The researchers are working on the relationship between body cell condition and aging. Their experiments have shown that the presence of stem cells or progenitor (undifferentiated) cells can have a beneficial effect on cells afflicted with either progeria or simple old age. Merely injecting the stem cells had an impact on cells in the brain and muscles. In experiments conducted with cells in a culture dish, the proximity of stem cells – close but not touching – had a beneficial effect on unhealthy cells.

Rather obviously this research begs a question: What do the stem cells do to the aging cells? This type of research is pretty much a ‘black box’ experiment. The cells are injected and the results observed, but the chemistry or molecular-level pathways are not known. Which is why further research is required. However, it should be noted that a lot of medicine is used in which the results are accepted without knowing the underlying mechanism. These days, however, as equipment and procedures for work at the molecular level improve, it should be possible to take this kind of top-level research and successfully look for low-level linkages to the aging process.

The cover page for downloading is at: NHS choices, Hope and hype: stem cells in the media.

The pituitary gland is tiny, about the size of a pea, but it has an extremely important set of roles in the body’s hormonal chemistry (which applies to almost all mammals including mice and men). As the key organ to the endocrine system, the pituitary glands secrete nine major hormones regulating growth, fertility, blood pressure, breast milk, temperature control and fluid management – among other things. When the pituitary gland is malfunctioning, a lot of bad things happen. The ability to repair and eventually replace pituitary glands with synthetic tissue is obviously a major achievement. But science is not there yet.

The big news from the Japanese researchers is that they have been able to culture the mice embryo stem cells into pituitary gland cells, which is no easy feat. It required that the cells be grown together with cells of the hypothalamus, a companion gland of the pituitary. These two glands have many symbiotic connections and it became obvious that functional pituitary cells could not be reproduced without the interaction of the hypothalamus. The researchers also pioneered new techniques for implanting the synthetic pituitary cells into living mice. This too was tricky and represents a future hurdle for applying the technique to human beings.

As is usually the case with breakthroughs accomplished with lab animals, mice in this case, there is always the caveat that a similar procedure for human beings may or may not work. Typically the biochemistry is compatible, but the scale change and complexity of the human brain sometimes make the transition from mice to men very difficult. Dr. Sasai at RIKEN believes that it will take about three years to produce human pituitary cells, but the technique for implanting them successfully might take much longer.

So far stem cells have been turned into synthetic liver, heart, muscle, eye, and other organs. The list grows. In some cases this has been done with embryonic stem cells, which has a controversial side, especially when it comes to humans. Most researchers try to do the same thing with pluripotent stem cells derived (through various techniques) from adult or differentiated stem cells, which gets around the controversy. The researchers at RIKEN would like to follow this path in the future.

When the Broad team discovered more than 3,500 unique lincRNAs in the human and mouse genomes in 2009, “the potential was enormous, and we wanted to know what they could be doing.”

[Source: Technology Review]

Here’s the scenario: A team of researchers at the Broad Institute (a joint operation of Harvard University and Massachusetts Institute of Technology, USA) discovered in 2009 that human and mouse genomes were encoded to produce thousands of a hitherto unknown form of RNA. The role of RNA, as commonly understood, is to carry the genetic code for protein production from the DNA to the locations of protein manufacture. Over the years, however, new forms of RNA were discovered – microRNA, mRNA, siRNA, RNAi, among others – and the range of function for RNA extended. In fact, the team at Broad Institute had discovered a form of RNA that didn’t appear to have any role in the coding and manufacturing of proteins. Called a lincRNA for ‘large intergenic non-coding RNA’, this form of RNA was found in all cells in great numbers. The team eventually identified over 3500 unique forms of lincRNAs. The question staring them in the face and making their adrenalin pump (so to speak) was, of course, what do all these lincRNAs do?

It turns out that at least one of the things lincRNAs do is coordinate and organize the assembly of proteins in embryonic stem cells – and probably all other cells as well. This is something of a revelation. It has always been thought that proteins themselves direct the development of cells, now it appears that lincRNAs provides the structure – a kind of chemical scaffold – on which cell protein is assembled. In stem cells, it is the role of lincRNAs to provide the all-important control of whether a stem cell remains pluripotent (able to turn into almost any other kind of cell) or differentiates into a specific type of adult cell. This role is so important to the development of life that discovery of a previously unknown agent of such influence as lincRNA is almost shocking. Except that for biochemists this is the kind of thing that makes a career, or even a Nobel.

