-facial benign tumours and to develop target-specific treatment."

Shi and his collaborators uncovered a cellular signalling pathway that converts healthy mesenchymal stem cells in the jaw into ossifying fibroma mesenchymal stem cells (OFMSC), lessening their ability to make healthy bone tissue and greatly increasing the rate at which they multiply. The tumour stem cells display increased signalling activity with TGF-beta (TGF-b), a signalling protein already shown to be heavily tied to other craniofacial malformations.

Epigenetic up-regulation – switching existing but inactive genes "on" – of the TGF-b cell signalling loop appears to increase the formation of ossifying fibroma tumours. Conversely, suppressing TGF-b signalling seems to quell the tumour's proliferation rate, Shi says.

While there is still much more investigation needed, Shi says hopes that the findings have shed light on a way to stop the harmful growth of the tumours before risky surgery is needed.

"With an increased understanding of the mechanism of OFMSC, we can induce them to turn into normal jawbone MSC," he says.

"But before we can put this into clinical use, more translational research is needed."

Source: University of Southern California 

Contact: Beth Newcomb

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ZenMaster

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Human Stem Cell Research in Europe

New Report Calls for Sustained Public Endorsement and Funding for Human Stem Cell Research

Wednesday, 06 November 2013

A strategic report from the European Science Foundation examines the key scientific questions for human stem cell research in the context of the rapidly emerging field of regenerative medicine. In parallel to the potential new treatments for incurable diseases resulting from stem cell research, heated ethical and legal debates have arisen across the world. This report presents a comparative view of the legislative framework on human stem cell research across Europe and provides a selection of success stories in frontier research and clinical trials that underpin the advances achieved in Europe to date.

In recent years, international research on regenerative medicine and stem cells has yielded some promising results and even greater expectations in society. In this medical field, human embryonic stem cells could be applied in a variety of ways, for example to identify new compounds for drug development, or as cell-based therapies. The potential to use human stem cells to repair or replace tissue or organ functions lost through age, disease, damage or birth defects may raise ethical issues that must be considered integrally with any research. Europe is currently witnessing developments and debates that will impact regulation and public funding of stem cell research and innovation for years to come.

The report observes that Europe plays a leading role in regenerative medicine research, with most countries featuring legislative frameworks that are globally favourable to human stem cell research. The 30 countries' position on human stem cell research was grouped into five broad categories; very permissive, permissive with restrictions, restrictive by default, very restrictive and unlegislated. The report found that 63% of the countries fell into the first two categories.

"Europe has a valuable track record in the area of stem cell research. The report highlights the need to continue to fund this research so that its full potential can be realised." said Professor Stig Slørdahl, Dean of the Faculty of Medicine at Norwegian University of Science and Technology, who chaired the report.

The authors recommend that sustained public endorsement and funding need to continue in order for further research to be carried out and public-private partnerships to develop, bringing safe and innovative therapies to the market, with a potential benefit to millions of patients worldwide.

Dr Vanessa Campo-Ruiz, ESF Science Officer to the Chief Executive and lead author of the report commented:

"We hope this report may help to inform future policy and funding decisions across Europe and thus contribute to ensure this continent's scientific leadership, social welfare and economic growth."

About The European Science Foundation

The European Science Foundation coordinates collaboration in research, networking, and funding of international research programmes, as well as carrying out strategic and science policy activities at a European level. Its members are 67 national research funding and performing organisations, learned societies and academies in 29 countries.

Source: European Science Foundation 

Contact: Emma Knott

Reference:

Human Stem Cell Research and Regenerative Medicine: Focus on European policy and scientific contributions

(available online, pdf)

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Riboswitches in Action

Scientists at SISSA investigate a mechanism that switches genes on and off 

Monday, 04 November 2013

Riboswitch RNA. 

Riboswitches are RNA segments that switch genes on and off, either during DNA transcription or during protein translation, but little is known about the precise workings of this process. A study at SISSA uncovers some of the basic steps in this complex mechanism and paves the way for future research.

A cell is a complex environment in which substances (metabolites) must maintain a correct state of equilibrium, which may vary depending on specific needs. Cells can maintain the proper concentrations of metabolites by regulating gene protein encoding through specific “switches”, called riboswitches, which are able to block or activate protein synthesis. The precise mechanism by which these short strands of RNA carry out this function is still poorly understood. However, a study conducted by SISSA scientists Giovanni Bussi, Francesco Colizzi and Francesco De Palma and published in the journal RNA, now provides some important insights. 

