Dynamic Changes in Methylation Can Determine Cell Fate

Dynamic Changes in Methylation Can Determine Cell Fate Wednesday, 28 September 2011

Scientists at Cold Spring Harbor Laboratory (CSHL) and the University of Southern California (USC) have uncovered intriguing new evidence helping to explain one of the ways in which a stem cell's fate can be determined.

The new data show how the "marking" of DNA sequences by groups of methyl molecules – a process called methylation – can influence the type of cell a stem cell will become. The cellular maturation process, called differentiation, has long been thought to be affected by methylation. Subtle changes in methylation patterns within subsets of a particular cell type have now been observed and closely scrutinized, and they reveal some intriguing mechanisms at work in the process.

A team led by postdoc Dr. Emily Hodges, working in the laboratory of CSHL Professor and HHMI Investigator Gregory Hannon, studied how methylation changes in blood stem cells can affect whether a given stem cell will differentiate into either a myeloid cell or a lymphoid cell. These are the two major lineages of mature blood cells. Sophisticated mathematical analyses of the data were performed under the direction of USC Professor Andrew D. Smith.

The study, which will appear in print October 7 in the journal Molecular Cell, generated some surprising findings that challenge currently held theories about how methylation operates. First, it demonstrated that methylation patterns are more dynamic than they are often thought to be.

"It's not a question of methylation being 'on' or 'off' at a given site in the genome," explains Hodges.

"We find, instead, an interesting fluctuation of the boundaries of regions that are free of methylation marks. This fact, in turn, can have a profound impact upon cell fate."

Areas lacking methylation, called hypomethylated regions, or HMRs, tend to coincide with so-called CpG islands, sites in the genome where adjacent "Cs" and "G's" – cytosine and guanine nucleotides – are seen in strings of repeats. These unmethylated regions tend to be ones associated with nearby genes that are capable of being expressed. In contrast, sites in the genome that are methylated are typically not expressed.

The new study, which looks at these areas at high resolution in cells of the different blood cell lineages and in blood stem cells, finds that in many cases, a core portion of the unmethylated region is shared in common, but that adjacent areas, sometimes called "CpG shores" – the outlying areas around CpG islands – differ markedly in breadth. The CSHL-USC team refines the notion of islands and shores, preferring to describe the narrowing and widening of the "shoreline" as a tidal phenomenon.

"We observed that the boundaries of these unmethylated regions goes in and out, like the tides," says Hodges.

"The key question is what drives these changes. We found that the width of these regions depends on the gene that is associated with the region. We showed in blood cells that the variation is lineage-specific."

The team deduced this after making close study of the methylation patterns in genomic regions containing genes known from other research to be expressed specifically in lymphoid cells, but not in myeloid cells, or vice versa. In these cases, all blood cells share a narrow "core" region of hypomethylation; but only in one lineage did the unmethylated region widen – a widening that opens the promoter of the "underlying" gene to the cellular machinery initiating gene expression. In other words, the lack of methylation over a wider area enables the underlying gene to be activated – only in the specified cell-type, but not in any of the others.

Another striking observation made from this data is the directional preference of this expansion. For example, in the widening of the unmethylated region seen in the case of the lymphoid cell, the direction of the widening was toward the area occupied by the underlying gene, which in this case was a gene encoding a B cell surface marker called CD22.

It has generally been thought that methylation is a stable epigenetic mark and that change in methylation are unidirectional; and further, that cells become increasingly methylated as they move through the differentiation process toward their mature identity. And in fact, the only known direction of active change is from an unmethylated state to a methylated state.

The new data suggests, however, that dynamic changes in methylation status may be possible. The relevant evidence comes from blood stem cells, which were observed to have methylation patterns that the team describes as "intermediately methylated," seemingly in dynamic equilibrium of the two extreme states of "methylated" and "unmethylated."

According to Hodges, this raises the possibility that methylation might in fact be bidirectional, and that there might be an as yet undiscovered, active mechanism that performs de-methylation. No known enzyme has this ability to remove methyl groups from DNA; DNA methyltransferase is the well-known enzyme that catalyzes the addition of methyl groups.