The researchers at Broad Institute, with first author Mitchell Guttman and senior staffers David Root and Eric Lander, decided in 2009 to concentrate on the activity of lincRNA in embryonic stem cells, which are heavily studied and of such obvious biological importance. In order to determine the role of lincRNA, they used genetic techniques to turn off and on the production of specific lincRNAs. Eventually they isolated about 100 forms of lincRNA that appeared to be at work in stem cells. From there they used biochemical analysis to follow the effect of lincRNA on cell protein.

The results of their work, published in the journal Nature [28 August 2011, paywalled, lincRNAs act in the circuitry controlling pluripotency and differentiation] is the first comprehensive view of lincRNA at work in a specific cell type. As one of the researchers put it, “lincRNAs are like team captains, bringing together the right [protein] players to get a job done.”

Sports analogies aside, the discovery of a whole new class of RNA – one with such a powerful role in the development of cells – opens the way to explore yet another massive complication in the processes of life. This might be overstating the case, but probably not. In any case, this is the kind of challenge that scientists live for, which is definitely not an overstatement. The opportunity to experiment and provide answers to big questions (even if the subject matter is very small) is rare enough.

Sixth in a series of posts inspired by ten topics in ‘Insights of the Decade’ from the December 17, 2010 special issue of Science Magazine The topics are: Inflammation, climatology, tricks of light, alien planets, the microbiome, cell reprogramming, Martian water, the DNA time machine, cosmology and epigenetics. The original articles are now behind a paywall; they won’t be reproduced here, but their gist is present. I’ll try to put them in context and also within the Impact Areas of SciTechStory.

“Researchers are also working to understand exactly how reprogramming works.” That statement from the Science Magazine article could tell you a lot about the state-of-the-art. One way of putting it: Mostly art, not much state. When a geneticist or cell biologist ‘reprograms’ a cell, what they mean is that they change the genetic instructions, in the DNA mostly, so that the cell changes into a different kind of cell. These days, a lot of the reprogramming has one goal – creation of stem cells. Especially taking an adult cell, let’s say a skin cell, and reprogramming it so that it is a cell that can become almost any other kind of cell (a pluripotent stem cell).

Stem cells have been in and out of the mainstream news, mostly because of the controversy over their origin, which used to be exclusively from the tissue of human embryos. In particular, the culture of the United States has a remarkably large portion of the population engaged in preserving the ‘right to life’ even for non-living embryos that in the usual course of things were destined for disposal but a few of their cells were salvaged for embryonic stem cell research. For religious and ethical reasons, the use of embryos for anything remains controversial and more or less illegal in several countries.

Stem cells have enormous medical promise. Because they are unformed and undeveloped, they can be and have been used to help repair many kinds of tissue (heart, muscle, liver, skin). The number of applications is growing exponentially. Unfortunately, the controversy and consequent difficulties in working with stem cells has made research and especially clinical trials difficult. When presented with this problem, scientists immediately started to wonder about ways of circumventing it. The answer, it seems, is reprogramming.

The idea, which is much easier to express than do, is to take an ‘adult’ cell (a cell already fully committed to being, say, a skin cell) and by tweaking its DNA, turn it back into a stem cell. Specifically such a cell is called an induced Pluripotent Stem Cell or iPSC. The foundation for this work was laid by John Gurdon in the 1960s and was part of the research on cloning. Cloning is almost as controversial as stem cells, but most of the work has been to develop genetic techniques, not produce clones per se. The techniques mostly involved inserting various elements of DNA either into a ‘blank’ (enucleated) cell or into the existing DNA of a cell.

In the mid-oughties Shinya Yamanaka and his team demonstrated that by adding just four genes to an adult mouse cell, they could produce a viable iPSC. Further refinements followed quickly. These days researchers are finding multiple ways to reprogram cells of various types not only into pluripotent stem cells but, skipping the development process, into other kinds of adult cells. Put another way, this could mean doing the work of stem cells without using stem cells – the post stem cell era.

