Scientists Distinguish Signatures of Old versus Young Stem Cells

Scientists Distinguish Signatures of Old versus Young Stem Cells

Friday, 28 June 2013

A chemical code scrawled on histones — the protein husks that coat DNA in every animal or plant cell — determines which genes in that cell are turned on and which are turned off. Now, Stanford University School of Medicine researchers have taken a new step in the deciphering of that histone code.

Professor of neurology Thomas Rando, 

MD, PhD.. Credit: Stanford University 

Medical Center. 

In a study to be published June 27 in Cell Reports, a team led by Thomas Rando, MD, PhD, professor of neurology and neurological sciences and chief of the Veterans Affairs Palo Alto Health Care System's neurology service, has identified characteristic differences in "histone signatures" between stem cells from the muscles of young mice and old mice. The team also distinguished histone-signature differences between quiescent and active stem cells in the muscles of young mice.

"We've been trying to understand both how the different states a cell finds itself in can be defined by the markings on the histones surrounding its DNA, and to find an objective way to define the 'age' of a cell," said Rando, who is also director of Stanford's Glenn Laboratories for the Biology of Aging and deputy director of the Stanford Center on Longevity.

While all cells in a person's body share virtually the same genes, these cells can be as different from one another as a nerve cell is from a fat cell. This is because only a fraction of a cell's genes are actually "turned on" — actively involved in the production of one or another protein. A muscle cell produces the proteins it uses to be a muscle cell, a liver cell produces those it needs in order to be a liver cell and so forth. Rando's team thinks the same kinds of on/off differences may distinguish old stem cells from young stem cells.

In human cells, the DNA in which genes are found doesn't float loose inside the cell nucleus but is, rather, packaged inside protein "husks" called histones. Chemical marks on the histones, which sheathe our chromosomal DNA in each cell's nucleus, act as "stop" and "go" traffic signals. These signals tell the complex molecular machinery that translates genes' instructions into newly produced proteins which genes to read and which ones to skip.

In 2005, Rando and his colleagues published a study in Nature showing that stem cells in several tissues of older mice, including muscle, seemed to act younger after continued exposure to younger mice's blood. Their capacity to divide, differentiate and repopulate tissues, which typically declines with an organism's advancing age, resembled those of their stem-cell counterparts in younger animals.

This naturally led to curiosity about exactly what is happening inside a cell to rejuvenate it, said Rando. One likely place to look for an answer was histones, to see if changes in the patterns of the chemical marks on them might reveal any secrets, at the cellular level, of the aging process we all experience — and, perhaps, whether there might be anything we can do about it. Rando and his colleagues also wanted to learn more about what kinds of difference in these patterns accompany a cell's transition from one level of activity to another.

To do that, Rando and his team looked at satellite cells, an important class of stem cells that serve as a reserve army of potential new muscle tissue. Under normal circumstances, these rather rare stem cells sit quietly adjacent to muscle fibres. But some signal provided by muscular injury or degeneration prompts satellite cells to start dividing and then to integrate themselves into damaged fibres, repairing the muscle tissue. The investigators profiled the histone markings of mice that are as old, in mouse years, as young human adults, as well as mice whose human counterparts would be 70 to 80 years old.

The researchers harvested satellite cells from both healthy and injured muscle tissue of young mice and from healthy tissue of old mice; extracted these cells' DNA with the histone coatings intact; and used tagged antibodies targeting the different kinds of marks to find which spots on those histones were flagged with either "stop" or "go" signals.

"Satellite cells can sit around for practically a lifetime in a quiescent state, not doing much of anything. But they're ready to transform to an activated state as soon as they get word that the tissue needs repair," Rando said.

"So, you might think that satellite cells would be already programmed in a way that commits them solely to the 'mature muscle cell' state." The researchers expected that in these quiescent stem cells, the genes specific for other tissues like skin and brain would be marked by "stop" signals.

Instead, they found, in quiescent satellite cells taken from the younger mice, copious instances in which histones in the vicinity of genes ordinarily reserved for other tissues were marked with both "stop" and "go" signals, just as genes associated with development to mature-muscle status were.

"We weren't looking for that, and we certainly weren't expecting it," Rando said.

"We figured all the muscle genes would be either poised for activity — marked with both 'on' and 'off' signals — or 'on,' and that all the other genes would be turned off. But when you look at these satellite cells the way we did, they seem ready to become all kinds of cells. It's a mystery," he said, suggesting that it could mean stem cells thought to be committed to a particular lineage may be capable of becoming other types of tissue entirely.

"Maybe their fates are not permanently sealed," he said.

"The door is not locked. Who knows what could happen if they're given the right signals?" The Rando lab is now beginning to test this proposition.

