Telomerase and Wnt signaling

Now that research into telomeres and telomerase has (finally) garnered a Nobel Prize, it's a good time to write about recent research on the subject.

Seminal work on telomeres by Elizabeth Blackburn, one of the Nobel winners, was published way back in 1978, and active studies have been going on ever since. So perhaps it's not surprising that the rate of new findings is not so rapid as occurs in newer areas – such as stem cells.

But fascinating new results on telomeres and telomerase do still appear, and one of them connects with more recent research areas – such as Wnt signaling and... stem cells.

Here's the press release:

Discovery pinpoints new connection between cancer cells, stem cells (7/1/09)

A molecule called telomerase, best known for enabling unlimited cell division of stem cells and cancer cells, has a surprising additional role in the expression of genes in an important stem cell regulatory pathway, say researchers at the Stanford University School of Medicine. The unexpected finding may lead to new anticancer therapies and a greater understanding of how adult and embryonic stem cells divide and specialize.

Don't bother getting excited about the "new anticancer therapies" bit. That's just boilerplate that about 77.3% of all press releases dealing with cell biology contain, presumably to impress the rubes. If you need something to get excited about, you might recall that telomerase is also being investigated intensively in connection with issues of aging and longevity, independently from cancer. However, while it's possible that something of medical significance may come from this research, that's probably way down the road.

Before discussing the new research, let's review some of the background on telomeres, telomerase, and Wnt signaling.

To begin with, a telomere is a series of short repeated segments of DNA found at the ends of chromosomes in all eukaryotic cells. In cells of vertebrate animals the repeated segment is TTAGGG (where the letters represent nucleobases: T=thymine, A=adenine, G=guanine).

The total number of nucleotides in these repeated segments varies a great deal from species to species, but in humans it is (initially) about 10,000 nucleotides (or ~1700 complete segments). I say "initially", because part of the telomere is lost every time a cell divides – perhaps 50 to 200 nucleotides per division. Obviously, this means that an adult cell newly derived from a stem cell can divide at most 50-200 times before the telomere is all gone. In practice, the number is a lot less than the maximum, perhaps 40 to 60 times, which is called the Hayflick limit. Cells are programmed to stop dividing on reaching this limit, since otherwise useful DNA would be lost or damaged upon further division.

Why does this loss occur? It seems to be somewhat of an accident of the nitty-gritty details of how DNA replication occurs during cell division. I won't go into that, since it's best explained with some diagrams; you can read about it at Wikipedia. In fact, in the early days of molecular biology (around 1972), what happens at the end of chromosomes during DNA replication was rather puzzling, and the puzzle was called the "end replication problem". Now it's pretty well understood, though somewhat messy.

In any case, the loss of DNA from the telomeres during cell division is a fact, and it conveniently explains the existence of the Hayflick limit, which had been recognized since 1965. What happens when the teleomere is all used up and the limit is reached? Cells that have reached the limit don't necessarily die (though they might), but they do stop dividing, and they enter a static phase of cell life known as senescence.

If you think about it, senescence can be a problem, especially in certain tissues that need to continually replenish their cells, such as skin and the lining of the intestines, as well as hair, fingernails, etc. How is senescence circumvented in such tissues? The answer is (adult) stem cells. It turns out that stem cells are not subject to the Hayflick limit. It's not clear whether they are subject to limits at all.

Like any other cell type, stem cells also divide (by the process technically known as mitosis). But there is one difference from the way "ordinary" cells divide. Stem cells can divide asymmetrically, where one daughter cell is another stem cell, but the other daughter is a "progenitor cell", which is close to a normal, fully-differentiated adult cell of a fixed tissue type. Progenitor cells differentiate further into the final form when they divide, and they are able to divide only a limited number of times.

So how is it that stem cells are able to escape the Hayflick limit, dividing an indefinite number of times, even though they are also subject to the same loss of telomere nucleotides with each division? The answer is the enzyme telomerase, which is able to rebuild shortened teleomeres. It's fortunate for the longevity of complex organisms that telomerase exists, otherwise stem cells would not be able to divide often enough to allow tissues exposed to harsh conditions (such as skin and intestinal lining) to be replenished.

In order to explain the results of the research to be described here we need to say more about telomerase, but first let's summarize the function of telomeres. At first it may seem that all they do is compensate for the sloppy way that DNA replication works, by providing long stretches of expendable DNA at the ends of chromosomes. But there is one other notable function: teleomeres are a useful "cap" at the end of a chromosome that is recognizable to cellular mechanisms responsible for detecting DNA damage. If it weren't for the telomeres, the ends of chromosomes would be indistinguishable from DNA damage, which can result from errors in the replication process, as well as damage due to external agents such as ionizing radiation or harmful chemicals. Cells have various mechanisms for repairing many kinds of damage, but even if repair isn't possible, other mechanisms exist that recognize the damage and prevent further cell division or cause programmed cell death (apoptosis).

Not all DNA damage can be repaired or compensated for by apoptosis or cessation of cell division. Unrepaired DNA damage (however it occurs) is the main cause of cancer (though not the only one). So the existence of the Hayflick limit as a result of teleomere shortening acts as one defense against cancer. Cancer can be defined as the uncontrolled proliferation of cells as a result of DNA damage (affecting existing mechanisms that normally control proliferation) or other causes. So fixed limits on the number of times a cell can divide is one of a number of mechanisms organisms have to guard against cancer.

Before moving on, here's a quick summary of the functions served by telomeres: (1) compensate for the chromosome "end replication problem"; (2) make it possible for cells to distinguish chromosome ends from damaged chromosomes; (3) provide natural limits to the number of times ordinary cells can divide, as protection against cancer.

As noted above, the third of these functions is a problem for stem cells that do need the ability to divide an indefinite number of times. Repair of tissues exposed to harsh conditions is not the only circumstance this ability is needed. Another very important case is that of embryonic development. Multicellular, sexually-reproducing organisms start from a single cell (zygote). Yet there are close to 10¹⁴ cells in an adult human.

It is true that a single cell that underwent 47 cycles of cell division could theoretically produce that many cells. But that's really pushing the limits, since some cell types are needed in much larger numbers than others. The bottom line is that embryonic development is the other main circumstance when limits on cell division need to be overcome.

Telomerase is what makes this overcoming of telomere limits possible. And it does it in a pretty straightforward way. Telomerase is a complex molecule with three distinct parts. Two of these are proteins: Telomerase Reverse Transcriptase (TERT) and dyskernin, which are coded for by distinct genes. TERT does most of the work. The other part is a short piece of RNA, called the telomerase RNA component (TERC), which contains and is somewhat longer than the repeat unit (TTAGGG in vertebrates). Like any other reverse transcriptase enzyme (other examples of which occur in RNA viruses such as HIV), TERT simply translates a piece of RNA into DNA and inserts it into a chromosome. In the case of telomerase, the RNA is carried inside the enzyme complex itself. Telomerase does its job simply by replacing the telomere DNA that is lost from chromosomes during mitosis.

So telomerase performs the function of rebuilding telomeres, and this is essential in tissues where cells must proliferate rapidly, such as in embryonic development and tissue regeneration. But as we noted above, if the restriction on cell division is circumvented, the risk of cancer goes up. Although telomerase serves an essential purpose in specialized contexts, it also makes it possible for cells to become cancerous. Consequently, telomerase is not expressed in most adult body cells. However, telomerase is expressed in about 90% of tumor cells.

It might seem as though one approach to controlling or even destroying cancer could involve either inhibiting telomerase or developing a vaccine using telomerase to induce an immune response against telomerase-rich cancer cells. There are in fact various clinical trials exploring both techniques. But this is tricky and rather risky, because as we've observed, telomerase is needed in stem cells required for tissue regneration, at least once the cells have begun proliferating. Those cells need to be protected while they are simply doing their job – replenishing skin and intestinal linings, for example. We need to understand how such cells are controlled so that they work without leading to cancer.