What’s important is that reprogramming cells isn’t just a way around the ethical controversy over embryonic stem cells; it is a different way of approaching genetics, cell biology, and medicine. It straddles the boundaries of permanent gene modification and therapeutic manipulation. Most of the reprogramming does not affect reproduction (cell meiosis). Scientists have mostly shied away from that. Some of the reprogramming doesn’t even carry over when the cell reproduces (mitosis). Nevertheless, this kind of genetic manipulation exposes a realm of experimentation into the why’s and wherefore’s of DNA and genetic reproduction. Most scientists envision this as a gateway to understanding the origin of many diseases (cancer always being the example).

Now let’s return to the opening point: Scientists don’t fully know how reprogramming works. This is another of those very important pieces of science and technology where we ‘just do it.’ What pops into my mind is how we ‘just did it’ with nuclear fission, but that’s an unfair comparison, I hope. In any case, there are some warning flags going up. Studies have demonstrated that some of the reprogramming approaches increase the risk of cancerous cell growth. Other studies point to problems with the quality (meaning potential for side effects) of the induced stem cells. Even the difficulty in producing reprogrammed cells (typically only very small percentage successfully reprogram) may indicate that there are fundamental issues that are not yet understood.

There is the suspicion that while reprogramming can produce obvious and useful changes in a cell, the underlying situation is more complicated. Research is discovering that while genes provide the ‘blue print’ for cell construction, there is an equally important process of transforming genetic instruction into the proteins and other materials of the cell. This includes new fields of study such as DNA configuration (the presentation of DNA elements for development), proteomics (the study of proteins), and especially epigenetics (which considers, among other things, how genetic instructions are adapted to fit the environment of the cell). This research work suggests that while genes can be tweaked and some results are obtained, the results may not be stable, predictable, or durable.

In short, what we don’t yet know about cell biology may well turn out to place limits on the effectiveness of cell reprogramming. This would not be the first time that a ‘highly promising’ development in biology and medicine turns out to be useful but not revolutionary (or whatever hyper-optimistic word). Actually this is mostly how advances in science proceed; the curve of experience over the years tends to flatten the perceived dramatic effect of a new technique or procedure.

Stem cells and cell reprogramming will continue to have a major impact on research. In fact, just getting beyond the need for embryonic stem cells would be a big improvement. We’ll track that work at SciTechStory in the areas of DNA Decoding, Genetic Modification, Stem Cells, Proteomics and Cell Biology. Occasionally there will also be clinically proven and reliable applications. However, this appears to be one of those areas of science where the betting on widely effective applications shouldn’t fall to the side of the over optimistic.

Previous leading-edge work by Shinya Yamanaka (Kyoto University, Japan) in 2008 made the dramatic leap of inducing pluripotent stem cells from skin fibroblast cells (the structural cells that hold the skin together). This was the first time that pluripotent stem cells (stem cells that can, in turn, become almost any kind of adult cell) were manufactured without using embryonic stem cells. The approach avoided the ethical and religious issues involved with anything embryonic. Significantly for the field of stem cell research, Dr. Yamanaka’s work also exposed the role of a particular protein, called OCT4, which is encoded by the POU5F1 gene and is an important factor in reprogramming the adult cells (now called Yamanaka factors).

Dr. Bhatia and his team started by collecting skin fibroblast cells from several volunteers. The fibroblast cells were injected with a virus that ‘inserted’ (really a form of controlled infection) the gene POU5F1 into the DNA of each cell. This causes the cells to produce the OCT4 protein. The protein provides the chemical pathway to controlling the growth and nature of the cell. The control was developed by growing the cells in a soup of appropriate chemicals, most of them cytokines, which are signaling proteins that direct cell activity for growth and immunity.

During the process, multipotent cells (or progenitor cells) are produced. These are cells that are restricted in what type of cell they can become (in this case, blood cells) but they can become several kinds of those cells. By tweaking the cytokine mixture, the research team was able to produce the three major classes of blood cells: White, red, and platelet.

To spell out the significance of directly converting one adult cell type to another adult cell type:

1. Bypassing the stage of pluripotent stem cell production obviously simplifies the procedure.
2. The cells produced are completely ‘adult’ cells, needing no further reprogramming.
3. Removing the pluripotent stage reduces the risk of introducing tumor causing mutations.
4. With no stem cells involved, there is no moral hazard.
5. A person’s own skin cells can be used to produce a variety of blood cells, removing the danger of rejection.
6. It demonstrates the potential for converting many types of cells.