A riboswitch is contained in a strand of messenger RNA, an RNA fragment that acts as a sort of template that “prints” the proteins that are needed for cell metabolism. However, unlike the rest of the messenger RNA unit, riboswitches don’t actually encode any portion of the protein (i.e., they form part of what geneticists call non-coding RNA) but they serve to activate or deactivate the protein printing process and are located, in bacteria, in a stretch preceding the coding sequence. Scientists know that this switching action is made possible by a change in shape, which occurs when a part of the riboswitch (the aptamer) binds to a molecule in the cell environment which acts as a signal. Bussi and colleagues used computer simulations to reproduce the dynamics of the process and understand how binding to the metabolite brings about the change in shape.

More specifically, Bussi and colleagues simulated the riboswitch that uses an adenine molecule as a signal, to regulate the gene expressing a protein involved in the metabolism of adenine itself. Their findings clarify how adenine stabilizes the active form of the riboswitch (the one triggering protein synthesis) to the detriment of the inactive conformation.

“We used molecular dynamics as a kind of ‘virtual microscope’ with which we observed the workings of the process”, explained Bussi.

“It’s very important to understand these regulatory mechanisms since they are present in many bacteria – as well as in multicellular organisms – and may be useful for developing new antibiotics in the future.”

The computer-based simulations carried out in the study relied on PLUMED software, the latest release of which was presented in another recent paper published by Bussi in Computer Physics Communications. PLUMED is a software program devised to create and analyse simulations.

“This more advanced version of the program was developed as a team of five young researchers from international institutions”.

It’s no coincidence that the researchers involved in this work are all young: the research project was in part funded by a special grant provided for young, non-permanent research fellows at SISSA and awarded to Bussi in 2011.

The research was carried out within the framework of a European Research Council (ERC)-funded project coordinated by  Bussi.

Source: Sissa Medialab

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For more on stem cells and cloning, go to CellNEWS at

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A New Model for Organ Repair

Kidney repair may not require stem cells

Saturday, 02 November 2013

Harvard Stem Cell Institute (HSCI) researchers have a new model for how the kidney repairs itself, a model that adds to a growing body of evidence that mature cells are far more plastic than had previously been imagined.

Microscopy of kidney tissue. Credit:
JW Schmidt. 

After injury, mature kidney cells dedifferentiate into more primordial versions of themselves, and then differentiate into the cell types needing replacement in the damaged tissue. This finding conflicts with a previously held theory that the kidney has scattered stem cell populations that respond to injury. The research appears online in PNAS Early Edition.

HSCI Kidney Diseases Program Leader Benjamin Humphreys, MD, PhD, a Harvard Medical School assistant professor at Brigham and Women's Hospital, was suspicious of the kidney stem cell repair model because his previous work suggested that all kidney cells have the capacity to divide after injury. He and his colleagues decided to test conventional wisdom by genetically tagging mature kidney cells in mice that do not express stem cell markers; the hypothesis being that the mature cells should do nothing or die after injury. The results showed that not only do these fully differentiated cells multiply, but they can multiply several times as they help to repair the kidney.

"What was really interesting is when we looked at the appearance and expression patterns of these differentiated cells, we found that they expressed the exact same 'stem cell markers' that these other groups claimed to find in their stem cell populations," said Humphreys.

"And so, if a differentiated cell is able to express a 'stem cell marker' after injury, then what our work shows is that that's an injury marker — it doesn't define a stem cell."

This new interpretation of kidney repair suggests a model by which cells reprogram themselves; similar to the way mature cells can be chemically manipulated to revert to an induced pluripotent state. The research echoes a study published last month by HSCI Principal Faculty member David Breault, MD, PhD, who showed that cells in the adrenal glands also regenerate by means of natural lineage conversion.

"One has to remember that not every organ necessarily is endowed with clear and well-defined stem cell populations, like the intestines or the skin," Humphreys explained.

"I'm not saying that kidney stem cells don't exist, but in tissues where cell division is very slow during homeostasis, there may not have been an evolutionary pressure for stem cell mechanisms of repair."

He plans to apply his kidney repair discovery to define new therapeutic targets in acute kidney injury. The goal would be to find drugs that accelerate the process of dedifferentiation and proliferation of mature kidney cells in response to injury, as well as slow down pathways that impair healing or lead to scar tissue formation.

Source: Harvard University 

Contact: B. D. Colen

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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at

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Study Finds a Patchwork of Genetic Variation in the Brain

Salk scientists find a surprising degree of variation among genomes of individual neurons from the same brain

Saturday, 02 November 2013

It was once thought that each cell in a person's body possesses the same DNA code and that the particular way the genome is read imparts cell function and defines the individual. For many cell types in our bodies, however, that is an oversimplification. Studies of neuronal genomes published in the past decade have turned up extra or missing chromosomes, or pieces of DNA that can copy and paste themselves throughout the genomes.