Anyhow, TERT is a protein that is an essential component of telomerase, which plays an important role in cell proliferation. The new research we're finally almost ready to discuss shows that, surprisingly, TERT also plays a role in a completely different aspect of cell proliferation having nothing to do with telomeres. That's where Wnt signaling comes in.

Wnt signaling is a subject we've looked at several times before. Some of the relevant articles are here, here, here, and here. Wnt was first noticed in connection with embryonic development and tissue regeneration. This article has many examples. The name Wnt alludes to a gene called "wingless", because the gene causes fruit flies to lack wings when the gene is mutated.

Recently Wnt's importance in stem cells has also received a lot of attention. Some of our discussion of that may be found here, here, here, and here. Wnt's relevance to cancer and cancer stem cells is often touched on in most of all the articles listed.

Basically, Wnt is the name of a family of proteins that play an important role in signals promoting cell proliferation, especially in the context of embryonic development and tissue renewal (skin, intestines, hair, and immune system cells). Since Wnt proteins promote proliferation, they also play a role in cancer.

The fact, then, that new research shows teleomerase can enhance Wnt signaling is signficant as a second, entirely separate route through which it plays a role in both normal stem cell function and cancer.

Here's the research abstract:

Telomerase modulates Wnt signalling by association with target gene chromatin (7/2/09)

Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/β-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/β-catenin signalling by serving as a cofactor in a β-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior–posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert^-/- mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/β-catenin signalling pathway.

That's a pretty good summary of the paper, but I imagine most people would like a bit more explanation of what's going on.

Previous research had disclosed some interesting "coincidences" involving stems cells and embryonic development, in which both telomerase and Wnt signaling seemed to have similar effects, even though no obvious connection was known. For example, epidermal (skin) stem cells spend most of their time in a quiescent (non-dividing) state. The main function of Wnt proteins is to carry signals between cells that inform target cells of a need to start dividing, for example during various stages of embryonic development or to heal wounds. The curious thing is that telomerase was known to have a similar effect as Wnt signaling on some stem cells. This coincidence is enough to motivate looking for a connection.

If you are really into this sort of thing, you might want to refer to this diagram of various cell signaling pathways, including that of Wnt. One of the things it illustrates is the position of β-catenin in the Wnt pathway. β-catenin operates at the end of the pathway, where it becomes a part of a protein complex that includes transcription factors (known as TCF/LEF), and the complex causes expression of a variety of Wnt-regulated genes, which go on to enable cell proliferation.

One of the main findings of the research is that TERT interacts with another protein called BRG1 (or SMARCA4), and together these become important parts ("cofactors") of the gene-regulating protein complex. It was also determined that TERT is the only component of telomerase that is involved with Wnt signaling.

Apparently TERT is more important for some instances of Wnt signaling than for others. For example, low levels of TERT in embryos of the frog Xenopus laevis resulted in very abnormal development of the frog embryos (in terms for the anterior-posterior axis of the body). But low levels of TERT at a later stage of development caused only somewhat more subtle defects in formation of ribs in the embryo. Similar somewhat minor effects also occured with low levels of TERT in mouse embryos, and these defects were much like the effects of low levels of β-catenin.

So what, then, is so interesting about this research? It's satisfying to know the reasons for what were previously just observed coincidences, such as the fact that telomerase is not only very important for the ability of stem cells to divide freely (during embryonic development and tissue regeneration) but that it also can help stimulate stem cell division.

But what makes this research especially significant is the importance of the biological processes in which both telomerase and Wnt signaling play major roles – namely embryonic development, tissue regeneration and repair, and cancer. The latter two processes are especially important for medical reasons, although we're still a long way from being able to use this knowledge about telomerase and Wnt signaling for therapeutic purposes.

As far as cancer is concerned, we need to understand much more about how telomerase and Wnt signaling work in various types of stem cells, given that stem cells may play a big role in some types of cancer (though not in others).

But tissue regeneration and repair are also of significant medical interest, since it is the eventual inability of various types of tissues to replenish themselves in old age that is responsible for the many debilities that appear in old age. Many people have speculated that telomerase could help alleviate this problem – provided it does not also lead to facilitating the development of cancer.

Finally, we are left with the puzzle of how it is that TERT happens to play important – yet quite distinct – roles in two very separate processes that are, neverthelesss, both important for cell proliferation. We don't know the answer to this, but we can speculate. Perhaps it's because TERT has to be around anyway for stem cells during embryogenesis and tissue regeneration. That being the case, perhaps at some point in evolution, TERT also happened to help boost Wnt signaling a little. The effect of amplifying Wnt signaling in the same contexts that telomerase was needed would be advantageous and worth conserving. This kind of double duty should lead to more efficient use of cell resources.

Park, J., Venteicher, A., Hong, J., Choi, J., Jun, S., Shkreli, M., Chang, W., Meng, Z., Cheung, P., Ji, H., McLaughlin, M., Veenstra, T., Nusse, R., McCrea, P., & Artandi, S. (2009). Telomerase modulates Wnt signalling by association with target gene chromatin Nature, 460 (7251), 66-72 DOI: 10.1038/nature08137

Further reading:

Cell biology: The not-so-odd couple (7/2/09) – expository article in Nature about the telomerase-Wnt research

Nobel in medicine honors discoveries of telomeres and telomerase (10/5/09) – news article in Science News

Nobel for insights into ageing and cancer (10/5/09) – news article in New Scientist

Chromosome protection scoops Nobel (10/5/09) – news article in Nature

Three Americans Win Physiology or Medicine Nobel (10/5/09) – news article at ScienceNOW.com

Work on Telomeres Wins Nobel Prize in Physiology or Medicine for 3 U.S. Genetic Researchers (10/5/09) – news article in Scientific American

Nobel Winners Isolate Protein Behind Immortality, Cancer (10/5/09) – news article at Wired.com

Tags:

telomeres, telomerase, Wnt signaling

Induced pluripotent stem cells with one transcription factor

Just under three years ago, in October 2006, some important stem cell research was announced by a Japanese scientific team led by Shinya Yamanaka. The team showed how ordinary mouse skin cells could be transformed into cells that turned out to be pluripotent, just like embryonic stem cells (ESCs). The new cells were called induced pluripotent stem cells (iPSCs). Although "ground-breaking" is an over-used term, this research genuinely deserved the description.

Aside from the fact that it could be done at all, the surprising thing was that the transformation could be effected by adding transcribable genes for just four transcription factors to the skin cell DNA. Those genes were Oct4, Sox2, c-Myc, and Klf4. And now very recent research shows that, under the right conditions, just the addition of Oct4 alone can accomplish the same feat.

We discussed some of the early research here, with additional reports here, here, and here.

In the three years since the original announcement, research has extended and improved the process in a number of ways. The ultimate goal is to be able to produce pluripotent human stem cells that are in all important respects equivalent to embryonic stem cells, by a process that meets several important criteria:

• Cells to be reprogrammed into a pluripotent state should be readily obtainable from human subjects (unlike embryonic cells or rare types of adult stem cells).
  
• No permanent changes to cellular DNA should be made, only changes to gene expression.
  
• The process should be relatively quick and efficient, so that reasonable number of pluripotent cells can be obtained for routine therapeutic or experimental uses.
  