With so many plusses – and so much potential – this is the kind of research that has earned the laudative ‘breakthrough.’

Of course, the story isn’t finished. So far the converted cells have only been tested in mice. It remains to be seen if the blood cells can be used in humans. There have been problems with the viability (especially long term) for some of the uses of stem cells. At this point, it is unknown if the reprogrammed cells have any peculiarities. A real concern is whether the artificially produced cells will react normally in the epigenetic environment of living human cells. That is the utterly complex and as yet poorly understood interaction between DNA and the proteins that guide its expression to adapt to existing conditions.

As Dr. Bhatia and his team are well aware, there are many steps ahead before even contemplating trials of blood in human beings. For one thing, they must develop a method for producing converted cells in quantity. The current procedure works well in a Petri dish, so to speak, but may run in complications with attempts to scale up production. Once the quantities are sufficient, then use in screening drugs and other experimental procedures are ahead.

The ultimate test would be transplanting the cells into humans, says Bhatia, but that isn’t on the cards — at least not yet. “The clinical side is going to be a lot of work,” he says. “At least from our estimation, this is the most encouraging result we’ve seen for using blood cells for cell-replacement therapy.”

[Source: Nature, News]

Just when stem cells are in the news in the United States because of a major ruckus in the court system, the first patient with a spinal injury is about to be injected with embryonic stem cells in hopes of regenerating damaged neurons and restoring some functionality. The patient, at the Shepherd Center in Atlanta, Georgia (USA) will be given a course of treatment developed by the biotech company Geron, based in California. As part of the trial, several other patients, all with spinal damage within 14 days of starting treatment, will be given the same stem cell protocol. First and foremost their condition will be closely monitored for initial problems and side-effects, secondarily the treatment will be evaluated for efficacy (did it help).

This trial, which has labored through a seemingly endless approval process, was finally given the green light by the U.S. Food and Drug Administration in January of 2009. This approval was met with considerable opposition from some religious groups because the stem cells used are derived from human embryos. This is the first use for human patients of what are known in the field as hESCs (human embryo stem cells). There have been other human trials with stem cells derived from adult human cells.

Millions of the stem cells, technically progenitor stem cells for neurons (oligodendrocytes), will be flooded into the damaged spinal area with a fine needle. It’s a bit of a blunderbuss approach (blast lots of cells into an area and hope for the best), but this is the very beginning for stem cell treatments. Stem cell researchers have hailed the trials as “a milestone” and “the dawn of the stem cell era.”

In the United States, stem cell research usually becomes tangled with legal issues stemming from a 1995 law forbidding federal funding for experiments that involve destruction of human embryos – precisely how the stem cells in this trial are derived. In August 2010 a federal court upheld the law and ordered a suspension of all embryonic stem cell research. An appeals court later overruled the decision, temporarily. Whether this is germane to the actual trials of stem cells, which it can be argued are quite a different kind of research, is unknown.

As could be expected, the actual start of this clinical trial is something of a morale builder for the U.S. stem cell research community. However, it will be years before the trials are completed and the results can be evaluated. It would be a bigger boost for stem cell research if the treatment works.

As covered in a SciTechStory post February 15, 2010, Induced stem cells not such good news efforts to create the equivalent of human embryonic stem cells starting with adult cells have not turned out so well. A study done at the University of Wisconsin (Madison, USA) compared five embryonic stem cell lines with twelve induced stem cell lines. They found that the induced pluripotent stem cells (iPSC) converted to neuron cells do not match all the differentiations made by embryonic stem cells. The study also showed that iPS cells created without using genes, which in theory should have resulted in ‘cleaner’ differentiation, did no better than gene induced cells.

Now a new study from the Massachusetts General Hospital Center for Regenerative Medicine and Harvard Stem Cell Institute has identified a gene whose silencing may be responsible for at least some of the induced pluripotent stem cells’ limitations. By using the latest genetic assay tests on the DNA of stems cells from mice, they compared the developmental potential of two natural embryonic stem cell lines with induced pluripotent stem cell lines. By comparing the RNA transcriptions (copies made from the DNA) of each line, they found that the natural stem cells produced viable mice, and the induced cells did not. The difference was an obvious reduction in the transcription of two genes in the induced cells. These genes are found in a cluster on chromosome 12 and are normally imprinted maternally (only the mother’s genes are expressed).