Oddly, activated satellite cells from injured muscle tissue featured far more gene-associated "stop" signals than did quiescent ones.

"As a cell goes from quiescent to activated state, you might expect to see more genes marked by 'on' signals," Rando said.

"We found the opposite. The dominant pattern when cells become activated is a big increase in repressive marks across the genome. Apparently it's not until then that a satellite cell makes the effort to turn off all of its non-muscle options."

The differences between quiescent and activated cells, Rando's team found, are mirrored by those between young and old quiescent satellite cells.

"With age, there's an uptick in repressive markers. A lot more genes are locked in the 'off' position," he said.

The meaning of this is not yet clear, he added.

"In a division-capable cell, as opposed to the non-dividing, differentiated muscle cells that activated satellite cells may someday become, it may be important to maintain a high level of repression with age. Maybe this increase in repression is a kind of tumour-suppression mechanism, keeping aging satellite cells — which could have accumulated some dangerous mutations over the passing months and years — in check."

The description of the histone-code differences between young and old cells constitutes a yardstick allowing investigators to ask which of these differences are important in aging and in rejuvenation, Rando said.

"We don't have the answers yet. But now that we know what kinds of changes occur as these cells age, we can ask which of these changes reverse themselves when an old cell goes back to becoming a young cell" — as appeared to be the case when tissues of older mice were exposed to blood from younger mice.

Rando's group is now looking to test whether the signatures they've identified in satellite cells generalize to other kinds of adult stem cells as well.

Source: Stanford University Medical Center 

Contact: Bruce Goldman

Reference:

Chromatin Modifications as Determinants of Muscle Stem Cell Quiescence and Chronological Aging

Ling Liu, Tom H. Cheung, Gregory W. Charville, Bernadette Marie Ceniza Hurgo, Tripp Leavitt, Johnathan Shih, Anne Brunet, and Thomas A. Rando
Cell Reports, 27 June 2013, 10.1016/j.celrep.2013.05.043

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Reading DNA, Backward and Forward

Reading DNA, Backward and Forward

Tuesday, 25 June 2013

MIT biologists have discovered a mechanism that allows cells to read their own DNA in the correct direction and prevents them from copying most of the so-called "junk DNA" that makes up long stretches of our genome.

Only about 15 percent of the human genome consists of protein-coding genes, but in recent years scientists have found that a surprising amount of the junk, or intergenic DNA, does get copied into RNA — the molecule that carries DNA's messages to the rest of the cell.

Scientists have been trying to figure out just what this RNA might be doing, if anything. In 2008, MIT researchers led by Institute Professor Phillip Sharp discovered that much of this RNA is generated through a process called divergent expression, through which cells read their DNA in both directions moving away from a given starting point.

In a new paper appearing in Nature on June 23, Sharp and colleagues describe how cells initiate but then halt the copying of RNA in the upstream, or non-protein-coding direction, while allowing it to continue in the direction in which genes are correctly read. The finding helps to explain the existence of many recently discovered types of short strands of RNA whose function is unknown.

"This is part of an RNA revolution where we're seeing different RNAs and new RNAs that we hadn't suspected were present in cells, and trying to understand what role they have in the health of the cell or the viability of the cell," says Sharp, who is a member of MIT's Koch Institute for Integrative Cancer Research.

"It gives us a whole new appreciation of the balance of the fundamental processes that allow cells to function."

Graduate students Albert Almada and Xuebing Wu are the lead authors of the paper. Christopher Burge, a professor of biology and biological engineering, and undergraduate Andrea Kriz are also authors.

Choosing direction

DNA, which is housed within the nucleus of cells, controls cellular activity by coding for the production of RNAs and proteins. To exert this control, the genetic information encoded by DNA must first be copied, or transcribed, into messenger RNA (mRNA).

When the DNA double helix unwinds to reveal its genetic messages, RNA transcription can proceed in either direction. To initiate this copying, an enzyme called RNA polymerase latches on to the DNA at a spot known as the promoter. The RNA polymerase then moves along the strand, building the mRNA chain as it goes.

When the RNA polymerase reaches a stop signal at the end of a gene, it halts transcription and adds to the mRNA a sequence of bases known as a poly-A tail, which consists of a long string of the genetic base adenine. This process, known as polyadenylation, helps to prepare the mRNA molecule to be exported from the cell's nucleus.

By sequencing the mRNA transcripts of mouse embryonic stem cells, the researchers discovered that polyadenylation also plays a major role in halting the transcription of upstream, noncoding DNA sequences. They found that these regions have a high density of signal sequences for polyadenylation, which prompts enzymes to chop up the RNA before it gets very long. Stretches of DNA that code for genes have a low density of these signal sequences.