Reprogramming of other cell types into pluripotent cells is important not just as a technical feat to prove it can be done. There are two other important objectives. The first is to develop human cell lines that model many types of pathology (cancer, Parkinson's disease, or whatever) to facilitate research into therapeutics for these diseases. The best way to develop such lines is first to obtain pluripotent cells with the appropriate pathology, derived from human subjects with the disease, which can't generally be done from embryonic sources. From there, several techniques can be used to produce appropriate cell cultures with the desired model pathology.

The second objective is longer-range but even more important: to manufacture cells, for patients with certain diseases, that can be used as therapeutic replacements for the patient's own malfunctioning tissue. This would be accomplished by obtaining pluripotent cells derived from the patient, correcting genetic problems in those cells, and then inducing the cells to differentiate into the required tissue type. Diseases that should be treatable in this way include Parkinson's disease, Type 1 diabetes, and heart disease. Starting with cells from the actual patient eliminates the problem of tissue incompatibility.

The criteria listed above that are imposed on the process are important for meeting both of these objectives.

In the three years since the original work was announced, dozens of research groups have set about testing improvements to the original procedures in order to progress towards the ultimate objectives. The improvements that have been made include:

• adapting the procedures to work in species other than mice – including pigs and fruit flies, as well as humans
  
• reducing the number of transcription factors that need to be introduced, or finding other suitable transcription factors
  
• finding other cell types besides skin cells to start with, generally various types of non-pluripotent stem cells – which makes other improvements in the process easier to accomplish
  
• changing the way that the transcription factors are introduced into the target cells, in order to avoid alteration of the original DNA (since such alterations may introduce risks of cancer or other cellular malfunction)
  
• finding other proteins or small molecule compounds that can be added to enhance the efficiency and speed of the process
  

Quite a few important improvements have been announced within the past several months, along with other related news. The most interesting related news is a demonstration that iPSCs really are not only equivalent to ESCs in terms of gene expression, but are in fact equally pluripotent. This latter fact was convincingly demonstrated by cloning several generations of live, healthy mice from iPSCs. (We'll discuss that in a separate article, but here's an overview.)

What I want to discuss here is how the list of transcription factors (or their genes) that need to be added to a non-pluripotent cell has been reduced to just one: Oct4. The work was done by a mostly German team led by Hans Schöler of the Max Planck Institute for Molecular Biomedicine.

So how was this accomplished? Well, the trick is, you have to start with the right kind of cells. In this case the researchers used human fetal neural stem cells (HFNSCs). While such cells aren't pluripotent, they are "multipotent", which means they can normally differentiate into various other cell types.

Back in February the researchers in this study reported that reprogramming with just Oct4 could be done in mouse neural stem cells (see here, here, or here). But would this also work with human cells?

Yes. The latest report shows that HFNSCs can be reprogrammed to a pluripotent state using only Oct4 and Klf4, and (generally) even with Oct4 alone. How is this possible? It is known that mouse neural stem cells already express Sox2, c-Myc, and Klf4. As for the human case, the paper says, cautiously, that "The feasibility to reprogram directly NSCs by OCT4 alone might reflect their higher similarity in transcriptional profiles to ES cells than to other stem cells like haematopoietic stem cells or than to their differentiated counterparts."

And the main indication of this is that the process works: "One-factor human NiPS cells resemble human embryonic stem cells in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency."

What this is saying is that there are several criteria for similarity to embryonic stem cells that the reprogrammed HFNSCs meet. At a molecular level the reprogrammed cells express the same genes and have the same epigenetic markers as ESCs. In addition, they can differentiate into many adult cell types both in vitro and in vivo (in the latter case, by forming teratomas (mixed masses of cell types) when implanted in mice).

There are still several drawbacks to this method for practical purposes, even of research. For one thing, human fetal neural stem cells are not exactly easily obtainable. And in addition, retroviruses were used (as in the original Yamanaka work) to introduce Oct4 into the cells. For therapeutic applications it would be absolutely necessary to use one of the other methods that have been explored and that do not disrupt the existing cell DNA or leave exogenous DNA in derived cells – since either alternative means the derived cells might revert to a more undifferentiated state. On top of all that, the process is still inefficient and slow.

Reprogramming methods that have been explored in other research include the introduction of genetic material in forms other than retroviruses, as well as direct delivery of the transcription factor proteins. The researchers in this study intend to investigate such possibilities, as well as use of other initial cell types: "Future studies will show if direct reprogramming is possible with small molecules or OCT4 recombinant protein alone. ... It will be interesting to extend this study to human NSCs derived from other sources, such as dental pulp, as well as to other stem-cell types."

Kim, J., Greber, B., Araúzo-Bravo, M., Meyer, J., Park, K., Zaehres, H., & Schöler, H. (2009). Direct reprogramming of human neural stem cells by OCT4 Nature DOI: 10.1038/nature08436

Further reading:

One step to human pluripotency (8/28/09) – blog post at The Scientist

Stem cells, down to one factor (8/28/09) – blog post at The Niche

Induced pluripotent stem cells, down to one factor (9/10/09) – excellent overview at Nature Reports Stem Cells

Direct reprogramming of human neural stem cells by OCT4 (8/28/09) – Nature research paper

One-gene method makes safer human stem cells (8/28/09) – New Scientist article

Tags: stem cells, pluripotency

Embryonic stem cell differentiaton

A typical mammal, like a human, has over 200 different cell types in its body, corresponding to tissue types such as liver, heart, brain, muscle, etc. Obviously, for each specialized type of cell to perform its function, rather different sets of genes have to be expressed. Yet the DNA of each cell type contains all the genes, whether they're needed or not. One may reasonably wonder what kind of mechanisms are used to keep unneeded (and unwanted) genes "out of the way" in fully differentiated cells.

Further, a multipotent stem cell, and especially a pluripotent stem cell, might be expected to manage its supply of genes somewhat differently than does a fully differentiated cell. If it does, the question of how is especially interesting with respect to "induced pluripotent stem cells" that are obtained by a relatively, and surprisingly, simple "reprogramming" of fully differentiated cells such as skin cells.

Some new research has begun to address these questions:

Unlocking Stem Cell, DNA Secrets To Speed Therapies (10/10/08)

In a groundbreaking study led by a molecular biologist at Florida State University, researchers have discovered that as embryonic stem cells turn into different cell types, there are dramatic corresponding changes to the order in which DNA is replicated and reorganized.

The findings bridge a critical knowledge gap for stem cell biologists, enabling them to better understand the enormously complex process by which DNA is repackaged during differentiation -- when embryonic stem cells, jacks of all cellular trades, lose their anything-goes attitude and become masters of specialized functions. ...

"Understanding how replication works during embryonic stem cell differentiation gives us a molecular handle on how information is packaged in different types of cells in manners characteristic to each cell type," said David M. Gilbert, the study's principal investigator. "That handle will help us reverse the process in order to engineer different types of cells for use in disease therapies."

"We know that all the information (DNA) required to take on the identity of any tissue type is present in every cell.... We must learn how cells lose pluripotency in the first place so we can do a better job of reversing the process without risks to patients.

"The challenge is, adult cells are highly specialized and over the course of their family history over many generations they've made decisions to be certain cell types rather than others," he said. "In doing so, they have tucked away the information they no longer need on how to become other cell types. Hence, all cells contain the same genetic information in their DNA, but during differentiation they package it with proteins into 'chromatin' in characteristic ways that define each cell type. The rules that determine how cells package DNA are complicated and have been difficult for scientists to decipher."

But, Gilbert noted, one time that the cell "shows its cards" is during DNA replication.

"During this process, which was the focus of our FSU research, it's not just the DNA that replicates," he said. "All the packaging must be replicated as well in each cell division cycle."

He explained that embryonic stem cells have many more, smaller "domains" of organization than differentiated cells, and it is during differentiation that they consolidate information.

"In fact, 'domain consolidation' is what we call the novel concept we discovered," he said.