Yet another of the team's unexpected findings concerns the position of HMRs relative to know genic regions. While unmethylated regions tend to be associated with nearby genes that are capable of being expressed, the team found, according to Hodges, "a lot of HMRs located far away from any annotated gene locus."

One notable thing about these regions, she says, "is that they were highly enriched for binding sites of specific regulatory molecules that are involved in chromatin organization."

Chromatin consists of DNA and the protein complexes called histones around which genomic DNA is packed. In a given cell, chromatin organization, like methylation, helps to determine whether specific genes can be expressed or not.

About CSHL

Founded in 1890, Cold Spring Harbor Laboratory (CSHL) has shaped contemporary biomedical research and education with programs in cancer, neuroscience, plant biology and quantitative biology. CSHL is ranked number one in the world by Thomson Reuters for impact of its research in molecular biology and genetics. The Laboratory has been home to eight Nobel Prize winners. Today, CSHL's multidisciplinary scientific community is more than 400 scientists strong and its Meetings & Courses program hosts more than 8,000 scientists from around the world each year. Tens of thousands more benefit from the research, reviews, and ideas published in journals and books distributed internationally by CSHL Press. The Laboratory's education arm also includes a graduate school and programs for undergraduates as well as middle and high school students and teachers. CSHL is a private, not-for-profit institution on the north shore of Long Island.

Source: Cold Spring Harbor Laboratory

Contact: Peter Tarr

Reference:

Directional DNA Methylation Changes and Complex Intermediate States Accompany Lineage Specificity in the Adult Hematopoietic Compartment
Emily Hodges, Antoine Molaro, Camila O. Dos Santos, Pramod Thekkat, Qiang Song, Philip J. Uren, Jin Park, Jason Butler, Shahin Rafii, W. Richard McCombie, Andrew D. Smith and Gregory J. Hannon
Molecular Cell October 7, 2011, doi:10.1016/j.molcel.2011.08.026
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Mice Stem Cells Guided into Myelinating Cells by the Trillions

Process paves way for research, possible treatments of multiple sclerosis and more
Sunday, 25 September 2011

Scientists at Case Western Reserve University School of Medicine found a way to rapidly produce pure populations of cells that grow into the protective myelin coating on nerves in mice. Their process opens a door to research and potential treatments for multiple sclerosis, cerebral palsy and other demyelinating diseases afflicting millions of people worldwide.

The findings will be published in the online issue of Nature Methods, Sunday, Sept. 25.

"The mouse cells that we utilized, which are pluripotent epiblast stem cells, can make any cell type in body," Paul Tesar, an assistant professor of genetics at Case Western Reserve and senior author of the study, explained.

"So our goal was to devise precise methods to specifically turn them into pure populations of myelinating cells, called oligodendrocyte progenitor cells, or OPCs."

Their success holds promise for basic research and beyond.

"The ability of these methods to produce functional cells that restore myelin in diseased mice provides a solid framework for the ability to produce analogous human cells for use in the clinic," said Robert H. Miller, vice dean for research at the school of medicine and an author of the paper.

Tesar worked with CWRU School of Medicine researchers Fadi J. Najm, Shreya Nayak, and Peter C. Scacheri, from the department of genetics; Anita Zaremba, Andrew V. Caprariello and Miller, from the department of neurosciences; and with Eric. C. Freundt, now at the University of Tampa.

Myelin protects nerve axons and provides insulation needed for signals to pass along nerves intact. Loss of the coating results in damage to nerves and diminished signal-carrying capacity, which can be expressed outwardly in symptoms such as loss of coordination and cognitive function.

Scientists believe that manipulating a patient's own OPCs or transplanting OPCs could be a way to restore myelin.

And, they have long known that pluripotent stem cells have the potential to differentiate into OPCs. But, efforts to push stem cells in that direction have resulted in a mix of cell types, unsuitable for studying the developmental process that produces myelin, or to be used in therapies.

Tesar and colleagues are now able to direct mouse stem cells into oligodendrocyte progenitor cells in just 10 days. The team's success relied upon guiding the cells through specific stages that match those that occur during normal embryonic development.