The researchers also found another factor that influences whether transcription is allowed to continue. It has been recently shown that when a cellular factor known as U1 snRNP binds to RNA, polyadenylation is suppressed. The new MIT study found that genes have a higher concentration of binding sites for U1 snRNP than noncoding sequences, allowing gene transcription to continue uninterrupted.

A widespread phenomenon

The function of all of this upstream noncoding RNA is still a subject of much investigation.

"That transcriptional process could produce an RNA that has some function, or it could be a product of the nature of the biochemical reaction. This will be debated for a long time," Sharp says.

His lab is now exploring the relationship between this transcription process and the observation of large numbers of so-called long noncoding RNAs (lncRNAs). He plans to investigate the mechanisms that control the synthesis of such RNAs and try to determine their functions.

"Once you see some data like this, it raises many more questions to be investigated, which I'm hoping will lead us to deeper insights into how our cells carry out their normal functions and how they change in malignancy," Sharp says.

Source: Massachusetts Institute of Technology 

Contact: Sarah McDonnell

Written by: Anne Trafton, MIT News Office

Reference:

Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Albert E. Almada, Xuebing Wu, Andrea J. Kriz, Christopher B. Burge & Phillip A. Sharp

Nature (2013) doi:10.1038/nature12349

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Enhancing RNA Interference

Enhancing RNA Interference

Tuesday, 25 June 2013

Nanoparticles that deliver short strands of RNA offer a way to treat cancer and other diseases by shutting off malfunctioning genes. Although this approach has shown some promise, scientists are still not sure exactly what happens to the nanoparticles once they get inside their target cells.

A new study from MIT sheds light on the nanoparticles' fate and suggests new ways to maximize delivery of the RNA strands they are carrying, known as short interfering RNA (siRNA).

Lipid nanoparticles (carrying siRNA) are 

shown as they are transported inside cells 

using endocytic vesicles. Credit: Daria 

Alakhova and Gaurav Sahay. 

"We've been able to develop nanoparticles that can deliver payloads into cells, but we didn't really understand how they do it," says Daniel Anderson, the Samuel Goldblith Associate Professor of Chemical Engineering at MIT.

"Once you know how it works, there's potential that you can tinker with the system and make it work better."

Anderson, a member of MIT's Koch Institute for Integrative Cancer Research and MIT's Institute for Medical Engineering and Science, is the leader of a research team that set out to examine how the nanoparticles and their drug payloads are processed at a cellular and subcellular level. Their findings appear in the June 23 issue of Nature Biotechnology. Robert Langer, the David H. Koch Institute Professor at MIT, is also an author of the paper.

One RNA-delivery approach that has shown particular promise is packaging the strands with a lipid like material; similar particles are now in clinical development for liver cancer and other diseases.

Through a process called RNA interference, siRNA targets messenger RNA (mRNA), which carries genetic instructions from a cell's DNA to the rest of the cell. When siRNA binds to mRNA, the message carried by that mRNA is destroyed. Exploiting that process could allow scientists to turn off genes that allow cancer cells to grow unchecked.

Scientists already knew that siRNA-carrying nanoparticles enter cells through a process, called endocytosis, by which cells engulf large molecules. The MIT team found that once the nanoparticles enter cells they become trapped in bubbles known as endocytic vesicles. This prevents most of the siRNA from reaching its target mRNA, which is located in the cell's cytosol (the main body of the cell).

This happens even with the most effective siRNA delivery materials, suggesting that there is a lot of room to improve the delivery rate, Anderson says.

"We believe that these particles can be made more efficient. They're already very efficient, to the point where micrograms of drug per kilogram of animal can work, but these types of studies give us clues as to how to improve performance," Anderson says.

Molecular traffic jam

The researchers found that once cells absorb the lipid-RNA nanoparticles, they are broken down within about an hour and excreted from the cells.

They also identified a protein called Niemann Pick type C1 (NPC1) as one of the major factors in the nanoparticle-recycling process. Without this protein, the particles could not be excreted from the cells, giving the siRNA more time to reach its targets.

"In the absence of the NPC1, there's a traffic jam, and siRNA gets more time to escape from that traffic jam because there is a backlog," says Gaurav Sahay, an MIT postdoc and lead author of the Nature Biotechnology paper.

In studies of cells grown in the lab without NPC1, the researchers found that the level of gene silencing achieved with RNA interference was 10 to 15 times greater than that in normal cells.

Lack of NPC1 also causes a rare lysosomal storage disorder that is usually fatal in childhood. The findings suggest that patients with this disorder might benefit greatly from potential RNA interference therapy delivered by this type of nanoparticle, the researchers say. They are now planning to study the effects of knocking out the NPC1 gene on siRNA delivery in animals, with an eye toward testing possible siRNA treatments for the disorder.