The open access paper is available here:

Global Reorganization of Replication Domains During Embryonic Stem Cell Differentiation

Author Summary

Microscopy studies have suggested that chromosomal DNA is composed of multiple, megabase-sized segments, each replicated at different times during S-phase of the cell cycle. However, a molecular definition of these coordinately replicated sequences and the stability of the boundaries between them has not been established. We constructed genome-wide replication-timing maps in mouse embryonic stem cells, identifying multimegabase coordinately replicated chromosome segments—“replication domains”—separated by remarkably distinct temporal boundaries. These domain boundaries were shared between several unrelated embryonic stem cell lines, including somatic cells reprogrammed to pluripotency (so-called induced pluripotent stem cells). However, upon differentiation to neural precursor cells, domains encompassing approximately 20% of the genome changed their replication timing, temporally consolidating into fewer, larger replication domains that were conserved between different neural precursor cell lines. Domains that changed replication timing showed a unique sequence composition, a strongly biased directionality for changes in resident gene expression, and altered radial positioning within the three-dimensional space in the cell nucleus, suggesting that changes in replication timing are related to the reorganization of higher-order chromosome structure and function during differentiation. Moreover, the property of smaller discordantly replicating domains may define a novel characteristic of pluripotency.

Tags: stem cells

Induced pluripotent stem cells IV

Induced pluripotent stem cells (iPS cells) may again be judged one of the most significant scientific developments this year, and the news keeps coming. (Of course, it was near the top last year also.)

Some of our previous discussions are here, here, and here.

The ability to turn nearly any type of adult cell into the equivalent of a pluripotent stem cell seems almost too good to be true. And so far, that goal is still elusive, as a practical matter, with respect to treating diseases.

There have been at least three principal difficulties with experimental processes reported so far.

1. The process depends on a kind of gene therapy that inserts a few desired additional genes into cellular DNA using certain types of viruses, and the process itself significantly raises the chances of affected cells becoming cancerous.
   
2. One or more of the genes that are added to cellular DNA to induce pluripotency can also raise the chances of a cell becoming cancerous.
   
3. Alternative methods that address the first two problems tend to be substantially less efficient, therefor slower and more expensive, for producing the desired iPS cells.

The research we'll consider here addresses the first of these problems.

Induced Pluripotent Stem Cells Generated Without Viral Integration

Pluripotent stem cells have been generated from mouse and human somatic cells by viral expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc. A major limitation of this technology is the use of potentially harmful genome-integrating viruses. Here, we generate mouse induced pluripotent stem cells (iPS) from fibroblasts and liver cells by using nonintegrating adenoviruses transiently expressing Oct4, Sox2, Klf4, and c-Myc. These adenoviral iPS (adeno-iPS) cells show DNA demethylation characteristic of reprogrammed cells, express endogenous pluripotency genes, form teratomas, and contribute to multiple tissues, including the germ line, in chimeric mice. Our results provide strong evidence that insertional mutagenesis is not required for in vitro reprogramming. Adenoviral reprogramming may provide an improved method for generating and studying patient-specific stem cells and for comparing embryonic stem cells and iPS cells.

Perhaps a little more explanation would be in order. We've discussed the four transcription factors, Oct4, Sox2, Klf4, and c-Myc, in earlier articles.

The "insertional mutagenesis" mentioned here refers to the use of a type of virus that inserts some of its genes directly into the cellular DNA. Such viruses include lentiviruses and other retroviruses. HIV is an example of a lentivirus. The genetic material of this kind of virus is in the form of RNA, which must first be translated into DNA (by an enzyme called reverse transcriptase) and then integrated into the host cell DNA. The transcription factor genes are artificially added to the virus RNA so that they are copied along with everything else.

That integration step is what enables genes for the transcription factors to be inserted into host DNA. Although genes for those factors are already present, of course, in non-stem cells, they are in a form that is less readily translated into proteins than in pluripotent cells. The newly-integrated genes, however, can be easily translated, and they produce the protein transcription factors that go on to turn the cell into a pluripotent stem cell.

The drawback of this method is that the new genes can be inserted at arbitrary points of the host DNA, and this can harmfully affect other genes, which may make the cell susceptible to becoming cancerous.

What the research in this latest work has done is to add the transcription factor genes to a different kind of virus, called an adenovirus. The significant difference of an adenovirus from a retrovirus is that the genetic material of the former consists of double-stranded DNA, like the DNA of the host cell. When an adenovirus infects a cell, its DNA floats freely within the cell, and it can be translated into proteins by the same process as for the host cell DNA.

So what the new work described in this study does is to add the transcription factor genes to the adenovirus DNA, and then allow the virus to infect normal adult cells. This has the advantage of not damaging the host DNA, because it does not get integrated into it.

Naturally, this is an obvious approach to try, and it has been attempted before, but not successfully. The reason it hasn't worked before, probably, is that the adenovirus DNA is diluted every time an infected cell divides, since there may be, at most, only a couple dozen copies of the virus DNA in each infected cell. It does not get copied reliably into daughter cells, and that's a good thing on the whole. (Otherwise an infection might never go away.)

Fortunately, it turns out that simply having the adenovirus-carried transcription factor genes in a cell for a sufficiently long time can trigger further gene expression that confers pluripotency – which remains even after the virus genes are no longer present. Persistence pays.

The research was carried out using various types of mouse cells – fetal liver cells, adult hepatocytes, and fibroblasts from the mouse tail tip. The last of these can, of course, be obtained quite easily.

There is, however, a downside. The efficiency of inducing pluripotency by this method is still very low. Typically, only 0.0001% to 0.001% (1 in a million to 1 in 100,000) of cells are converted. This compares with 0.01% to 0.1% when DNA-integrating viruses are used.

The research proceeded to compare the adenovirus-induced pluripotent cells with natural pluripotent cells. The similarities were quite close:

• Pluripotency genes of the reprogrammed cells lack methylation (a chemical modification that inhibits expression), just like the genes of natural pluripotent cells.
  
• The pluripotency genes (including Oct4, Sox2, Klf4, c-Myc, and Nanog) of normal pluripotent cells are also expressed in the reprogrammed cells, even after all traces of adenovirus DNA are gone.
  
• The iPS cells formed teratomas (cell masses consisting of many different cell types) when injected into adult mice.
  
• When the iPS cells were injected into mouse blastocysts, which then developed into mostly normal, but "chimeric", young mice, evidence of descendants of the iPS cells turned up in many different tissue types.

The next step for research in this direction will be to find out whether the low efficiency of adenoviral reprogramming can be improved by techniques similar to those used to improve the efficiency of retroviral reprogramming.

News reports on this research:

• Important new step toward producing stem cells for human treatment (9/25/08) – Harvard University press release
  
• A New, Improved Stem Cell Recipe (9/26/08) – ScienceNOW
  
• Safer iPS cells (9/25/08) – The Scientist
  
• Cell 'rebooting' technique sidesteps risks (9/25/08) – Nature
  
• Safer Creation of Stem Cells (9/25/08) – Science News
  
• New way to make stem cells is safe: research (9/25/08) – Reuters
  
• Stem cells created without cancer-causing viruses (9/25/08) – New Scientist
  

M. Stadtfeld, M. Nagaya, J. Utikal, G. Weir, K. Hochedlinger (2008). Induced Pluripotent Stem Cells Generated Without Viral Integration Science DOI: 10.1126/science.1162494

Tags: stem cells, embryonic stem cells, induced pluripotent stem cells

Induced pluripotent stem cells III

There is still some news from here that we need to look at. It has to do with reducing the risk of tumorigenicity by using a signaling protein Wnt3a – a member of the Wnt family of proteins – in place of the c-Myc transcription factor for inducing pluripotency in differentiated adult cells.