In an examination of more than 60 other iPSC lines, the same pattern of silenced (non-expressing) genes was found in the vast majority. Tissues produced from these cell lines were genetically correct reproductions, but the gene silenced cells could not fully develop into live mice. Another experiment, this time reactivating the silenced genes in a few of the iPSC lines, and the cells were able to produce live animals. This may have been the first time that live animals were produced from induced pluripotent stem cells.

“The activation status of this imprinted cluster allowed us to prospectively identify iPSCs that have the full developmental potential of embryonic stem cells,” says Matthias Stadtfeld, PhD, a co-lead author of the report. “Identifying pluripotent cells of the highest quality is crucial to the development of therapeutic applications, so we can ensure that any transplanted cells function as well as normal cells. It’s going to be important to see whether iPSCs derived from human patients have similar differences in gene expression and if they can be as good as embryonic stem cells – which continue to be the gold standard – in giving rise to the 220 functional cell types in the human body.”

[Source: EurekAlert]

Pluripotent stem cells can also be created by nuclear transfer – removing the nucleus of an adult cell and replacing it with the nucleus of an embryonic cell…essentially the same technique used to clone animals. This approach also produces viable embryonic stem cells, but is difficult to perform in quantity. Consequently, the ability to make viable stem cells using induced methods may open the door to producing a greater quantity of high quality cells. Moving on from mouse stem cells, the next step is to perform similar experiments with human stem cells.

One of the things that stem cell research can do is develop replacement cells, for example heart cells grown from human embryonic stem cells are already being tested for repair of the heart. After many years of work by Stefan Heller, professor of otolaryngology at the Stanford University School of Medicine (Oakland, USA), as now reported in the journal Cell, it looks like something similar may be possible for replacing damaged human cochlear and vestibular hair cells.

This initial research used natural and induced (artificially stimulated) pluripotent stem cells from mice. (The research has to start with living cells from something; human cells are a goal, not a starting place.) The process of guiding these stem cells through the many phases of development – not in the mice but in vitro (in a Petri dish) – was difficult. A lot of trial and error with various cell development chemistries was necessary. Eventually, the researchers were able to turn the stem cells into embryonic cells like those of the skin and nerves (ectodermal). From there, various growth factors were used to coax the cells into the form known as ‘otic-progenitor’ – primitive ear cells. Finally, with more chemical stimulus, the cells were further differentiated to develop clusters of hair cells with the characteristic stereociliary bundles.

The stereocilia, tiny clumps of hair-like projections on the cell, were crucial. Sound vibrations transmitted through the air cause the stereocilia to bend, setting off mechanical vibrations at their base that are converted into an electrochemical (nerve) signal that is then interpreted by the brain as sound. When the researchers were able to verify by further testing and microscopic observation that these artificially induced stereocilia could indeed produce the electrochemical signals, they knew that the project was a success.

Heller, a leader in stem-cell based research on the inner ear, has recently been focused on two paths for possible cures for deafness: drug therapy — which could be as simple as an application of ear drops — and stem cell transplantation into the inner ear.

Both paths could be further advanced by the ability to develop hair-cell-like cells, he said. “We could now test thousands of drugs in a culture dish,” he explained. “It is impossible to achieve such a scale in animals. Within a decade or so we could reap the benefits of this type of screening.”

[Source: EurekAlert]

Mostly, science requires patience. Scientists like Dr. Heller think in terms of a lifetime of research – and maybe, just maybe, they will accomplish a major goal – in this case, to find a cure (or cures, or approaches to cures) for deafness.

“You can think of it this way,” said Ren. “Neurons and skin cells share the identical set of genetic material – DNA – yet their structure and function are very different. The difference can be attributed to differences in their epigenome. This is analogous to computer hardware and software. You can load the same computer with distinct operating systems, such as Linux or Windows, or with different programs and the computer will run very different types of operations.

“Similarly, the unique epigenome in each cell directs the cell to interpret its genetic information differently in response to common environmental factors. Understanding the differences of epigenomic landscapes in different cell types, especially between pluripotent and lineage-committed cells, is essential for us to study human development and mechanisms of human diseases.”