First, stem cells in a petri dish are treated with molecules to direct them to become the most primitive cells in the nervous system. These cells then organize into structures called neural rosettes reminiscent of the developing brain and spinal cord.

To produce OPCs, the neural rosettes are then treated with a defined set of signaling proteins previously known to be important for generation of OPCs in the developing spinal cord.

After this 10 day protocol, the researchers were able to maintain the OPCs in the lab for more than a month by growing them on a specific protein surface called laminin and adding growth factors associated with OPC development.

The OPCs were nearly homogenous and could be multiplied to obtain more than a trillion cells.

The OPCs were treated with thyroid hormone, which is key to regulating the transition of the OPCs to oligodendrocytes. The result was the OPCs stopped proliferating and turned into oligodendrocytes within four days.

Testing on nerves lacking myelin, both on the lab bench and in diseased mouse models, showed the OPCs derived from the process flourished into oligodendrocytes and restored normal myelin within days, demonstrating their potential use in therapeutic transplants.

Because they are able to produce considerable numbers of OPCs – a capability that up until now has been lacking – the researchers have created a platform for discovering modulators of oligodendrocyte differentiation and myelination. This may be useful for developing drugs to turn a patient's own cells into myelinating cells to counter disease.

Source: Case Western Reserve University

Contact: Kevin Mayhood
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Proteomics: Critical Similarity Between Embryonic and iPS Cells

Proteomics: Critical Similarity Between Embryonic and iPS Cells
Monday, 12 September 2011

Ever since human induced pluripotent stem cells were first derived in 2007, scientists have wondered whether they were functionally equivalent to embryonic stem cells, which are sourced in early-stage embryos.

Both cell types have the ability to differentiate into any cell in the body, but their origins – in embryonic and adult tissue – suggest that they are not identical.

Although both cell types have great potential in basic biological research and in cell- and tissue-replacement therapy, the newer form, called iPS cells, have two advantages. They face less ethical constraint, as they do not require embryos. And they could be more useful in cell replacement therapies: growing them from the patient's own cells would avoid immune rejection.

But until iPS cells are proven to have the same traits as embryonic stem cells, they cannot be considered to be identical.

In a study published today in Nature Methods (Sunday, Sept. 11), researchers at the University of Wisconsin-Madison report the first full measurement of the proteins made by both types of stem cells. In a study that looked at four embryonic stem cells and four iPS cells, the proteins turned out to be 99 percent similar, says Joshua Coon, an associate professor of chemistry and biomolecular chemistry who directed the project.

"We looked at RNA, at proteins, and at structures on the proteins that help regulate their activity, and saw substantial similarity between the two stem-cell types," he says.

Proteins are complex molecules made by cells for innumerable structural and chemical purposes, and the new study measured more than 6,000 individual proteins using highly accurate mass spectrometry, a technique that measures mass as the first step of identifying proteins.

The study is the first comprehensive comparison of proteins in the two stem cell types, says Doug Phanstiel, who is now at Stanford University, and worked with Justin Brumbaugh on the project as graduate students at UW-Madison.

"From a biological standpoint, what is novel is that this is the first proteomic comparison of embryonic stem cells and iPS cells," says Phanstiel, referring to the study of which proteins a cell produces.

In essence, every cell in the body has the genes to make any protein the body might need, but cells make only the proteins that further their own biological role. Cells regulate the formation and activity of proteins in three ways: first, by controlling the production of RNA, a molecule that transfers the DNA code to protein-making structures; second, by controlling the quantity of each protein made; and third, by adding structures to the protein that regulate when it will be active.

The new study measured each of these activities, Phanstiel says.

"And because we compared four lines of each type of stem cell, and the comparisons were run three times, the statistics are extremely robust," he adds.

The new report, Coon says, suggests that embryonic stem cells and iPS cells are quite similar. According to some measurements, the protein production of an embryonic stem cell was closer to that of an iPS cell than to a second embryonic stem cell.

The ability to measure proteins in such detail emerged from improved ways to measure mass, Coon says.

