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

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

Induced Pluripotent Stem Cells II

In this article from the April 4 Science, which I mentioned here, several research reports dealing with induced pluripotent stem cells were discussed. One of these I covered in the post I just noted.

Another just as important report apparently has not yet been formally published, but is (at least temporarily) available online since February 14 at Science Express:

Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells

Induced pluripotent stem (iPS) cells have been generated from mouse and human fibroblasts by the retroviral transduction of four transcription factors. However, the cell origins and molecular mechanisms of iPS cell induction remain elusive. This report describes the generation of iPS cells from adult mouse hepatocytes and gastric epithelial cells. These iPS cell clones appear to be equivalent to ES cells in gene expression and are competent to generate germ-line chimeras.

It's not surprising that this is significant research, as it's from the same team of Shinya Yamanaka that was the first to report successful creation of induced pluripotent stem cells. (See here.)

So what is this research about? Well, the investigators used the same four transcription factors (Oct3/4, Sox2, Klf4, and c-Myc) as employed in the majority of previous iPS studies. However, instead of applying the transcription factors to fibroblast cells, they were applied to two types of epithelial cells instead.

Fibroblasts are part of a body's connective tissue. They are involved in structure and support for other tissues and contain large amounts of the protein collagen. They do not divide for the most part, and so it is especially significant that it was possible to reprogram them into a stemcell-like state at all.

Epithelial cells, on the other hand, line the inner and outer surfaces of various body structures, including skin and the gastrointestinal tract. Such cells divide more frequently. They have to, in order to replace other cells of the same kind that are exposed to hostile environments. Epithelial cells also tend to be more adherent to other cells, because they more highly express an adherence protein called E-cadherin.

In some sense, then, epithelial cells are a little more like stem cells to begin with, so one might expect better results when attempting to reprogram them.

This expectation seems to have been met. One of the key differences the researchers found is that reprogrammed epithelial cells had less tendency to form cancerous tumors in mice into which they were included. Certainly not an inconsiderable advantage. This characteristic may be related to the finding that c-Myc seems to play a less essential role in reprogramming epithelial cells.

Specifically, reprogramming of epithelial cells was almost as efficient when c-Myc was not used as when it was included with the other three transcription factors. Yet it was not possible to accomplish reprogramming if any of the other three factors was omitted. In contrast, the efficiency of reprogramming fibroblasts dropped by 90% when c-Myc was omitted.

Another intriguing difference was that reprogrammed epithelial cells contained higher levels of expression of β-catenin than reprogrammed fibroblasts did. (You may recall – see here – that β-catenin is an important part of the Wnt signaling pathway.) In this regard, the reprogrammed epithelial cells are more like true embryonic stem cells than reprogrammed fibroblasts are. It's probably not a coincidence that expression of Nanog is stimulated by β-catenin, (see here), since Nanog is considered important for maintaining stem cell pluripotency.

A further advantage of the use of epithelial cells is that many fewer retroviral "integration sites" were needed to include the transcription factor genes into the cell genome, in comparison with fibroblasts. This is another way the risk of cancer is reduced.

Further reading:

• Stomach and liver cells reprogrammed – excellent 2/28/08 open access overview of the research from Nature Reports Stem Cells
  
  
• Epithelial cells made pluripotent – 2/14/08 blog post at The Scientist giving a good summary of the research findings
  
  
• Epithelial cells made pluripotent – blog post at Wisconsin Stem Cell Now on the preceding article, but noted mainly because it's by the owner of another interesting stem cell blog: Stem Cell Nation
  

Tags: stem cells, induced pluripotent stem cells

Pluripotency and Lin28

As we discussed here, pluripotent stem cells have been obtained by "reporogramming" various kinds of adult cells. In one case, a set of 4 transcription factors – Oct3/4, Sox2, c-Myc, and Klf4 – were used for the reprogramming. Another research team used a slightly different set – Oct3/4, Sox2, Nanog, and Lin28.

Two of the transcription factors are the same in these two sets. Of those that are different, c-Myc and Nanog are very familiar to molecular biologists for a variety of reasons. (We discussed some of what's known about c-Myc here, and Klf4 is discussed here.)

So what, if anything, is special about Lin28? Quite a lot, it turns out. Apparently Lin28 not only promotes pluripotency, but it also interacts with a very well-known type of microRNA called let-7. As we saw here, let-7 does several things that help suppress cancer. For one thing, let-7 regulates the oncogene Ras, apparently by binding to the mRNA encoding Ras, thereby inhibiting protein expression. (See here.) For another thing, and more to the point, let-7 tends to negate some of the "stemness" of stem cells, and pushes them onto a path for differentiation into more specialized cell types. (See here.) This helps inhibit cancer by reducing the ability of suspected cancer stem cells to proliferate. Let-7 has also been mentioned as an inhibitor of oncogenicity of c-Myc.

