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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, Parkinsons 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 cant 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 patients 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 Parkinsons 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. (Well discuss that in a separate article, but heres 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 arent 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

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 well 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. Weve 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 hasnt 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 thats 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

If youve ever taken up running as a form of exercise, or even thought about it, theres a certain paradox that may have occurred to you. The health benefits of aerobic exercise are well-documented. (See here, for example.) In particular such exercise has been shown to reduce risks of cardiovascular disease, diabetes, and some forms of cancer. Beneficial physiological effects include reduction of high blood pressure, better control of blood sugar, and reducing blood levels of low-density lipoprotein while raising levels of high-density lipoprotein.

On the other hand, exercise necessarily increases a persons rate of metabolism, as food is processed to provide energy expended through exercise. An inevitable side-effect of metabolism is the production of reactive oxygen species (ROS) and "free radicals" that can damage DNA and other cellular constituents. This cellular damage can lead to either cancer or accelerated aging due to cell senescence and cell death.

The paradox, then, is that the health benefits of exercise do not seem to be canceled out by the side-effects of higher rates of metabolism. Its an important issue not just for humans who are trying to stay healthy, but even more important in animals like birds that may need to expend energy continuously over significant periods of time.

So whats going on here? Perhaps this research has some of the answer:

The effect of marathon on mRNA expression of anti-apoptotic and pro-apoptotic proteins and sirtuins family in male recreational long-distance runners

Background

A large body of evidence shows that a single bout of strenuous exercise induces oxidative stress in circulating human lymphocytes leading to lipid peroxidation, DNA damage, mitochondrial perturbations, and protein oxidation.

In our research, we investigated the effect of physical load on the extent of apoptosis in primary cells derived from blood samples of sixteen healthy amateur runners after marathon (a.m.).

Results

Blood samples were collected from ten healthy amateur runners peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and bcl-2, bax, heat shock protein (HSP)70, Cu-Zn superoxide dismutase (SOD), Mn-SOD, inducible nitric oxide synthase (i-NOS), SIRT1, SIRT3 and SIRT4 (Sirtuins) RNA levels were determined by Northern Blot analysis. Strenuous physical load significantly increased HSP70, HSP32, Mn-SOD, Cu-Zn SOD, iNOS, GADD45, bcl-2, forkhead box O (FOXO3A) and SIRT1 expression after the marathon, while decreasing bax, SIRT3 and SIRT4 expression (P < 0.0001).

Conclusion

These data suggest that the physiological load imposed in amateur runners during marathon attenuates the extent of apoptosis and may interfere with sirtuin expression.

There are two main findings here, related to apoptosis and sirtuin expression. Lets take them in order.

Apoptosis is a form of programmed cell death that has several purposes. The invocation of a cells apopotosis program isnt necessarily an indication that something is wrong. For example, it occurs normally during embryonic development. Early in the development process embryos of all tetrapods have tissues between what will become the fingers and toes of their hands and feet. But since animals that have left an aquatic environment are usually better off without this extra tissue, evolution has led to signals at a certain stage of embryonic development that cause apoptosis in the cells of the relevant tissue. This is an example of whats known as the "extrinsic" apoptotic pathway.

But for our present purposes theres a second pathway – the "intrinsic" pathway – which is used whenever a cell either detects internal damage (usually to its DNA) or some stressful condition, such as an excessive level of reactive oxygen species. A ROS is a chemically-reactive molecule containing oxygen, including what are sometimes called "free radicals".

This condition of excess ROS is called oxidative stress. It can occur for various reasons, including exposure to high levels of heat or ultraviolet radiation – or abnormally rapid cell metabolism due to vigorous exercise. Cells recognize the condition of oxidative stress indirectly though signaling involving various other molecules that are produced in response to particular ROS molecules. Among such indicators are proteins called heat shock proteins. Two members of this family that were measured in the research under discussion were HSP70 and HSP32.

Signals of oxidative stress trigger the second, "intrinsic" apoptotic pathway, which involves a cells energy-producing organelles, the mitochondria. The main players in the intrinsic pathway are proteins called, generically, "caspases" – short for "cysteine-rich aspartate proteases". Caspases are enzymes that cleave proteins at aspartate units. (Cysteine and aspartate are two of the 21 amino acids that normally make up proteins.)