[Source: EurekAlert]

This quote is from Professor Bing Ren, University of California San Diego, an author of a paper, Distinct Epigenomic Landscapes of Pluripotent and Lineage-Committed Human Cells published May 7 in Cell: StemCell. Not all geneticists would agree with what he said, or at least would disagree in emphasis. Professor Ren is making a case for the importance of epigenetics.

Epigenetics is a relatively new field of study that has a number of definitions. Here’s a good one:

“The development and maintenance of an organism is orchestrated by a set of chemical reactions that switch parts of the genome off and on at strategic times and locations. Epigenetics is the study of these reactions and the factors that influence them.”

[Source: University of Utah – Learn Genetics]

Epigenetics addresses questions like this: How is it that humans, with about 24,000 genes in their genome, are so much more complicated than the round worms, with 20,000 genes in their genome? Or the question addressed in the paper mentioned above: How much do the epigenomes of human embryonic stem cells differ from adult (lineage-committed) cells?

Epigenetics (‘epi’ meaning ‘over’ or ‘above’… genetics) understands that the principle mechanism of heredity is DNA and that the genome (the sequence of genes) provides the blueprint on which life is built. However, it also recognizes that DNA alone doesn’t really explain how human beings develop and become so much more complex than, say, the round worm, since the genetic code between the two isn’t all that different. The answer, according to the epigeneticist, is that the genome is surrounded (physically and figuratively) by an epigenetic landscape of amino acids, proteins, enzymes, RNA (in several forms), and probably non-coding portions of the genome itself (the introns) that interact with the genes of the DNA and also with the environment to guide the expression of genes.

Sometimes the epigenetic landscape is also called the epigenome, as if it were a counterpart to the genome. In a way it is. To use a crude analogy: If the genome is the blueprint, then the epigenome is the general contractor. The design of cells and the inherited characteristics of an organism are carried in the DNA. To a certain extent, the DNA also provides the design for the elements of the epigenome; but once the elements are in place, then the epigenome takes over the implementation of the DNA designs. It guides the chemistry of life (largely using RNA to guide the building of proteins) through the various stages of development, for example, from the original human embryonic stem cells as they go through the process of differentiating into the many kinds of adult body cells (blood, neuron, skin, etc.). Like a general contractor, the epigenome marshals resources, controls the rate of growth, determines what gets built where and when, and also acts as an intermediary between the blueprint of the DNA and the demands of the environment. Controlling immediate adaptation is a very important part of the epigenome.

Don’t get too carried away with an embodiment of an epigenome. Molecular biologists are really just beginning its exploration. They know it is organic chemistry – very complicated organic chemistry. They know that some of the epigenome comes not from the DNA in the nucleus but from the DNA of the mitochondria (inherited only from the female). They’re beginning to discover that large segments of the genome, the so called ‘junk DNA’, also contain information used by the epigenome to regulate the expression of genes. Yet this is obviously only the beginning.

Many epigenetic researchers, such as Professor Ren, are interested in how the epigenome guides the development of cells. To a certain extent it is the measure of how little is known about epigenetics that the question he addresses is: What’s the difference in the epigenetic landscape of the human embryonic stem cell, compared to the landscape of an adult (differentiated) cell? The answer, as it turns out, is – a lot.

For this particular study, the researchers looked at the structures that support DNA (kind of like scaffolding). These are called chromatin structures, and chromatin is built from specific proteins (histones). Histones are known for their adaptive properties; they are often affected by enzymes that are the principle tools of epigenetic processes. It is known that the chromatin structures interact with the genes and play a part in gene expression. For the study, chromatin structures in embryonic stem cells were compared with fibroblasts, the cell commonly found in animal connective tissue. It was found that nearly a third of the genome differs in chromatin structure between the two. Most of the changes were the result of dramatic repression of chromatin through the histone protein – a result of epigenetic influence.

Sometimes the steps toward scientific knowledge seem very small. On the other hand, when walking into unknown territory…

Small steps or not, the exploration of epigenetics has a kind of liberating feel to it. It seems like it’s the wide world of opportunity, change, and adaptation compared to the more narrow and limited commands of DNA genetics. It is also, of course, a hugely complicating factor. If we thought genetics was complicated (and it is), then the influence of epigenetics makes it more complicated probably by orders of magnitude. Of course, it took nature many millions of years to work out the complications – we ought to have the patience of a few decades to figure them out.