Lin28, on the other hand, seems to regulate let-7, and therefore it helps preserve "stemness", but at the same time it may raise the risk for development of cancer. A paper in the April 4 issue of Science describes the research that indicates such activity:

Selective Blockade of MicroRNA Processing by Lin28

Here we show that Lin28, a developmentally regulated RNA binding protein, selectively blocks the processing of pri-let-7 miRNAs in embryonic cells. Using in vitro and in vivo studies, we found that Lin28 is necessary and sufficient for blocking Microprocessor-mediated cleavage of pri-let-7 miRNAs. Our results identify Lin28 as a negative regulator of miRNA biogenesis and suggest that Lin28 may play a central role in blocking miRNA-mediated differentiation in stem cells and in certain cancers.

Some people are suggesting that perhaps at least some "cancer stem cells" are actually more ordinary cancer cells that have been reprogrammed (in part by Lin28) to be capable of more stemcell-like behavior. What's really going on here still seems a bit speculative at this point.

This blog post of 3/25/08 goes into a lot of detail on mircoRNA, let-7, Lin28, and the whole ball of wax: bring 'em all together: cancer, stem cells, miRNAs.

Further reading:

Deconstructing Pluripotency – overview article in April 4, 2008 Science that discusses two stem cell papers, including the one cited above

Let7 miRNAs, Lin-28, Cancer and Stem Cells – 3/24/08 blog post that discusses this research

Lin-28 is Master of Let-7 miRNA Processing – 3/25/08 tongue-in-cheek blog post that discusses the previous blog post and the subject more generally

Tid Bits – 3/28/08 blog post on related topics

Tags: stem cells, induced pluripotent stem cells, microRNA, cancer

Embryonic stem cells and Klf4

There's now some additional information on one of the transcription factors written about here, which are able to reprogram adult skin cells into embryonic stem cells. To review, one of the teams responsible for this research used Oct3/4, Sox2, c-Myc, and Klf4 for the reprogramming, while another team used Oct3/4, Sox2, Nanog and Lin28.

Of the transcription factors in the first list, all but Klf4 have been well-studied. So it is of some interest to know more about Klf4, and why it seems to be somewhat less essential than the others.

Some of the interesting details are reported on here: Molecular Alliance That Sustains Embryonic Stem Cell State Identified.

Klf4 is normally active in real embryonic stem cells. To investigate the role Klf4 might be playing in the reprogramming of skin cells, the researchers investigated embryonic stem cells that had been artificially depleted of Klf4. To their surprise, the team found that the cells maintained their pluripotency.

The question then was how to explain this. What was found is that two closely-related transcription factors – Klf2 and Klf5 – took over the role of Klf4:

"Most important, the data showed that the other Klfs were bound to the target sites when one of them was depleted." said Dr. Ng. "These Krüppel-like factors form a very powerful alliance that work together on regulating common targets. The impact of losing one of them is masked by the other two sibling molecules."

This family of transcription factors, called Kruppel-like factors, gets its name from a homology to the Drosophila Krüppel protein. Members of this family have been studied for their roles in cell proliferation, differentiation and survival, especially in the context of cancer.

Interestingly enough, according to the research press release,

Klfs were found to regulate the Nanog gene and other key genes that must be active for ES cells to be pluripotent, or capable of differentiating into virtually any type of cells. Nanog gene is one of the key pluripotency genes in ES cells.

"We suggest that Nanog and other genes are key effectors for the biological functions of the Klfs in ES cells," Dr. Ng said.

"Together, our study provides new insight into how the core Klf circuitry integrates into the Nanog transcriptional network to specify gene expression unique to ES cells.

Nanog, of course, is one of the transcription factors in the set of transcription factors which was found to be an alternative, for reprogramming adult cells, to the set that contained Klf4.

The Nanog protein, too, is known to be critically important in pluripotent stem cells. It is a homeobox transcription factor that appears to play an essential role in self-renewal of undifferentiated embryonic stem cells. It also appears to be connected with cancer, because (according to Wikipedia) "It has been shown that the tumour suppressor p53 binds to the promoter of NANOG and suppresses its expression after DNA damage in mouse embryonic stem cells. p53 can thus induce differentiation of embronic stem cells into other cell types which undergo efficient p53-dependent cell-cycle arrest and apoptosis."