The researchers are also looking for other factors involved in nanoparticle recycling that could make good targets for possibly slowing down or blocking the recycling process, which they believe could help make RNA interference drugs much more potent. Possible ways to do that could include giving a drug that interferes with nanoparticle recycling, or creating nanoparticle materials that can more effectively evade the recycling process.

Source: Massachusetts Institute of Technology 

Contact: Sarah McDonnell

Written by: Anne Trafton, MIT News Office

Reference:

Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling

Gaurav Sahay, William Querbes, Christopher Alabi, Ahmed Eltoukhy, Sovan Sarkar, Christopher Zurenko, Emmanouil Karagiannis, Kevin Love, Delai Chen, Roberto Zoncu, Yosef Buganim, Avi Schroeder, Robert Langer & Daniel G Anderson

Nature Biotechnology (2013), doi:10.1038/nbt.2614

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Researchers Demonstrate Use of Stem Cells to Analyse Causes, Treatment of Diabetes

Using patient-specific stem cells to correct deficient insulin-producing cells

Tuesday, 18 June 2013

A team from the New York Stem Cell Foundation (NYSCF) Research Institute and the Naomi Berrie Diabetes Center of Columbia University has generated patient-specific beta cells, or insulin-producing cells, that accurately reflect the features of maturity-onset diabetes of the young (MODY).

The researchers used skin cells of MODY patients to produce induced pluripotent stem (iPS) cells, from which they then made beta cells. Transplanted into a mouse, the stem cell-derived beta cells secreted insulin in a manner similar to that of the beta cells of MODY patients. Repair of the gene mutation restored insulin secretion to levels seen in cells obtained from healthy subjects. The findings were reported today in the Journal of Clinical Investigation.

Patient-specific iPSC-derived β cells.
Credit: NYSCF

Previous studies have demonstrated the ability of human embryonic stem cells and iPS cells to become beta cells that secrete insulin in response to glucose or other molecules. But the question remained as to whether stem cell-derived beta cells could accurately model genetic forms of diabetes and be used to develop and test potential therapies.

"We focused on MODY, a form of diabetes that affects approximately one in 10,000 people. While patients and other models have yielded important clinical insights into this disease, we were particularly interested in its molecular aspects — how specific genes can affect responses to glucose by the beta cell," said co-senior author Dieter Egli, PhD, Senior Research Fellow at NYSCF, who was named a NYSCF–Robertson Stem Cell Investigator in 2012.

MODY is a genetically inherited form of diabetes. The most common form of MODY, type 2, results in a loss-of-function mutation in one copy of the gene that codes for the sugar-processing enzyme glucokinase (GCK). With type 2 MODY, higher glucose levels are required for GCK to metabolize glucose, leading to chronic, mildly elevated blood sugar levels and increased risk of vascular complications.

MODY patients are frequently misdiagnosed with type 1 or 2 diabetes. Proper diagnosis can not only change the patient's course of treatment but affect family members, who were previously unaware that they, too, might have this genetic disorder.

NYSCF scientists took skin cells from two Berrie Center type 2 MODY patients and "reprogrammed" — or reverted — them to an embryonic-like state to become iPS cells. To examine the effect of the GCK genetic mutation, they also created two genetically manipulated iPS cell lines for comparison: one fully functional (two correct copies of the GCK gene) and one with complete loss of function (two faulty copies of the GCK gene). They then generated beta cell precursors from the fully functional and loss-of-function iPS cell lines and transplanted the cells for further maturation into immune-compromised mice.

"Our ability to create insulin-producing cells from skin cells, and then to manipulate the GCK gene in these cells using recently developed molecular methods, made it possible to definitively test several critical aspects of the utility of stem cells for the study of human disease," said Haiqing Hua, PhD, lead author on the paper, a postdoctoral fellow in the Division of Molecular Genetics, Department of Pediatrics and Naomi Berrie Diabetes Center at Columbia University and the New York Stem Cell Foundation Research Institute.

When given a glucose tolerance test three months later, mice with MODY beta cells had decreased sensitivity to glucose but a normal response to other molecules that stimulate insulin secretion. This is the hallmark of MODY. Mice with two faulty copies of the GCK gene secreted no additional insulin in response to glucose. When the researchers repaired the GCK mutation using molecular techniques, cells with two restored copies of GCK responded normally to the glucose stress test. Unlike other reported techniques, the researchers' approach efficiently repaired the GCK mutation without introducing any potentially harmful additional DNA.