"New technical developments in both our ability to measure a protein's mass – accurate to the third or fourth decimal place – and to compare the proteins from up to eight different cell lines at a time -- permitted this important comparison for the first time," says Coon.

The study is not the last word in determining the similarity of the two types of pluripotent stem cells, says Coon, who worked with UW-Madison stem-cell pioneer James Thomson, on the project.

Because clinical uses of either type of stem cells will require that they be transformed into more specialized cells, researchers still need to know more about protein production after a stem cell is differentiated into, for example, a neuron or heart muscle cell.

This technology, Coon says, "is now well-positioned to study how closely molecules contained in these promising cells change after they are differentiated into the cells that do the work in our bodies – a critical next step in regenerative medicine."

Source: University of Wisconsin-Madison

Contact: Joshua Coon

Reference:

Proteomic and phosphoproteomic comparison of human ES and iPS cells
Douglas H Phanstiel, Justin Brumbaugh, Craig D Wenger, Shulan Tian, Mitchell D Probasco, Derek J Bailey, Danielle L Swaney, Mark A Tervo, Jennifer M Bolin, Victor Ruotti, Ron Stewart, James A Thomson & Joshua J Coon
Nature Methods 11 September 2011, doi:10.1038/nmeth.1699
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Researchers Overcome Major Obstacle for Stem Cell Therapies and Research

Researchers Overcome Major Obstacle for Stem Cell Therapies and Research
Thursday, 08 September 2011

Jeanne Loring, Ph.D., is a professor of

Developmental Neurobiology, The Scripps

Research Institute. Credit: Photo courtesy

of The Scripps Research Institute.

Stem cells show great potential to enable treatments for conditions such as spinal injuries or Lou Gehrig's disease, and also as research tools. One of the greatest problems slowing such work is that researchers have found major complications in purifying cell mixtures, for instance to remove stem cells that can cause tumors from cells developed for use in medical treatments. But a group of Scripps Research scientists, working with colleagues in Japan, have developed a clever solution to this purification problem that should prove more reliable than other methods, safer, and perhaps 100 times cheaper.

The work appears in the current edition of the journal Cell Research.

Effective tricks for separating stem cells from other types are essential for many emerging medical treatments. These techniques begin with researchers inducing stem cells to take specific forms, or differentiate, for instance into nerve cells. These differentiated cells might then be used to repair a spinal cord injury. Other cells might enable a diabetic's body to produce adequate insulin.

A key problem is that in the differentiation process, at least some stem cells inevitably remain in their undifferentiated, or pluripotent, state. These cells can grow to form tumors in patients if injected along with differentiated cells, a concern that has already led the US Food and Drug Administration (FDA) to delay clinical trials for promising stem cell-based therapies.

A New Approach

To date, almost all attempts at purification have focused on developing antibodies — immune system attack cells — that can remove or destroy stem cells in mixtures. But this approach has had shortcomings. Effective antibodies are difficult and expensive to develop, and their use in medical therapies raises safety issues because they are produced in animals.

The Scripps Research team, led by Professor of Developmental Neurobiology Jeanne Loring, was looking for a new route to solve the purification and safety problems. The group recently began experimenting with chip-based tools known as lectin arrays. At various points on these devices, plant-produced proteins called lectins are attached. These lectins bind with specific sugars including some found on the surface of cells.

Working in the lab with cellular components, rather than whole cells, the Loring team first found that specific combinations of sugars and proteins known as glycoproteins on stem cells reliably bind to certain lectins. They were then able to exploit this connection to purify cell mixtures.

"When we discovered there was a specific binding pattern, we decided we should just go for it and see whether we could use the lectins to purify cells," said Yu-Chieh Wang, the first author of the research article.

"We tested the idea and it works very well, and lectins are readily available and inexpensive."

After identifying the lectin that bound best with stem cells, the group took the work to the next level to show that they could actually separate out stem cells. To accomplish this, they first attached the lectin to tiny beads. Then they exposed these beads to mixtures of stem cells along with non-stem cells.