The connection of Klf proteins with cancer is not only through Nanog. According to Wikipedia, "Klf4 also interacts with the p300/CBP transcription co-activators." The closely-related p300 and CBP "interact with numerous transcription factors and act to increase the expression of their target genes." And they too are involved with cancer:

Mutations in the p300 gene have been identified in several other types of cancer. These mutations are somatic, which means they are acquired during a person's lifetime and are present only in certain cells. Somatic mutations in the p300 gene have been found in a small number of solid tumors, including cancers of the colon and rectum, stomach, breast and pancreas. Studies suggest that p300 mutations may also play a role in the development of some prostate cancers, and could help predict whether these tumors will increase in size or spread to other parts of the body. In cancer cells, p300 mutations prevent the gene from producing any functional protein. Without p300, cells cannot effectively restrain growth and division, which can allow cancerous tumors to form.

Another intriguing connection of p300 is that it can be inhibited by the action of the sirtuin deacetylase Sirt1. (See here.)

P300/CBP themselves are targets of intense research activity. Their physical structure has only very recently been determined. (See here, here, here.)

Finally (for now), it's interesting that p300 plays a role in stem cell signaling through one of our favorite signaling pathways – Wnt (see here). According to this report: Stem Cell Signaling Mystery Solved, a small molecule called IQ-1 interferes with Wnt signaling via p300:

What IQ-1 does, Kahn explains, is to block one arm of a cell-signaling pathway called the Wnt pathway, while enhancing the signal coming from the other arm of the Wnt pathway. The Wnt pathway is known to have dichotomous effects on stem cells i.e. both proliferative and differentiative. More specifically, IQ-1 blocks the coactivator p300 from interacting with the protein ß-catenin; this prevents the stem cells from being 'told' to differentiate into a more specific cell type.

Additional reading:

A core Klf circuitry regulates self-renewal of embryonic stem cells – research abstract published online 2/10/08

Molecular Alliance Identified that Sustains Embryonic Stem-Cell State – another summary of the Klf4 study

Tags: stem cells, cancer

Induced pluripotent stem cells

It was one of the top science stories of 2007: number 2 on Science's list – reprogramming ordinary adult body cells (of mice and humans) to act like embryonic stem cells. (See here for summary.) As Science put it,

The riddle of Dolly the Sheep has puzzled biologists for more than a decade: What is it about the oocyte that rejuvenates the nucleus of a differentiated cell, prompting the genome to return to the embryonic state and form a new individual? This year, scientists came closer to solving that riddle. In a series of papers, researchers showed that by adding just a handful of genes to skin cells, they could reprogram those cells to look and act like embryonic stem (ES) cells.

The story really began in October 2006, when a team at Kyoto University in Japan, led by Shinya Yamanaka, announced that they had reprogrammed mouse skin cells into cells that closely resembled embryonic stem cells, based on certain characteristic genes that were expressed. The reprogramming was done by introducing genes for four important stem cell transcription factors (Oct4 (or sometimes the similar Oct3), Sox2, c-Myc, and Klf4) into the skin cells with the help of a genetically engineered retrovirus. (See here for more about Oct4.)

But the team could not at that time demonstrate that these reprogrammed cells would differentiate into a variety of adult cells after having been introduced into a mouse embryo which then developed into an adult mouse. Being able to do this would verify the pluripotency of the reprogrammed cells. (Pluripotency is the ability of a cell to develop into any type of fetal or adult cell. It is characteristic of embryonic stem cells.) The reprogrammed cells are called induced pluripotent stem (iPS) cells.

However, in June 2007 Yamanaka's team, along with two others, reported that they had been able to provide the missing demonstration of pluripotency. The second team that joined in reporting this accomplishment was led by Rudolf Jaenisch at MIT's Whitehead Institute for Biomedical Research. The third team was led jointly by Konrad Hochedlinger of the Harvard Stem Cell Institute and Kathrin Plath of the UCLA Institute for Stem Cell Biology and Medicine. (References: here, here, here, here, here, here, here, here, here.)

The crucial step, of course, was being able to reprogram adult human cells in the same way. For all anyone knew, this might be quite difficult. However, just five months later, in November 2007, Yamanaka's team, together with another led by James Thomson of the University of Wisconsin, announced that this objective had been accomplished. (References: here, here, here, here, here, here, here, here, here, here.)