"Generation of patient-derived beta cells with gene correction could ultimately prove to be a useful cell-replacement therapy by restoring patients' ability to regulate their own glucose. This result is truly exciting," said Susan L. Solomon, Chief Executive Officer of The New York Stem Cell Foundation.

The researchers also used an electron microscope to assess beta cells for insulin content by counting granules — packages that store insulin for release. Even though all beta cell types had a similar number of granules, complete loss of function of the GCK gene was associated with decreased beta-cell production.

"These studies provide a critical proof-of-principle that genetic characteristics of patient-specific insulin-producing cells can be recapitulated through use of stem cell techniques and advanced molecular biological manipulations. This opens up strategies for the development of new approaches to the understanding, treatment, and, ultimately, prevention of more common types of diabetes," said co-senior author Rudolph Leibel, MD, Christopher Murphy Memorial Professor of Diabetes Research, Columbia University Medical Center, and Director, Division of Molecular Genetics, and Co-Director of the Naomi Berrie Diabetes Center.

Source: New York Stem Cell Foundation 

Contact: David McKeon

Reference:

iPSC-derived β cells model diabetes due to glucokinase deficiency

Haiqing Hua, Linshan Shang, Hector Martinez, Matthew Freeby, Mary Pat Gallagher, Thomas Ludwig, Liyong Deng, Ellen Greenberg, Charles LeDuc, Wendy K. Chung, Robin Goland, Rudolph L. Leibel, Dieter Egl

Journal of Clinical Investigation, June 17, 2013, doi:10.1172/JCI67638

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Fingernails Reveal Clues to Limb Regeneration

Researchers discover biochemical pathway that links nail growth to fingertip regeneration

Wednesday, 12 June 2013

Mammals possess the remarkable ability to regenerate a lost fingertip, including the nail, nerves and even bone. In humans, an amputated fingertip can sprout back in as little as two months, a phenomenon that has remained poorly understood until now. In a paper published today in the journal Nature, researchers at NYU Langone Medical Center shed light on this rare regenerative power in mammals, using genetically engineered mice to document for the first time the biochemical chain of events that unfolds in the wake of a fingertip amputation. The findings hold promise for amputees who may one day be able to benefit from therapies that help the body regenerate lost limbs.

"Everyone knows that fingernails keep growing, but no one really knows why," says lead author Mayumi Ito, PhD, assistant professor of dermatology in the Ronald O. Perelman Department of Dermatology at NYU School of Medicine. Nor is much understood about the link between nail growth and the regenerative ability of the bone and tissue beneath the nail.

Now, Dr. Ito and team have discovered an important clue in this process: a population of self-renewing stem cells in the nail matrix, a part of the nail bed rich in nerve endings and blood vessels that stimulate nail growth. Moreover, the scientists have found that these stem cells depend upon a family of proteins known as the "Wnt signalling network" — the same proteins that play a crucial role in hair and tissue regeneration — to regenerate bone in the fingertip.

"When we blocked the Wnt-signalling pathway in mice with amputated fingertips, the nail and bone did not grow back as they normally would," says Dr. Ito. Even more intriguing, the researchers found that they could manipulate the Wnt pathway to stimulate regeneration in bone and tissue just beyond the fingertip.

"Amputations of this magnitude ordinarily do not grow back," says Dr. Ito.

These findings suggest that Wnt signalling is essential for fingertip regeneration, and point the way to therapies that could help people regenerate lost limbs. An estimated 1.7 million people in the U.S. live with amputations.

The team's next step is to zoom in on the molecular mechanisms that control how the Wnt signalling pathway interacts with the nail stem cells to influence bone and nail growth.

Source: New York University School of Medicine 

Contact: Christopher Rucas

Reference:

Wnt activation in nail epithelium couples nail growth to digit regeneration

Makoto Takeo, Wei Chin Chou, Qi Sun, Wendy Lee, Piul Rabbani, Cynthia Loomis, M. Mark Taketo & Mayumi Ito

Nature 12 June 2013 (2013), doi:10.1038/nature12214

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Rewinding Development

A step forward for stem cell research

Thursday, 06 June 2013

Scientists at the Danish Stem Cell Center, DanStem, at the University of Copenhagen have discovered that they can make embryonic stem cells regress to a stage of development where they are able to make placenta cells as well as the other foetal cells. This significant discovery, published in the journal Cell Reports today, has the potential to shed new light on placenta related disorders that can lead to problematic pregnancies and miscarriages.

The picture shows a 9.5-day-old mouse
embryo including extra-embryonic tissue.
The red region marks embryonic stem cells
in the extra-embryonic yolk sac. Embryonic
stem cells are not normally able to do this,
but when cells are pushed backwards in
development as described in Morgani et al..
Credit: Sophie Morgani, University of
Copenhagen. 

