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]

Another approach taken by some labs is to use RNA (ribonucleic acid) as the medium for reprogramming adult cells. In a study published September 30, 2010 in the journal Cell: Stem Cell [Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA] Dr. Derrick J. Rossi (Harvard Medical School, USA) and team created synthetic RNA, which they call modifiedRNA, that encodes the creation of the appropriate proteins to produce stem cells (iPSCs).

This method has several advantages, not the least of which is that it doesn’t alter the DNA of the cell. This greatly reduces the risk of problems with mutation. Another advantage is found when the stem cells are applied to patient-specific therapy, such as the regeneration of certain kinds of tissue. Again, using a modified RNA, the stem cell can very efficiently be reprogrammed into a specific type of adult cell.

It is the efficiency of this approach that surprised the researchers:

Repeated administration of the modified RNAs resulted in robust expression of the reprogramming proteins in mature skin cells that were then converted to iPSCs with startling efficiency.

“We weren’t really expecting the modified RNAs to work so effectively, but the reprogramming efficiencies we observed with our approach were very high,” says Dr. Rossi.

[Source: Cell News]

The reprogramming of cells via modified RNA is promising, but still at a very early stage of development. As with all stem cell research, there are many steps between proof of concept and the approved application in human beings. Many questions must be answered, such as: How robust are stem cells produced in this way? Are there any complications? Is it practical to re-administer RNA continuously?

Relevant previous SciTechStory posts:
Induced stem cells not such good news
New method: Creating stem cells from fat cells

In work done at the University of Georgia (USA) by Stephen Dalton and colleagues and published September 2, 2010 in the journal Cell Stem Cell [Myc Represses Primitive Endoderm Differentiation in Pluripotent Stem Cells], it is becoming clear that Myc is involved in critical gene expression (which genes are translated into protein) within stem cells. In previous work the Dalton team had established that Myc was important for stem cell maintenance, but the new work revealed something unexpected.

For most living things stem cells are the basis of growth and development. Pluripotent stem cells have the ability to transform into almost any kind of cell – muscle, heart, skin and so forth. Gestation from an egg to a living embryo is a constant process of converting stem cells into more differentiated cells – a process that continues throughout most of life. Consequently a reserve of pluripotent stem cells needs to be retained. How are these reserve cells maintained?

It has been known for some time that stem cell differentiation is triggered and perhaps guided by a crucial gene called GATA6 – the master regulator. What regulates the regulator? That is, if GATA6 is always active then every stem cell will be transformed into a differentiated cell. In order to retain a supply of stem cells, something inhibits GATA6. That inhibitor is Myc. In fact, the mechanism for inhibition involves two forms of Myc: c-Myc and N-Myc. The scientists confirmed this by experiment. They found that deactivating both of the Myc forms (but not one) triggers GATA6.

The process is similar in reverse, that is, when turning differentiated cells back into stem cells. Again, Myc plays a role in preventing GATA6 from establishing the differentiation of cells that have been reprogrammed.

“During the reprogramming of cells, Myc represses genes associated with the differentiated state and primes them for the expression of stem cell genes,” he said. “We now speculate that during the early reprogramming stage, Myc serves to change the cell cycle so that stem cells can divide for long periods of time without aging. This is also what Myc does in cancer cells.”

Dalton said that there is an intriguing relationship between normal stem cells and cancer cells. Since Myc is crucial for maintenance of stem cells and for the development of cancer, pluripotent stem cells represent a good model for tumor biologists. Cancer is thought to be initiated by rogue stem cells found in different tissues, further highlighting the link between stem cell biology, cancer and Myc.

[Source: EurekAlert]

It is this relationship between Myc and the mechanisms of cell aging – either normal or in cancer that is the golden find in this research. Not that Dalton or anyone else has the full explanation for how it works. Digging into the molecular level of cell biology for explanations will take many (many) years. However, any research work that can bring insight into both fundamental cell processes, such as stem cell differentiation, and into the malign realm of cancer (where it is believed that rogue stem cells may be the ultimate cause) – for scientists this is perhaps the best of times – years of work ahead (and dreams of Nobel prizes dancing in their head).

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.