Yamanaka's team used the same four transcription factors for the human cells as they used for the mouse cells. The cells they reprogrammed were adult human fibroblasts. Thomson's team also used Sox2 and Oct3/4, but instead of c-Myc and Klf4 they used other transcription factors: Nanog and Lin28. The cells they reprogrammed were fetal or newborn fibroblasts.

Very soon thereafter, the Japanese team announced that they could also dispense with c-Myc. That was good, because c-Myc is linked with cancer, as we discussed here. But the downside was that the process was much slower and less efficient. (References: here, here.)

In early December the Jaenisch tean from the Whitehead Institute collaborated with a team led by Tim Townes of the University of Alabama to show that their mouse iPS cells could treat a mouse model of sickle cell anemia. Specifically, they started with skin cells from sickle cell mice and made iPS cells. They also added a corrected hemoglobin gene, and then let the cells differentiate into blood-producing stem cells. When these cells were placed in mice whose defective blood stem cells had been killed, healthy red blood cells were eventually produced, alleviating the symptoms of sickle cell disease. (References: here, here, here, here, here, here, here.)

Of course, it's far too early to attempt such an experiment with humans. All concerns about the possiblity of cancer developing from the iPS cells would need to be cleared away. Then the procedure would need to be tested carefully using a lengthy series of clinical trials.

In late December, a team led by George Daley from Harvard Medical School and Children's Hospital in Boston announced thay they had also been able to convert ordinary human skin cells into embryonic-like stem cells. (See here.) The team had also programmed iPS cells from mesenchymal stem cells (adult stem cells found in bone marrow that can differentiate into fat, bone and cartilage).

Recently (mid-February), Kathrin Plath's team at UCLA has also announced success in reprogramming human skin cells, using the same techniques as previously reported. They have also verified that the induced pluripotent cells are very similar to embryonic stem cells:

Human Skin Cells Reprogrammed Into Embryonic Stem Cells (2/11/08)

The reprogrammed cells were not just functionally identical to embryonic stem cells. They also had identical biological structure, expressed the same genes and could be coaxed into giving rise to the same cell types as human embryonic stem cells.

As we've noted, there have been some potential problems with the work already mentioned. First, any process that activates c-Myc (directly or indirectly) runs risks of promoting cancerous tumors. Second, the processes have used retroviruses to introduce the necessary genetic material into cells to be reprogrammed. This also runs the risk of inducing cancer.

So Konrad Hochedlinger's team has come along with work in mice to reduce or remove these cancer-causing risks:

Discovery Could Help Reprogram Adult Cells To Embryonic Stem Cell-like State (2/15/08)

Harvard Stem Cell Institute (HSCI) and Massachusetts General Hospital (MGH) researchers have taken a major step toward eventually being able to reprogram adult cells to an embryonic stem cell-like state without the use of viruses or cancer-causing genes.

In a paper released online today by the journal Cell Stem Cell, Konrad Hochedlinger and colleagues report that they have discovered how long adult cells need to be exposed to reprogramming factors before they convert to an embryonic-like state, and have “defined the sequence of events that occur during reprogramming.”

This work on adult mouse skin cells should help researchers narrow the field of candidate chemicals and proteins that might be used to safely turn these processes on and off. This is particularly important because at this stage in the study of these induced pluripotent (iPS) cells, researchers are using cancer-causing genes to initiate the process, and are using retroviruses, which can activate cancer genes, to insert the genes into the target cells. As long as the work involves the use of either oncogenes or retroviruses, it would not be possible to use these converted cells in patients.

And hard on their heels, other teams are announcing similar findings:

Stem cell breakthrough may reduce cancer risk (2/27/08)

The main obstacle to using "reprogrammed" human stem cells – the danger that they might turn cancerous – has been solved, claims a US company.

PrimeGen, based in Irvine, California, says that its scientists have converted specialised adult human cells back to a seemingly embryonic state – using methods that are much less likely to trigger cancer than those deployed previously.

The company also claims to be able to produce reprogrammed cells faster and much more efficiently than other scientists.

More: here.

Additional reading:

• Redefining What is Embryonic Stem Cell: Nuclear Reprogramming and Epigenetic Remodeling of Fibroblasts – announcement of a talk on March 3, 2008 by Kathrin Plath, describing her reprogramming work.
  