The researchers used a range of different types of both embryonic stem cells and induced pluripotent cells, which are embryonic stem cell-like cells that are produced by inserting certain genes into skin cells. They included cell lines from both Scripps Research and the labs of their collaborators in Japan and the United States.

In every case, the team found that the stem cells bound remarkably well to the beads, while the cells that washed past were almost all non-stem cells; this meant that both cell types could be collected separately for use in research or in treatments.

Purity's Potential

Possible uses for the new technique are essentially as numerous as those for stem cells themselves. Lectin purification could be used with any of a huge range of therapies currently in development. In addition to low cost and reliability, the lectins used are plant products, so they do not introduce the type of safety concerns that could arise from using antibodies that are produced by animal cells.

Even in more basic research, effective studies using stem or differentiated cells generally requires purification so that effects can be identified and tracked without introducing complications from impurities in a group of cells.

Loring's group, for instance, is studying the production of nerve cells that might be used to treat a specific type of autism caused by a known genetic mutation. Producing the nerve cells needed is a laborious process that will be more efficient with better purification.

The Loring team is also working to identify different binding patterns that would allow them to similarly purify mixtures of specific types of non-stem cells.

"In theory, this should allow us to pull any cell type out of any mixture," she said of the basic lectin technique.

At the more basic research level, because all the different stem cell lines from both humans and animals seem to produce similar glycoproteins binding to the lectins, it is possible these glycoproteins infer some basic qualities fundamental to the pluripotent state. Loring and her colleagues are exploring this possibility in hopes of better understanding stem cells' still mysterious abilities to transform into any type of cell.

"We may have uncovered something really fundamental about pluripotency," said Loring.

Source: Scripps Research Institute

Contact: Mika Ono

Reference:

Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis

Yu-Chieh Wang, Masato Nakagawa, Ibon Garitaonandia, Ileana Slavin, Gulsah Altun, Robert M Lacharite, Kristopher L Nazor, Ha T Tran, Candace L Lynch, Trevor R Leonardo, Ying Liu, Suzanne E Peterson, Louise C Laurent, Shinya Yamanaka and Jeanne F Loring

Cell Research, September 6, 2011; doi:10.1038/cr.2011.148

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Generation of Haploid Mouse Stem Cells

Scientists create mammalian cells with single chromosome set
Thursday, 08 September 2011

Researchers have created mammalian cells containing a single set of chromosomes for the first time in research funded by the Wellcome Trust and EMBO. The technique should allow scientists to better establish the relationships between genes and their function.

Mammal cells usually contain two sets of chromosomes – one set inherited from the mother, one from the father. The genetic information contained in these chromosome sets helps determine how our bodies develop. Changes in this genetic code can lead to or increase the risk of developing disease.

Scientists at the University of Cambridge bred

mice with fluorescent green cells derived from

haploid (single chromosome set) embryonic

stem cells. Credit: Anton Wutz and Martin Leeb,

University of Cambridge/Nature.

To understand how our genes function, scientists manipulate the genes in animal models – such as the fruit fly, zebra fish and mice – and observe the effects of these changes. However, as each cell contains two copies of each chromosome, determining the link between a genetic change and its physical effect – or 'phenotype' – is immensely complex.

Now, in research published today in the journal Nature, Drs. Anton Wutz and Martin Leeb from the Wellcome Trust Centre for Stem Cell Research at the University of Cambridge report a technique which enables them to create stem cells containing just a single set of chromosomes from an unfertilized mouse egg cell. The stem cells can be used to identify mutations in genes that affect the cells' behavior in culture. In an additional step, the cells can potentially be implanted into the mouse for studying the change in organs and tissues.

The technique has previously been used in zebra fish, but this is the first time it has been successfully used to generate such mammalian stem cells.

Dr. Wutz, a Wellcome Trust Senior Research Fellowship, explains:

"These embryonic stem cells are much simpler than normal embryonic mammalian stem cells. Any genetic change we introduce to the single set of chromosomes will have an easy-to-determine effect. This will be useful for exploring in a systematic way the signalling mechanisms within cell and how networks of genes regulate development."