The new study, led by Su-Chun Zhang, compared five embryonic stem cell lines with twelve induced stem cell lines. They found that the induced pluripotent stem cells (iPS) 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. As Dr. Zhang explains…

It was predicted that the absence of exotic genetic factors would result in cells essentially identical to embryonic stem cells. “It is totally surprising that doesn’t happen at all,” says Zhang. “It tells us the techniques for generating induced pluripotent stem cells are still not optimal. There is room for improvement.”

Despite their unpredictability, Zhang notes that induced stem cells can still be used to make pure populations of specific types of cells, making them useful for some applications such as testing potential new drugs for efficacy and toxicity. He also noted that the limitations identified by his group are technical issues likely to be resolved relatively quickly.

“It appears to be a technical issue,” he says. “Technical things can usually be overcome.”

The key, he explains, is determining what things are at play that make the induced cells different.

[Source: EurekAlert]

It is unclear whether not knowing ‘what things are at play’ – possibly some fundamental information is missing – constitutes a technical issue.

Of course, turning fat cells into stem cells is not simple. In the case of this research, it resulted from a fortunate combination of skills and knowledge.

The finding brings together disparate areas of Stanford research. Kay’s laboratory invented the minicircles several years ago in a quest to develop suitable gene therapy techniques. At the same time, Longaker was discovering the unusual prevalence and developmental flexibility of stem cells from human fat. Meanwhile, Wu was searching for ways to create patient-specific cell lines to study some of the common, yet devastating, heart problems he was seeing in the clinic.
…
“This is a great example of collaboration,” said Longaker. “This discovery represents research from four different departments: pediatrics, surgery, cardiology and radiology. We were all doing our own things, and it wasn’t until we focused on cross-applications of our research that we realized the potential.”

“About three years ago Mark gave a talk and I asked him if we could use minicircles for cardiac gene therapy,” said Wu. “And then it clicked for me, that we should also be able to use them for non-viral reprogramming of adult cells.”

[Source: EurekAlert]

Kay’s ‘minicircles’ are DNA elements arranged in microscopic rings. These can be injected into the body of a cell to look and work somewhat like the cell’s own plasmids (circular DNA molecules found outside of the cell nucleus). The minicircles then direct the cell’s RNA to produce DNA, RNA, or other proteins for therapeutic effect. This is a proven technique that has a great virtue in not using viruses to reprogram DNA/RNA (viruses being difficult to safely filter and control). However, the technique had not been used before to reprogram adult cells into stem cells.

The minicircles were applied to fat cells because Wu’s and Longaker’s research had shown this type of adult cell to have a good DNA configuration for reprogramming and was relatively easy to isolate.

The final experiments with minicircles and fat cells, done in vitro (in a Petri dish), showed that stem cells were created at the rate of about 0.005% of cells – a low rate compared to other techniques, but given the plenitude of fat cells, not a problem for production. The stem cells produced appear to have no differences from pluripotent cells from other sources.

As time will tell, if this method for producing stem cells is viable and scalable (can be done in large quantities), then it is indeed a major step toward making stem cells available for many kinds of diagnostic and therapeutic applications.

The underlying targets of these ‘genomic snapshots’ was to map the changes in DNA methylation. Chemically, this is the process whereby a methyl (CH3, related to methane CH4) is attached to genes. In most cases, methylation turns off the gene, that is, suppresses its expression. Once a gene has been methylated, it typically remains that way throughout the life of the cell and is also passed on to any cell created from it. In short, this is how cells become differentiated – how a stem cell becomes a skin cell – various genes are shut down, and the end result is a skin cell.

In reviewing the data produced by the study—information on the methylation of three billion base pairs of DNA—the scientists were able to identify previously unknown patterns of DNA methylation. They identified cases where DNA methylation appeared to enhance, rather than repress, the activity of the surrounding DNA, and found evidence to suggest a role for DNA methylation in the regulation of mRNA splicing.

“We produced a very large amount of data,” said Loring [Scripps Research Professor Jeanne Loring, co-author of the paper], “but it actually simplifies the picture. We identified patterns of many genes that are methylated or de-methylated during differentiation. This will allow us to better understand the exquisitely choreographed changes that cells undergo as they develop into different cell types.”

[Source: EurekAlert]

The data from this study are being made publicly available. For one thing, there is an enormous amount of data, and it will take many researchers many years to work through it. For another, it is usually in collateral studies based on genomic analysis that eventually lead to the most revealing information. This has been the case with the human genome and will be expected for this genome study of stem cell development.