This press release gives the executive summary:

Embryonic-like Stem Cells Can Be Created Without Cancer-causing Gene (8/6/08)

Currently, IPS cells can be created by reprogramming adult cells through the use of viruses to transfer four genes (Oct4, Sox2, c-Myc and Klf4) into the cells' DNA. The activated genes then override the adult state and convert the cells to embryonic-like IPS cells.

However, this method poses significant risks for potential use in humans.

First, the viruses employed in the process, called retroviruses, are associated with cancer because they insert DNA anywhere in a cell's genome, thereby potentially triggering the expression of cancer-causing genes, or oncogenes. Second, c-Myc is a known oncogene whose overexpression can also cause cancer. For IPS cells to be employed to treat human diseases such as Parkinson's, researchers must find safe alternatives to reprogramming with retroviruses and oncogenes.

Earlier research has shown that c-Myc is not strictly required for the generation of IPS cells. However, its absence makes the reprogramming process time-consuming and highly inefficient.

To bypass these obstacles, the Whitehead researchers replaced c-Myc and its retrovirus with a naturally occurring signaling molecule called Wnt3a. When added to the fluid surrounding the cells being reprogrammed, Wnt3a promotes the conversion of adult cells into IPS cells.

What amounts to a crude form of gene therapy has been used to make IPS cells. The idea is to insert extra copies of genes for 4 different transcription factors into a cell's DNA in order to raise the expression level of those factors. The problem is that every insertion of a gene into a cell's DNA risks damage to some other random gene in the DNA. Wnt3a, on the other hand, is a signaling protein that normally affects cells only by attaching to receptors on the cell surface.

So what has been accomplished here is that the number of transcription factor genes that need to be inserted into the DNA is reduced from 4 to 3. In addition, the factor that is eliminated, c-Myc, has tumorigenicity risks of its own. Therefore, this research represents a small but useful improvement. However, it is probably only a first step.

More about the present research: here

We discussed earlier research aimed at eliminating use of c-Myc in making IPS cells here.

In fact, just a little bit earlier than the research discussed above, a team from Germany reported, on July 31 in Nature, making IPS cells by adding just two transcription factors. However, they didn't start with adult somatic cells, but with neural stem cells that already had higher expression levels of Sox2 and c-Myc. Given that, they needed to add only Oct4 and either Klf4 or more c-Myc. Here's the abstract:

Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors

Here we show that adult mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than embryonic stem cells, and that exogenous Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells. These two-factor iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germ line, and form chimaeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors.

Keep in mind that there's a further variable that's important here: the efficiency of the process, i. e. the yield of IPS cells obtained as a percentage of original cells at the beginning. It should be obvious that a fair amount of work still needs to be done to find a method of making IPS cells that's both efficient and produces cells that are potentially safe to use in therapeutic applications (as opposed to pure research).

OK, enough of that. Let's move on to something new.

One of the interesting questions about IPS cells is about exactly how close they are to actual embryonic stem cells, which are pluripotent by definition. The best way to measure the degree of closeness is by comparing gene expression levels between embryonic stem cells and IPS cells.

The next research has done exactly that. In fact, it studies gene expression levels for stem-like cells obtained from a wide variety of sources:

A new test distinguishes embryonic stem cells and those with equal therapeutic potential (8/24/08)

To distinguish adult stem cells from pluripotent cells, Loring’s team compared the gene activity of about 150 stem cell samples of various types, including reprogrammed cells, embryonic stem cells and neural stem cells. Out of this comparison popped 299 interacting genes that form what the researchers call a pluripotency network, or PluriNet. Measuring the activity of these genes could reliably distinguish the different kinds of stem cells, the team reports.

Here's the abstract for this research:

Regulatory networks define phenotypic classes of human stem cell lines

We report here the creation and analysis of a database of global gene expression profiles (which we call the 'stem cell matrix') that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent and differentiated cell types. Using an unsupervised clustering method to categorize a collection of ∼150 cell samples, we discovered that pluripotent stem cell lines group together, whereas other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis we uncovered a protein–protein network (PluriNet) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas and induced pluripotent cells). Analysis of published data showed that the PluriNet seems to be a common characteristic of pluripotent cells, including mouse embryonic stem and induced pluripotent cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotency and self-renewal are under tight control by specific molecular networks.

Tags: stem cells, embryonic stem cells, induced pluripotent stem cells

MicroRNA and stem cells II

MicroRNA and stem cells are both pretty hot topics these days. But curiously, there haven't been a whole lot of reports that involve the two together. My last discussion of both, back in March, is here.

However, the combination of microRNA and stem cells just was back in the news, as briefly noted here.

As you recall, microRNA refers to small single-stranded RNA molecules, generally about 21 to 23 nucleotides in length. Each different microRNA (miRNA for short) is transcribed from a DNA gene like any other gene, but the resulting RNA isn't translated into a protein. Instead, the typical miRNA functions by downregulating the expression of another gene that codes for a protein.

An embryonic stem cell (ESC) has the property of pluripotency, which means that it is capable of giving rise to essentially any type of cell in the body of a multicellular organism. Whenever an ESC divides, the resulting daughter cells may also by ESCs (hence pluripotent) or they may be more specialized cells that will eventually give rise to some type of adult body cell.

In any particular ESC, the determination of remaining pluripotent or instead heading down the path to a more specialized cell type depends on what set of genes are expressed. Since miRNAs downregulate gene expression, they can keep an ESC in its pluripotent state, if they are active and suppress a gene that would make an ESC more specialized. But then if such a miRNA is blocked from being expressed, the ESC can start to become more specialized.

On the other hand, as we shall see, some miRNAs may block transcription factors that are needed to maintain a pluripotent state. Such an miRNA needs to be silent in an ESC, so some other protein needs to suppress its expression. (The miRNA called miR-21, discussed later, is an example.)

So the name of the game in studying ESCs, as far as miRNA is concerned, is to figure out what causes a miRNA gene to be expressed or not. Like other genes in an ESC, which genes are expressed is strongly controlled by a few master transcription factors.

There are four such transcription factors which seem to be especially important in ESCs: Oct4, Sox2, Nanog, and Tcf3. As discussed here, the first three of these factors have been found capable of playing a role in turning an ordinary adult cell into a pluripotent stem cell (called an "induced pluripotent stem cell").

Currently there are 336 mature mouse miRNAs known, and 441 mature human miRNAs. It is simple (given the known, complete sequences of mouse and human genomes) to locate the genes for each miRNA. However, in order to determine when a transcription factor regulates the miRNA gene, the promoter for the gene (a separate portion of DNA) must also be located. In order for a gene to be expressed, the right transcription factors have to bind to the gene's promoter.

Finding promoters is a lot harder, but there are techniques that involve searching for methylation of histone proteins that make up the nucleosomes around which cellular DNA is wrapped.

It was known, before the recent research we're discussing, that there were 14,230 sites in the genome where all four of the named master transcription factors could bind simultaneously. Most of those sites were not promoters of some miRNA, but it was straightforward to identify those that were. Of those miRNAs that appeared to be regulated by Oct4, Sox2, Nanog, and Tcf3, it was found that most are in fact preferentially expressed in ESCs. This set of miRNAs would seem to be good candidates for maintaining ESC pluripotency by downregulating other genes.

On the other hand, some of the miRNAs mediated by the transcription factors are silent in ESCs. Subsequent research found that another type of proteins (polycomb proteins) also bind to the miRNA promoters. These proteins were already known to block transcription by binding to gene promoters. But it turns out that some of these silenced miRNAs become active once the ESC loses its pluripotency and begins to differentiate.