The researchers hope that this technique will help advance mammalian genetics and our understanding of the gene-function relationship in the same way that a similar technique has helped geneticists understand the simpler zebra fish animal model.

Understanding how our genetic make-up functions and how this knowledge can be applied to improve our health is one of the key strategic challenges set out by the Wellcome Trust. Commenting on this new study, Dr. Michael Dunn, Head of Molecular and Physiological Sciences at the Wellcome Trust, says:

"This technique will help scientists overcome some of the significant barriers that have so far made studying the functions of genes so difficult. This is often the first step towards understanding why mutations lead to disease and, ultimately, to developing new drugs treatments."

Source: Wellcome Trust

Contact: Craig Brierley

Reference:

Derivation of haploid embryonic stem cells from mouse embryos
Martin Leeb & Anton Wutz
Nature (2011), doi:10.1038/nature10448
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Key Protein Reveals Secret of Stem Cell Pluripotency

Key Protein Reveals Secret of Stem Cell Pluripotency
Monday, 05 September 2011

A protein that helps maintain mouse stem cell pluripotency has been identified by researchers at the RIKEN Omics Science Center. The finding, published in the August issue of Stem Cells (first published online July 26, 2011), points the way to advances in regenerative medicine and more effective culturing techniques for human pluripotent stem cells.

Diagram of the Ccl2 and leukemia inhibitory
factor (LIF) signal pathways integrating into
the transcription network. Known LIF signal
pathway is shown with black arrow. Our finding
of Ccl2 signal pathway for promoting
pluripotency is shown as dot black arrow.
Abbreviations: IL-6R, interleukin-6 receptor;
LIF, leukemia inhibitory factor; LIFR,
leukemia inhibitory factor receptor; NC,
negative control; PI(3)K, phosphoinositide
3-kinase.

Through their capacity to differentiate into any other type of cell, embryonic stem cells (ES cells) and induced-pluripotent stem cells (iPS cells) promise a new era of cell-based treatments for a wide range of conditions and diseases. Cultivating such cells, however, commonly relies on the use of so-called "feeder" cells to maintain pluripotency in cell culture conditions. Feeder cells keep stem cells in their undifferentiated state by releasing nutrients into the culture medium, but they have the potential to introduce contamination which, in humans, can lead to serious health risks.

Previous research has shown that mouse pluripotent stem cells can be cultured without feeder cells through the addition of a cytokine called Leukemia Inhibitory Factor (LIF) to the culture media ("feeder-free" culture). LIF is secreted by mouse feeder cells and activates signal pathways reinforcing a stem cell regulatory network. The researchers discovered early in their investigation, however, that the amount of LIF secreted from feeder cells is much less than the amount needed to maintain pluripotency in feeder-free conditions. This points to other, as-of-yet unknown contributing factors.

To clarify these factors, the research group analyzed differences in gene expression between mouse iPS cells cultured on feeder cells and those cultured in feeder-free (LIF treated) conditions. Their results revealed 17 genes whose expression level is higher in feeder conditions. To test for possible effects on pluripotency, they then selected 7 chemokines (small proteins secreted by cells) from among these candidates and overexpressed them in iPS cells grown in feeder-free conditions. They found that one chemokine in particular, CC chemokine ligand 2 (CCL2), enhances the expression of key pluripotent genes via activation of a well-known signal pathway known as Jak/Stat3.

While CCL2 is known for its role in recruiting certain cells to sites of infection or inflammation, the current research is the first to demonstrate that it also helps maintain iPS cell pluripotency. The findings also offer broader insights applicable to the cultivation of human iPS/ES cells, setting the groundwork for advances in regenerative medicine.

Source: RIKEN
Contact: Harukazu Suzuki

Reference:
CC Chemokine Ligand 2 and Leukemia Inhibitory Factor Cooperatively Promote Pluripotency in Mouse Induced Pluripotent Cells
Yuki Hasegawa, Naoko Takahashi, Alistair R. R. Forrest, Jay W. Shin, Yohei Kinoshita, Harukazu Suzuki and Yoshihide Hayashizaki.
Stem Cells, 2011, DOI: 10.1002/stem.673
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