Embryonic stem cells can make all kinds of adult cells in the human body such as muscle, blood or brain cells. However, these embryonic stem cells are created at a point when the embryo has already lost the ability to make extra-embryonic tissue such as placenta and yolk sac. Extra-embryonic tissues are formed at the very earliest stage of development right after fertilization and are essential for the growth of the embryo and its implantation in the womb.

A team of scientists at the Danish Stem Cell Center, DanStem, at the University of Copenhagen have shown that it is possible to rewind the developmental state of embryonic stem cells. By maintaining mouse embryonic stem cells under certain conditions, they found that cells appear to regress and resemble extremely early embryo cells that can form any kind of cell including placenta and yolk sac cells.

"It was a very exciting moment when we tested the theory," says Professor Josh Brickman from DanStem.

"We found that not only can we make adult cells but also placenta, in fact we got precursors of placenta, yolk sac as well as embryo from just one cell."

"This new discovery is crucial for the basic understanding of the nature of embryonic stem cells and could provide a way to model the development of the organism as a whole, rather than just the embryonic portion. In this way we may gain greater insight into conditions where extra-embryonic development is impaired, as in the case of miscarriages," added Sophie Morgani, PhD student at DanStem and first author of the paper.

LIF protein plays a crucial role

Brickman and colleagues grew their embryonic stem cells in a solution containing LIF, which is a protein known to somehow support embryonic stem cells but also for its role in implantation of the embryo into the uterus. As implantation is stimulated by the cells that will become the placenta, not the embryo, these roles appeared to be contradictory. The DanStem study resolved this contradiction by revealing that LIF helps maintain the cells in their regressed, early stage of development.

"In our study we have been able to see the full picture unifying LIF's functions: What LIF really does, is to support the very early embryo state, where the cells can make both embryonic cells and placenta. This fits with LIFs' role in supporting implantation," Josh Brickman says.

Source: University of Copenhagen 

Contact: Joshua Brickman

Reference:

Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

Sophie M. Morgani, Maurice A. Canham, Jennifer Nichols, Alexei A. Sharov, Rosa Portero Migueles, Minoru S.H. Ko, and Joshua M. Brickman
Cell Reports, 06 June 2013, 10.1016/j.celrep.2013.04.034

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A New Gene Essential for Nuclear Reprogramming

The article describes the role of TRF1, a protector of telomeres in cell pluripotency

Wednesday, 05 June 2013

Researchers are still fascinated by the idea of the possibility of reprogramming the cells of any tissue, turning them into cells with the capacity to differentiate into cells of a completely different type — pluripotent cells — and they are still striving to understand how it happens.

A group from the Spanish National Cancer Research Centre (CNIO), headed by researcher Ralph P. Schneider, from the Telomeres and Telomerase Group led by María A. Blasco, publishes this week an article in Nature Communications on the discovery of a new gene called TRF1 that is essential for nuclear reprogramming.

It is also known that TRF1 is indispensable for protecting telomeres, the ends of chromosomes. Existing evidence suggests that the length of telomeres and pluripotency — the capacity of a cell to differentiate into multiple cell types — are related. Pluripotent cells, for example, have very long telomeres — a previous finding at CNIO — but until now no protective protein for the telomeres had been found that was essential for pluripotency.

To investigate the connection between telomeres and pluripotency, researchers generated a 'reporter' mouse: they linked together the TRF1 gene and the gene coding for a green fluorescent protein and created a lineage of mice carrying this new genetic baggage. In these animals, the green fluorescent protein acts as a label to show expression of TRF1.

They discovered that TRF1 is an excellent marker for stem cells, both in adult stem cells — those that are found in tissues and the different organs of the body — and embryonic stem cells. It is also the case with 'induced pluripotent' stem cells (iPS cells), which are pluripotent cells that come from artificially reprogrammed specialised cells.

In the case of tissues, the authors write:

"TRF1 distinguishes adult stem cells and is indispensable for their functioning".

The discovery is useful for both identifying and eventually isolating the stem cell population in tissues, something that is important for the development of regenerative medicine. The cells in which TRF1 is expressed are also the most pluripotent.

In iPS cells, the same thing happens. The authors explain that:

"The expression of TRF1 is an indicator of pluripotency. Those iPS cells that express the highest levels of TRF1 are also the most pluripotent. Furthermore, we demonstrate that TRF1 is necessary for the induction and maintenance of pluripotency, inhibiting the triggering of DNA damage responses and apoptosis ('cell suicide')".