The next step will be to figure out what each of these miRNAs regulated by Oct4, Sox2, Nanog, and Tcf3 actually does – either in the ESC or a differentiated cell. That should be very interesting, as the press release suggests:

Putting microRNAs on the stem cell map (8/7/08)

“We now have a list of what microRNAs are important in embryonic stem cells,” says Alex Marson, co-lead author on the paper and an MD/PhD student in the Young lab. “This gives us clues of which microRNAs you might want to target to direct an embryonic stem cell into another type of cell. For example, you might be able to harness a microRNA to help drive an embryonic stem cell to become a neuron, aiding with neurodegenerative disease or spinal cord injury.”

Moreover, the results give scientists a better platform for analyzing microRNA gene expression in cancer and other diseases. “We and others are finding that the overall gene circuitry for embryonic stem cells and cancer cells is very similar,” notes Marson. “Now that we have connected the circuitry to microRNAs, we can begin to compare microRNAs that are regulated in embryonic stem cells to those in cancer cells.”

Here's a somewhat more detailed description of the research: Stem Cell microRNA, Transcription Factor Interplay Uncovered (8/8/08)

Other research on miRNA and ESCs that has appeared since the previous discussion (here) gives a small taste of what may be learned about the miRNAs silenced in ESCs:

Protein Protects Embryonic Stem Cells' Versatility And Self-renewal (3/23/08)

A protein known as REST blocks the expression of a microRNA that prevents embryonic stem cells from reproducing themselves and causes them to differentiate into specific cell types, scientists at The University of Texas M. D. Anderson Cancer Center report in the journal Nature.

Researchers show RE1-silencing transcription factor (REST) plays a dual role in embryonic stem cells, said senior author Sadhan Majumder, Ph.D., professor in M. D. Anderson's Department of Cancer Genetics. "It maintains self-renewal, or the cell's ability to make more and more cells of its own type, and it maintains pluripotency, meaning that the cells have the potential to become any type of cell in the body."

The details are particularly interesting:

In studies using mouse embryonic stem cells, the researchers found that REST disarms a specific microRNA called microRNA-21 or miR-21. MicroRNAs are tiny pieces of RNA that control gene expression by binding to the gene's messenger RNA.

The team found that MiR-21 suppresses embryonic stem cell self-renewal and is associated with a corresponding loss of expression of critical self-renewal regulators, such as Oct4, Nanog, Sox2 and c-Myc. REST counters this by suppressing miR-21 to preserve the cells' self-renewal and pluripotency.

The researchers discovered the roles of REST and miR-21 in a series of experiments using cultured mouse embryonic stem cells in either a self-renewal state or a differentiating state. They found that REST expression was significantly higher in the self-renewal state. Withdrawing REST reduced the stem cells' ability to reproduce themselves and started differentiation -- even when the cells were grown under conditions conducive to self-renewal. Adding REST to differentiating cells maintained their self-renewal.

These experiments also revealed that REST is bound to the gene chromatin of a set of microRNAs with the potential to target self-renewal genes. REST controls transcription of 11 microRNAs.

Is anything special known about miR-21? Yes, in fact – it is known to play a role in cancers of the colon, liver, and thyroid. (See here.)

Tags: microRNA, stem cells, embryonic stem cells

Stem cell news deluge

There's been a flood of news in the stem cell area during the past two weeks. A lot of it is important, and most of it builds on several themes related to stem cells that we've discussed before. In this post I'll just summarize the results and reference a press release. Watch for more details to follow.

Making new neurons from induced pluripotent stem cells

This is the only story here that's been reported more than a week ago (and just barely). Skin cells from an ALS (amyotrophic lateral sclerosis) patient have been reprogrammed into induced pluripotent stem cells, and then pushed towards development into motor neuron cells – containing the same defect that is manifested in ALS. These cells cannot be used to treat the disease. Instead, they will enable much more detailed research into the disease pathology.

Our most recent discussion of induced pluripotent stem cells is here.

First Neurons Created From ALS Patient's Skin Cells (7/31/08)

Harvard and Columbia scientists have for the first time used a new technique to transform an ALS ... patient's skin cells into motor neurons, a process that may be used in the future to create tailor-made cells to treat the debilitating disease.

The research – led by Kevin Eggan, Ph.D. of the Harvard Stem Cell Institute – will be published July 31 in the online version of the journal Science.

This is the first time that skin cells from a chronically-ill patient have been reprogrammed into a stem cell-like state, and then coaxed into the specific cell types that would be needed to understand and treat the disease.

Cell lines for genetic disease research

Just like the work described above, cell lines from 9 additional genetic disease have been derived from induced pluripotent stem cells, which were in turn derived from fully differentiated cells, skin cells in most cases. These cell lines will also be used for research into disease pathology. Most of the diseases in question are considered to result from mutations in more than a single gene, so that understanding the disease necessitates research into how several defective genes interact with each other or with environmental influences.

The better-known of the diseases involved are muscular dystrophy, Parkinson's disease, Huntington's disease, type 1 diabetes, and Down syndrome. There's quite a bit of news hype surrounding this announcement – although it's extremently important from a research standpoint – because the cells cannot be used as a treatment, since they carry the disease's genetic defect.

Twenty Disease-specific Stem Cell Lines Created (8/7/08)

A set of new stem cell lines will make it possible for researchers to explore ten different genetic disorders—including muscular dystrophy, juvenile diabetes, and Parkinson's disease—in a variety of cell and tissue types as they develop in laboratory cultures.

Harvard Stem Cell Institute researcher George Q. Daley, MD, PhD, also associate director of the Stem Cell Program at Children's Hospital Boston, and HSCI colleagues Konrad Hochedlinger and Chad Cowan have produced a robust new collection of disease-specific stem cell lines, all of which were developed using the new induced pluripotent stem cell (iPS) technique.

Improved technique for making induced pluripotent stem cells

You may recall from our discussion of induced pluripotent stem cells (here) that the resulting cells are vulnerable to becoming cancerous (as has been confirmed in experiments on mice), because they depend on overexpression of the gene for the c-Myc transcription factor. The new research shows that it is possible to obtain induced pluripotent stem cells by overexpressing a member of the important Wnt family of signaling proteins. (We've covered Wnt several times, most recentely here.)

Embryonic-like Stem Cells Can Be Created Without Cancer-causing Gene (8/6/08)

A drug-like molecule called Wnt can be substituted for the cancer gene c-Myc, one of four genes added to adult cells to reprogram them to an embryonic-stem-cell-like state, according to Whitehead researchers.

Researchers hope that such embryonic stem-cell-like cells, known as induced pluripotent (IPS) cells, eventually may treat diseases such as Parkinson's disease and diabetes.

Controling embryonic stem cell development

For all the future potential that may come from research into induced pluripotent stem cells, they are sufficiently different from true embryonic stem cells that we can't fully understand the latter by studing the former. In particular, true embryonic stem cells may be easier to push into any differentiated cell type, without the same cancer risk. New research illuminates development of endoderm cells from embryonic stem cells. Specific types of endoderm cells include cells of the lungs and pancreas (among many others).

Our most recent discussion of embryonic stem cells is here.

Scientists uncover the key to controlling how stem cells develop (8/8/08)

The results of a new study involving a McMaster University researcher provide insight into how scientists might control human embryonic stem cell differentiation.

In collaboration with researchers from SickKids and Mount Sinai hospitals, Dr. Jon Draper, a scientist in the McMaster Stem Cell and Cancer Research Institute, focused on producing early endoderm cells from human embryonic stem cells. ...

The researchers focused on generating stable progenitor cells capable of producing all endoderm cell types. The cells were able to maintain their distinct profiles through many stages of cell culture without losing their ability to self renew.