Caspases are fairly active enzymes, so they dont ordinarily occur at significant concentrations within cells. Instead, they are produced when needed from other protein enzymes called procaspases. One of these, procaspase-9 is found normally within mitochondria, along with another protein, cytochrome c. Most of the time these proteins are confined within the mitochondria. However, under certain conditions some channels in a mitochondrions membrane can open and allow the release of procaspase-9 and cytochrome c. Once these proteins enter the cytosol (cell fluid) outside a mitochondrion, they can team up with another protein (Apaf-1: "apoptotic protease activating factor 1") to convert the procaspase-9 into the caspase known as caspase-9. The latter is an active enzyme that leads to the production of other caspases, with cell apoptosis as the eventual result.

Since a cell does not want to have apoptosis going on normally, the process must be tightly regulated. This is done (partly) by another pair of proteins, Bcl-2 and Bax. These two proteins have structural similarities and are considered to be in the same family, the Bcl-2 family. They are always present in the cytosol, and the relative concentration between Bcl-2 and Bax is what controls whether mitochondrial membrane channels will allow release of procaspase-9 and cytochrome c. If the ratio favors Bcl-2, the channels are essentially closed – the normal case – but if the ratio favors Bax, the channels open... and apoptosis may follow.

The present research measured the levels of certain proteins in 10 individuals before and after a marathon run. (The measurement was done indirectly by measuring levels of mRNA transcripts of the associated genes.) A key finding was that the ratio of Bcl-2 to Bax shifted in favor of Bcl-2 from the before to the after measurement. In other words, there was an anti-apoptotic effect, which countered the pro-apoptotic effects of ROS molecules produced by vigorous exercise. Although ROS levels were not measured (since there was no corresponding mRNA), levels of superoxide dismutase (SOD) antioxidants (Mn-SOD and Cu-Zn-SOD) increased after the marathons, reflecting ROS production.

Analysis of the results indicates that apoptosis actually was inhibited, though less in some experimental subjects than others. An increase in levels of procaspase-9 was not observed. Further, in 7 of the 10 experimental subjects, there was little evidence of DNA fragmentation (a consequence of apoptosis). In the other 3 subjects, there was some evidence of DNA fragmentation, but also smaller changes in the Bcl-2 to Bax ratios.

Most interestingly, there was a significant positive correlation in after marathon measurements between levels of Bcl-2 and both HSP70 and HSP32. This suggests that the expected increases of HSP70 and HSP32 may play some part in increased Bcl-2 levels. There was also a positive correlation post-marathon between HSP70 and Mn-SOD levels.

These findings, especially given the small sample size, certainly arent conclusive. But, as the paper says, "Here, we have found a significant relationship between HSP70 and bcl-2 RNA ... following marathon, but the underlying cellular and molecular mechanisms involved in this [sic] exercise induced adaptations in apoptosis and HSP70 are unknown and require further investigation."

Expression of the sirtuins SIRT1, SIRT3, and SIRT4 pre- and post-marathon were also measured. (Weve discussed the sirtuins on a number of occasions.) Theres an extensive history of research on SIRT1, concerning its connections with such things as cellular metabolism, cell survival under stress, and antioxidant activity. Research on other sirtuins like SIRT3 and SIRT4 is less extensive. However, members of this family have various things in common. All are enzymes. SIRT1 and SIRT3 are histone deacetylases (HDACs), so have epigenetic roles in affecting gene expression. SIRT3 and SIRT4 occur in mitochondria.

Although its possible to make various speculations about how sirtuins could be involved with apoptosis and metabolic consequences of exercise, not all that much is known about specific molecular mechanisms. Nevertheless, its interesting that the present research does show an effect of strenuous exercise on SIRT1, SIRT3, and SIRT4 expression. The paper notes that "the RNA contents of SIRT1 increased substantially in the group after marathon.... On the other hand, the RNA contents of SIRT3 and SIRT4 decreased in the group after marathon."

Further research into these connections could be very interesting.

Marfe, G., Tafani, M., Pucci, B., Di Stefano, C., Indelicato, M., Andreoli, A., Russo, M., Sinibaldi-Salimei, P., & Manzi, V. (2010). The effect of marathon on mRNA expression of anti-apoptotic and pro-apoptotic proteins and sirtuins family in male recreational long-distance runners BMC Physiology, 10 (1) DOI: 10.1186/1472-6793-10-7

Further reading:

Running a marathon halts cellular suicide (5/11/10)


Articles related to sirtuins:

Sirtuin proteins (11/16/07)

The discovery of sirtuins, part 1 (11/17/07)

The discovery of sirtuins, part 2 (11/20/07)

Sirtuin news (1/21/08)

SIRT1 and cancer (10/26/08)