Source: Centro Nacional de Investigaciones Oncologicas (CNIO) 

Reference:

TRF1 is a stem cell marker and is essential for the generation of induced pluripotent stem cells

Ralph P. Schneider, Ianire Garrobo, Miguel Foronda, Jose A. Palacios, Rosa M. Marión, Ignacio Flores, Sagrario Ortega, Maria A. Blasco

Nature Communications, doi:10.1038/ncomms2946

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Enhancer RNAs Alter Gene Expression

New class of RNA molecules may be key emerging 'enhancer therapy'

Tuesday, 04 June 2013

In a pair of distinct but complementary papers, researchers at the University of California, San Diego School of Medicine and colleagues illuminate the functional importance of a relatively new class of RNA molecules. The work, published online this week in the journal Nature, suggests modulation of "enhancer-directed RNAs" or "eRNAs" could provide a new way to alter gene expression in living cells, perhaps affecting the development or pathology of many diseases.

Enhancers are sequences in the genome that act to boost or "enhance" the activity or expression of nearby genes. They "often behave in a cell-specific manner and play an important role in establishing a cell's identity and functional potential," said Christopher Glass, MD, PhD, a professor in the department of Medicine and Cellular and Molecular Medicine at UC San Diego and principal investigator of one of the papers.

Although enhancers have been recognized for more than 25 years, scientists have laboured to fully flesh out the breadth and complexity of what enhancers do and how they do it. In 2010, it was discovered that enhancers directed expression of RNA on a broad scale in neurons and macrophages, a type of immune system cell. Dubbed eRNAs, they were different from other classes of nuclear non-coding RNAs, and raised new questions about their potential roles in the functions of enhancers. The two Nature papers attempt to answer some of these questions.

In the first, principal investigator Glass and colleagues investigated a pair of related transcriptional repressors called Rev-Erb-alpha and Rev-Erb-beta (proteins with important roles in regulating the circadian rhythm in many cell types) in mouse macrophages. Using genome-wide approaches, they found that the Rev-Erb proteins repressed gene expression in macrophages primarily by binding to enhancers. Collaboration with researchers at the Salk Institute for Biological Studies revealed that the repressive function of Rev-Erbs was highly correlated with their ability to repress the production of eRNAs.

In the second paper, principal investigator Michael G. Rosenfeld, MD, a professor in the UC San Diego Department of Medicine and Howard Hughes Medical Institute investigator, and colleagues looked at oestrogen receptor binding in human breast cancer cells – and its impact on enhancer transcription. In contrast to the repressive functions of Rev-Erbs, oestrogen receptors (ERs) activate gene expression; but, like Rev-Erbs, they primarily function by also binding to enhancers. ER binding was shown to be associated with increases in enhancer-directed eRNAs in the vicinity of oestrogen-induced genes, and to exert roles on activation of coding target genes.

Both papers offer new evidence that eRNAs significantly contribute to enhancer activity, and therefore to expression of nearby genes.

"Because many broadly expressed genes that play key roles in essential cellular functions are under the control of cell-specific enhancers, the ability to affect enhancer function by knocking down eRNAs could potentially provide a new strategy for altering gene expression in vivo in a cell-specific manner," said Glass, noting that in his research, anti-sense oligonucleotides were developed in conjunction with Isis Pharmaceuticals, which suppressed enhancer activity and reduced expression in nearby genes.

Source: University of California - San Diego

Contact: Scott LaFee

References:

Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription

Michael T. Y. Lam, Han Cho, Hanna P. Lesch, David Gosselin, Sven Heinz, Yumiko Tanaka-Oishi, Christopher Benner, Minna U. Kaikkonen, Aneeza S. Kim, Mika Kosaka, Cindy Y. Lee, Andy Watt, Tamar R. Grossman, Michael G. Rosenfeld, Ronald M. Evans &Christopher K. Glass

Nature (2013), doi:10.1038/nature12209

Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation

Wenbo Li, Dimple Notani, Qi Ma, Bogdan Tanasa, Esperanza Nunez, Aaron Yun Chen, Daria Merkurjev, Jie Zhang, Kenneth Ohgi, Xiaoyuan Song, Soohwan Oh, Hong-Sook Kim, Christopher K. Glass & Michael G. Rosenfeld

Nature (2013), doi:10.1038/nature12210

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A Step Closer to Artificial Liver

A Step Closer to Artificial Liver

Monday, 03 June 2013

Prometheus, the mythological figure who stole fire from the gods, was punished for this theft by being bound to a rock. Each day, an eagle swept down and fed on his liver, which then grew back to be eaten again the next day.

Modern scientists know there is a grain of truth to the tale, says MIT engineer Sangeeta Bhatia: The liver can indeed regenerate itself if part of it is removed. However, researchers trying to exploit that ability in hopes of producing artificial liver tissue for transplantation have repeatedly been stymied: Mature liver cells, known as hepatocytes, quickly lose their normal function when removed from the body.