MicroRNA and embryonic stem cells

The last time we discussed microRNA in connection with embryonic stem cells (here) we saw that microRNA controlled genes that enabled the cells' self-renewal and differentiation properties. The latest research takes a much closer look at how microRNA and conventional transcription factors work together to regulate differentiation of embryonic stem cells, and also how they play a role in making induced pluripotent stem cells from differentiated cells.

Putting MicroRNAs On The Stem Cell Map (8/7/08)

Embryonic stem cells are always facing a choice—either to self-renew or begin morphing into another type of cell altogether.

It's a tricky choice, governed by complex gene regulatory circuitry driven by a handful of key regulators known as "master transcription factors," proteins that switch gene expression on or off.

In the past few years, scientists in the lab of Whitehead Member Richard Young and their colleagues have mapped out key parts of this regulatory circuitry, but the genes that produce the tiny snippets of RNA known as microRNAs have until now been a missing piece of the map. Since microRNAs are a second set of regulators that help to instruct stem cells whether to stay in that state, they play key roles in development.

Young and colleagues have now discovered how microRNAs fit into the map of embryonic stem cell circuitry. With this map, the scientists have moved one step closer to understanding how adult cells can be reprogrammed to an embryonic state and then to other types of cells, and to understanding the role of microRNAs in cancer and other diseases.

Tags: stem cells, embryonic stem cells, induced pluripotent stem cells

Cancer stem cells II

Since we've just had a discussion of generalities about cancer stem cells (here), it seems like it would be fun to have a summary of research on CSCs that was presented at the recent meeting of the American Association for Cancer Research in San Diego, or reported since then. So here it is.

As general background, keep in mind that cell surface proteins CD44 and CD24 are considered to be markers of cancer stem cells in various (but not necessarily all) types of cancer. Also, certain cell signaling pathways are thought to be especially important in the activity of cancer stem cells. The list includes Wnt, Sonic hedgehog, Notch, and Bmi1.

Stem Cell-Like Cancer Cells Resistant To Standard Therapy, Responsive To Targeted Therapy (4/29/08)

Previous research had identified a subset of cells in breast tumors that have the ability to form colonies in culture and give rise to tumors in mouse models. Such cells are thought to be cancer stem cells. They express the cell surface glycoprotein CD44, but not CD24, and they appear to be resistant to standard chemotherapeutic agents. However, the drug lapatinib, which inhibits the HER2 pathway, seems to selectively kill these cells.

Getting To The Roots Of Breast Cancer (4/29/08)

This is another report on the research described in the previous item. It provides additional details on the research protocol.

Stem Cell Type Supposed To Be Crucial For Angiogenesis And Cancer Growth Does Not Exist? (4/22/08)

This study casts doubt on the existence of a certain type of bone-marrow derived stem cell that has been suspected of circulating in the blood and acting as a precursor to endothelial cells that make up blood vessel walls. Such cells, if they existed, would be an important target for inhibiting angiogenesis in tumors. The researchers showed, using advanced techniques with mouse models, that endothelial differentiation is not a typical function of bone-marrow derived stem cells.

Ovarian Cancer Stem Cells Identified, Characterized (4/17/08)

Researchers have identified, characterized and cloned ovarian cancer stem cells and have shown that these stem cells may be the source of ovarian cancer's recurrence and its resistance to chemotherapy. They isolated cells from samples of either peritoneal fluid or solid tumors. The cancer stem cells that were identified had traditional cancer stem cell markers including CD44 and MyD88 (which interacts with toll-like receptors to activate NF-κB).
The cells also showed a high capacity for repair and self-renewal. Such cells, when isolated, were capable of forming tumors 100 percent of the time. Within those tumors, 10 percent of the cells were CD44 positive, while 90 percent were CD44 negative, indicating that some cancer stem cells had undergone differentiation.

Stem Cells: The Role Of Cancer-initiating Cells In Diagnosis And Treatment (4/15/08)

This press release describes research presented at the AACR meeting related to stem cells and pancreatic, bladder, ovarian, and breast cancer, and glioma.

Research in pancreatic cancer found that in addition to CD44 and CD24, the enzyme aldehyde dehydrogenase was expressed in a small population of tumor cells. Cells expressing aldehyde dehydrogenase had greater growth capacity than those that didn't, and they were also associated with poorer overall survival.

In a study of breast cancer and glioma, surface markers were not found be sufficient as markers of stem cell activity. However, cells with low proteasome activity did have notably greater capacity for self-renewal and tumor production capacity. (Proteasomes are large protein complexes that degrade unneeded or damaged proteins.)

Researchers studying bladder transitional cell carcinomas found, in 40% of cases, CD44+ cells with other stem cell self-renewal patterns. In these cells, 85% had active Gli1, a part of the Hedgehog pathway, originally discovered in human glioblastoma. A relatively small percentage had active Bmi1, Stat3, or β-catenin (part of the Wnt pathway). None had active Oct4 or Nanog (pluripotent stem cells markers).

The research on ovarian cancer (noted above) involving CD44 and MyD88 markers is referenced again here.

Stem Cell Marker Controls Two Key Cancer Pathways (4/14/08)

Research into breast cancer stem cells has identified, for the first time, another gene that may be involved, Msi1. The investigators showed that Msi1 activated Wnt and Notch signaling. Other studies have shown that Msi1 is a marker of human adult stem cells in general because it has been found in human breast, colon, brain, skin, and other cells. Msi1 was found to affects mammary cells to influence whether they develop into muscle, milk duct linings, etc. Further, Msi1 was found to be expressed in particularly aggressive tumors.

Stem Cells And Cancer: Scientists Investigate A Fine Balancing Act (4/11/08)

This is a report of a general talk about how the mechanisms normally involved in balancing different functions of stem cells may also contribute to cancer. For example, research shows that Bmi1 is important for maintaining stocks of stem cells, and without it stocks of stem cells are depleted. But also Bmi1 is overactive in various cancers including brain tumors.

Secrets of cellular signaling shed light on new cancer stem cell therapies (4/10/08)

Researchers are beginning to study inhibition of signaling pathways that seem to be active in tumors fed by cancer stem cells. In this case, inhibition of the Notch pathway is being investigated as part of a treatment, together with chemotherapy, for metastatic breast cancer. An important question is whether cancer stem cells are sufficiently different from normal adult stem cells so that inhibition of Notch signaling is not harmful. Results of testing in mice indicate that Notch signals are not required for the maintenance of blood-forming stem cells in adult mice.

Stem cells and cancer: cancer pathways that also control the adult stem cell population (4/10/08)

Apc (adenomatosis polyposis coli) is a tumor-suppressing protein that controls β-catenin and hence affects Wnt signaling. When intestinal crypts are damaged and need to be regenerated, Wnt signaling directs stem cells to generate replacement cells. Apc normally turns off Wnt signaling of stem cells when it is no longer needed. The research here showed that if Apc is lost or damaged, Wnt signaling may continue and result in tumor formation

Cancer Stem Cells Created With New Technique (4/9/08)

One of the most important unresolved questions about cancer stem cells is how they originate to begin with. For instance, are they mutations of existing stem cells, or instead precancerous cells that have acquired stem-cell-like capabilities? The research here supports the latter scenario. Starting with normal skin cells, the researchers activated three genes associated with embryonic stem cells. The result closely resembled known cancer stem cells. And they also had more resemblance to normal embryonic stem cells than to normal adult stem cells. One of the genes was Myc, which has also been used to create pluripotent stem cells from skin cells. In addition to the scientific significance of this work, it should also facilitate study of cancer stem cells, which are otherwise hard to locate.