The only way to know for sure that neurons from the same person harbour unique DNA is by profiling the genomes of single cells instead of bulk cell populations, the latter of which produce an average. Now, using single-cell sequencing, Salk Institute researchers and their collaborators have shown that the genomic structures of individual neurons differ from each other even more than expected. The findings were published November 1 in Science.

This is Salk scientist Fred H. Gage, professor in

the Laboratory of Genetics. Credit: Courtesy

of the Salk Institute for Biological Studies. 

"Contrary to what we once thought, the genetic makeup of neurons in the brain aren't identical, but are made up of a patchwork of DNA," says corresponding author Fred Gage, Salk's Vi and John Adler Chair for Research on Age-Related Neurodegenerative Disease.

In the study, led by Mike McConnell, a former junior fellow in the Crick-Jacobs Center for Theoretical and Computational Biology at the Salk, researchers isolated about 100 neurons from three people posthumously. The scientists took a high-level view of the entire genome – looking for large deletions and duplications of DNA called copy number variations or CNVs – and found that as many as 41 percent of neurons had at least one unique, massive CNV that arose spontaneously, meaning it wasn't passed down from a parent. The CNVs are spread throughout the genome, the team found.

The miniscule amount of DNA in a single cell has to be chemically amplified many times before it can be sequenced. This process is technically challenging, so the team spent a year ruling out potential sources of error in the process.

"A good bit of our study was doing control experiments to show that this is not an artefact," says Gage.

"We had to do that because this was such a surprise – finding out that individual neurons in your brain have different DNA content."

This is research collaborator Michael McConnell

of the University of Virginia. Credit: Courtesy

of the Salk Institute for Biological Studies.

The group found a similar amount of variability in CNVs within individual neurons derived from the skin cells of three healthy people. Scientists routinely use such induced pluripotent stem cells (iPSCs) to study living neurons in a culture dish. Because iPSCs are derived from single skin cells, one might expect their genomes to be the same.

"The surprising thing is that they're not," says Gage.

"There are quite a few unique deletions and amplifications in the genomes of neurons derived from one iPSC line."

Interestingly, the skin cells themselves are genetically different, though not nearly as much as the neurons. This finding, along with the fact that the neurons had unique CNVs, suggests that the genetic changes occur later in development and are not inherited from parents or passed to offspring.

It makes sense that neurons have more diverse genomes than skin cells do, says McConnell, who is now an assistant professor of biochemistry and molecular genetics at the University Of Virginia School Of Medicine in Charlottesville.

"The thing about neurons is that, unlike skin cells, they don't turn over, and they interact with each other," he says.

"They form these big complex circuits, where one cell that has CNVs that make it different can potentially have network-wide influence in a brain."

Spontaneously occurring CNVs have also been linked to risk for brain disorders such as schizophrenia and autism, but those studies usually pool many blood cells. As a result, the CNVs uncovered in those studies affect many if not all cells, which suggests that they arise early in development.

The purpose of CNVs in the healthy brain is still unclear, but researchers have some ideas. The modifications might help people adapt to new surroundings encountered over a lifetime, or they might help us survive a massive viral infection. The scientists are working out ways to alter genomic variability in iPSC-derived neurons and challenge them in specific ways in the culture dish.

This is research collaborator Ira Hall of the

University of Virginia. Credit: Courtesy of the

Salk Institute for Biological Studies. 

Cells with different genomes probably produce unique RNA and then proteins. However, for now, only one sequencing technology can be applied to a single cell.

"If and when more than one method can be applied to a cell, we will be able to see whether cells with different genomes have different transcriptomes (the collection of all the RNA in a cell) in predictable ways," says McConnell.

In addition, it will be necessary to sequence many more cells, and in particular, more cell types, notes corresponding author Ira Hall, an associate professor of biochemistry and molecular genetics at the University of Virginia.

"There's a lot more work to do to really understand to what level we think the things we've found are neuron-specific or associated with different parameters like age or genotype," he says.

Source: Salk Institute 

Contact: Kat Kearney

Reference:

Mosaic Copy Number Variation in Human Neurons

Michael J. McConnell, Michael R. Lindberg, Kristen J. Brennand, Julia C. Piper, Thierry Voet, Chris Cowing-Zitron, Svetlana Shumilina, Roger S. Lasken, Joris R. Vermeesch, Ira M. Hall, Fred H. Gage

Science 1 November 2013:Vol. 342 no. 6158 pp. 632-637, DOI: 10.1126/science.1243472

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