"It's a paradox because we know liver cells are capable of growing, but somehow we can't get them to grow" outside the body, says Bhatia, the John and Dorothy Wilson Professor of Health Sciences and Technology and Electrical Engineering and Computer Science at MIT, a senior associate member of the Broad Institute and a member of MIT's Koch Institute for Integrative Cancer Research and Institute for Medical Engineering and Science.

Now, Bhatia and colleagues have taken a step toward that goal. In a paper appearing in the June 2 issue of Nature Chemical Biology, they have identified a dozen chemical compounds that can help liver cells not only maintain their normal function while grown in a lab dish, but also multiply to produce new tissue.

Cells grown this way could help researchers develop engineered tissue to treat many of the 500 million people suffering from chronic liver diseases such as hepatitis C, according to the researchers.

Lead author of the paper is Jing (Meghan) Shan, a graduate student in the Harvard-MIT Division of Health Sciences and Technology. Members of Bhatia's lab collaborated with researchers from the Broad Institute, Harvard Medical School and the University of Wisconsin.

Large-scale screen

Bhatia has previously developed a way to temporarily maintain normal liver-cell function after those cells are removed from the body, by precisely intermingling them with mouse fibroblast cells. For this study, funded by the National Institutes of Health and Howard Hughes Medical Institute, the research team adapted the system so that the liver cells could grow, in layers with the fibroblast cells, in small depressions in a lab dish. This allowed the researchers to perform large-scale, rapid studies of how 12,500 different chemicals affect liver-cell growth and function.

The liver has about 500 functions, divided into four general categories: drug detoxification, energy metabolism, protein synthesis and bile production. David Thomas, an associate researcher working with Todd Golub at the Broad Institute, measured expression levels of 83 liver enzymes representing some of the most finicky functions to maintain.

After screening thousands of liver cells from eight different tissue donors, the researchers identified 12 compounds that helped the cells maintain those functions, promoted liver cell division, or both.

MIT researchers have generated mature liver cells 

from induced pluripotent stem cells. In this image, 

the cell nuclei are stained blue. The green stain 

identifies liver cells, and the red stain identifies cells 

that are actively dividing. Image: Shan et al., Nature 

Chemical Biology, 2013.

Two of those compounds seemed to work especially well in cells from younger donors, so the researchers — including Robert Schwartz, an IMES postdoc, and Stephen Duncan, a professor of human and molecular genetics at the University of Wisconsin — also tested them in liver cells generated from induced pluripotent stem cells (iPSCs). Scientists have tried to create hepatocytes from iPSCs before, but such cells don't usually reach a fully mature state. However, when treated with those two compounds, the cells matured more completely.

Bhatia and her team wonder whether these compounds might launch a universal maturation program that could influence other types of cells as well. Other researchers are now testing them in a variety of cell types generated from iPSCs.

In future studies, the MIT team plans to embed the treated liver cells on polymer tissue scaffolds and implant them in mice, to test whether they could be used as replacement liver tissues. They are also pursuing the possibility of developing the compounds as drugs to help regenerate patients' own liver tissues, working with Trista North and Wolfram Goessling of Harvard Medical School.

Making connections

Bhatia and colleagues have also recently made progress toward solving another challenge of engineering liver tissue, which is getting the recipient's body to grow blood vessels to supply the new tissue with oxygen and nutrients. In a paper published in the Proceedings of the National Academy of Sciences in April, Bhatia and Christopher Chen, a professor at the University of Pennsylvania, showed that if preformed cords of endothelial cells are embedded into the tissue, they will rapidly grow into arrays of blood vessels after the tissue is implanted.

To achieve this, Kelly Stevens in the Bhatia lab worked with Peter Zandstra at the University of Toronto to design a new system that allows them to create 3-D engineered tissue and precisely control the placement of different cell types within the tissue. This approach, described in the journal Nature Communications in May, allows the engineered tissue to function better with the host tissue.

"Together, these papers offer a path forward to solve two of the longstanding challenges in liver tissue engineering — growing a large supply of liver cells outside the body and getting the tissues to graft to the transplant recipient," Bhatia says.

Source: Massachusetts Institute of Technology 

Contact: Anne Trafton, MIT News Office

Reference:

Identification of small molecules for human hepatocyte expansion and iPS differentiation

Jing Shan, Robert E Schwartz, Nathan T Ross, David J Logan, David Thomas, Stephen A Duncan, Trista E North, Wolfram Goessling, Anne E Carpenter & Sangeeta N Bhatia

Nature Chemical Biology, 02 June 2013, doi:10.1038/nchembio.1270

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ZenMaster

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