Module Map Links Embryonic Stem Cells And Cancer Stem Cells (4/9/08)

The researchers involved in the work described in the preceding report have additional related findings. They systematically compared gene expression patterns between embryonic stem cells and multiple types of human cancer cells. Gene expression patterns in diverse human epithelial cancers were much like patterns in embronic stem cells. Further, presence of these patterns in cancer cells strongly predicted metastasis and death. On the other hand, normal adult tissue stem cells had an opposite pattern, which was repressed in various human cancers compared to normal tissues. The researchers additionally demonstrated that c-Myc, but not other oncogenes, was sufficient to reactivate the ESC-like program in normal and cancer cells.

And here's some earlier research that features the Nanog and Bmi1 proteins:

To Evade Chemotherapy, Some Cancer Cells Mimic Stem Cells (9/19/07)

Anti-cancer treatments often effectively shrink the size of tumors, but some might have an opposite effect, actually expanding the small population of cancer stem cells believed to drive the disease, according to new findings.

"Our experiments suggest that some treatments could be producing more cancer stem cells that then are capable of metastasizing, because these cells are trying to find a way to survive the therapy," said one of the study's investigators, Vasyl Vasko.

When the researchers applied various anti-cancer drugs to experimental cancer cells, they found that surviving cells expressed more Nanog and Bmi1:

They selected a rare form of cancer, mesenchymal chondrosarcoma (MCS), which has not been well described and for which there is no effective treatment. The researchers first determined that Nanog and BMI1 stem cell markers were more highly expressed in metastatic tumors compared to primary tumors. ...

They then applied various therapies - from VEGF inhibitors such as Avastin to the proteasome inhibitor Velcade - in mice implanted with human MSC, and analyzed the effects on tumors. Some of the treatments seemed to work, because they led to a dramatic decrease in the size of the tumors, Dr. Vasko said. But analysis of stem cell expression before and after treatment revealed that even as some anti-cancer treatments shrank tumors, they increased expression of Nanog and BMI1. "These treatments were not enough to completely inhibit tumor growth, and the cancer stem cell markers were still present," Dr. Vasko said.

Tags: cancer, stem cells, cancer stem cells

Cancer stem cells

Cancer stem cells (CSCs) have been mentioned here before in passing – recently here, for example. But it's now time to direct particular attention to them.

One reason is that a number of new experimental results concerning CSCs were presented last month at the American Association for Cancer Research meeting in San Diego.

Another reason is that CSCs, like other stem cells, happen to utilize a number of the signaling pathways that are very interesting in connection with cancer, embryonic development, and various other cellular processes. The list includes Myc, Nanog, Notch, Sonic hedgehog (Shh), TOR, and Wnt. This is a rapidly developing field of research, and CSCs are right in the thick of things.

There is also some overlap with the recent active work going on with induced pluripotent stem cells (IPSCs). For example, Myc and Nanog turn up in IPSC research.

Finally, CSCs are also controversial (as is to be expected of any field that's in rapid flux) – and controversies are inherently interesting to read about.

One of the controversies concerns whether CSCs really exist and are important to the extent their principal investigators tend to believe. A reason to be skeptical of their importance is that there are some indications that CSCs should not be too rare, and that they should also be largely resistant to standard chemotherapy drugs. Yet in many cancers, chemotherapy can kill at least 99% of tumor cells, which would therefore include most CSCs. But this reasoning, too, is controversial.

But let's go back to first principles and explain just what a CSC is. Even that is tricky, since there are several models hypothesized for CSC behavior. In most general terms, however, a CSC is much like other adult stem cells, in that when a CSC divides, one daughter cell is an essentially similar CSC, while the other daughter is a more ordinary tumor cell, which does not have as much ability to proliferate by repeated division.

This is already somewhat different from the older picture of cancer. In that picture, one "renegade" cell first acquires somehow a mutation of its DNA which defeats one of the numerous cellular safeguards against uncontrolled cell proliferation. As time goes on, descendants of that cell accumulate further mutations that defeat other safeguards. Eventually, a significant population of cells exists which have a similar set of mutations that can evade most of the safeguards, and at that point, a dangerous tumor starts to grow. In effect, most of the cells of the tumor are stem cells, which continually produce more of their kind, without serious inhibition.

But it is gradually became clear that such a naive picture couldn't be generally correct. The main reason is that many cancerous cells stop dividing after awhile, and they are not capable of seeding a new tumor if (for instance) they are introduced into another part of an experimental animal having the tumor, or into a tumor-free animal. Most cells cannot divide an arbitrarily large number of times. There is a limit to the number of cell divisions, called the Hayflick limit. This limit, typically, is about 52 divisions. When the limit is reached, a cell does not necessarily die, but it does enter a new phase of life called the senescence phase, in which it ceases to divide.

The main reason for the Hayflick limit is a structure, called a telomere, found at the ends of all chromosomes. Telomeres consist of repeated short segments of DNA and protect the chromosome from damage during cell division. But a telomere shortens each time a cell divides, and once it becomes too short, the cell becomes senescent. This is because the mechanisms that protect a cell's DNA normally do not permit the cell cycle to function when telomeres are too short. Even if these mechanisms could be evaded, the cell's DNA would be damaged in the division process, and the resulting malfunctions would likely cause the cell to die.

However, stem cells need to be able to divide far beyond the Hayflick limit. Embryonic stem cells need this ability in order for an embryo to grow from a single cell into an organism with trillions of cells. Adult stem cells also need this ability to be able to replace cells in certain tissues that must be frequently renewed, such as in the skin and the lining of the intestines. In order to make this possible, stem cells express an enzyme, called telomerase, which has the specific function of rebuilding shortened telomeres.

Many, but not all, cancer cells also express telomerase. Only those that do are capable of indefinitely dividing. So such cancer cells have the production of telomerase in common with stem cells. That still doesn't mean that they are CSCs, because they may lack other attributes of stem cells. In particular, they may not be able to produce at least one exact copy of themselves when dividing. Instead, both daughter cells may be more differentiated and specialized than the mother cell, and (among other things) lose the ability to produce telomerase. Once that happens, the daughter cells' fate is eventual senescence, at best.

There are several possible ways in which CSCs might arise. One possibility is that all mutations necessary for a growing cancer occur, over a period of time, in a population of actual stem cells. (Once the mutations first occur, they of course are inherited by all descendants.) Another possibility is that only some of the mutations occur in the stem cell phase, while other mutations necessary for cancer occur after the full "stemness" is lost (but while the cells are still capable of repeated division). Perhaps a late stage of cancer development is that cells with enough oncogenic mutations to become cancerous reacquire an ability to divide repeatedly, if that was lost along the way.

In fact, there is evidence that both of these models, and possibly others, actually occur. It all boils down to the time sequence of mutation events in a particular type of cancer, and the sequence is likely to be different in different types of cancer. In any case, one of the main tasks of cancer research now is to figure out not only what mutations occur in a particular type of cancer, but also the order in which they typically occur. The hope is that sufficient knowledge of this process will make it possible to devise ways to stop it.

Here are some other things to read for additional background:

• Stemming the tumorous tide (4/17/08) – article at Economist.com, with recent information from the American Association for Cancer Research meeting in San Diego
  
  
• Scientists Weigh Stem Cells’ Role as Cancer Cause (12/21/07) – New York Times article by Gina Kolata; gives some history and CSC skeptic opinions
  
  
• Stem Cells: The Real Culprits in Cancer? (7/1/06) – detailed Scientific American article, one of whose authors, Michael F. Clarke, has been involved in significant CSC research
  
  
• Stem Cells May Be Key to Cancer (2/21/06) – New York Times article by Nicholas Wade
  

Update, 5/17/08: I've written up some summaries of recent research on cancer stem cells here.

Additional links:

The Cancer Stem Cells Project

Tags: cancer, stem cells, cancer stem cells