• Reprogramming Battle: Egg Vs. Virus – an editorial with review of technical information, published online in Stem Cells, November 29, 2007
  
• Field Leaps Forward With New Stem Cell Advances – November 23, 2007 news article in Science about the first reported successes of reprogramming human skin cells into cells that act like embryonic stem cells
  
• Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells – research article in Science that is described in the preceding news article
  
• Teams Reprogram Differentiated Cells--Without Eggs – June 8, 2007 news article in Science about the reprogramming of mouse cells
  
• Reprogramming, Take Two – June 8, 2007 news article in Science about research on another approach to reprogramming, which achieved reprogramming of mouse somatic cells using zygotes whose nuclear DNA has been removed
  

Tags: stem cells, induced pluripotent stem cells

What's special about embryonic stem cells?

Researchers discover key to embryonic stem-cell potential

CAMBRIDGE, Mass. (September 8, 2005) - What exactly makes a stem cell a stem cell? The question may seem simplistic, but while we know a great deal of what stem cells can do, we don't yet understand the molecular processes that afford them such unique attributes.

Now, researchers at Whitehead Institute for Biomedical Research working with human embryonic stem cells have uncovered the process responsible for the single-most tantalizing characteristic of these cells: their ability to become just about any type of cell in the body, a trait known as pluripotency.

When a sperm cell fuses with an egg, the cell that results is called a zygote. The zygote proceeds to divide into additional cells through the normal process of cell division. After a few days, when there are about 40 to 150 cells (of a mammalian embryo), a central fluid-filled cavity develops and the embryo is referred to as a blastocyte.

At first, all of the cells in the developing embryo are (embryonic) stem cells, of a type referred to as totipotent, because they can develop (directly or indirectly) into any other type of cell, including other totipotent stem cells. But by the blastocyte stage, the stem cells have lost the ability to remain totipotent when they divide. Yet they can still become any of the other more than 200 types of possible mammalian cells. Stem cells at this stage are referred to as pluripotent.

Human pluripotent stem cells are assumed to have the greatest therapeutic potential, because they can develop into any type of body tissue, and because they can also be cultured indefinitely as independent cells.

By the time the embryo has developed into an adult, there remain many stem cells -- adult stem cells -- but all have lost their pluripotency, as far as we can tell. Such stem cells can develop into certain limited types of cells -- different kinds of blood cells, for example -- but that's all.

So one important question is: What causes a pluripotent cell in an embryo to lose that property and to head down a path toward a cell that makes up a specific type of tissue? And the other side of the question is just as important: What allows a cell cultured outside an embryo to remain pluripotent?

The answer depends entirely on which of the 25,000 or so genes (in the case of the human genome) are "expressed", that is, capable of directing the production of one or more proteins. Some genes are always expressed, because the proteins they are responsible for are needed for the proper function of any cell. But other genes are not expressed unless their proteins are needed at a given time. Some of the genes of this latter kind determine (among other things) the type of cell one has. A liver cell, for instance, is a liver cell because certain specific genes are expressed.

So it is to be expected that there must be genes which, when expressed, maintain the initial pluripotency of an embryonic stem cell. Prior to the latest research findings, it had been determined that in humans there are (at least) three such genes, which are named (along with their associated proteins) Oct4, Sox2, and Nanog. It was known that all of these proteins are necessary for pluripotency, because if any one of the genes is not expressed, pluripotency is lost. It was also known that the three proteins do not play a direct role in cell function, such as forming part of the internal machinery of the cell. Instead, they were known to be transcription factors, which affect the expression of other genes.

What remained unclear was how these proteins did their job. A transcription factor can either facilitate or inhibit the expression of a gene, and several transcription factors usually work together to do this, by binding to specific locations in the cell's DNA. The finding of the present research is that when the three genes are expressed and all three proteins are produced, then if the proteins bind together at specific places they can inhibit the expression of 353 other genes. And so, when any of these three genes is silenced, 353 other genes can be expressed. All of these genes are responsible for other transcription factors, which in turn affect many more genes in a large cascade effect. Eventually new proteins are produced which lead to the cell's loss of plutipotency and its differentiation into some more specific type of cell.

The interaction of genes in a living cell is a lot like a computer program. Starting from some particular initial state, a program, whether it's in a computer or a cell, takes "input" from outside. Then the program state, influenced by the relevant external inputs, transitions to another state in the next "clock cycle". Ultimately, what is to be determined, is the overall "state diagram" of the genome. That is, what combination of expressed genes and external influences lead to a new set of expressed genes, new transcription factors, and new cell behavior? What has been figured out so far is just the very earliest stage of the puzzle.

This is still just the beginning of learning what's going on. Yet to be determined is what first inhibits one of more of Oct4, Sox2, and Nanog from being expressed. Presumably it's something in the cell's environment. And beyond that, what determines the path that the cell will eventually take, towards becoming a blood cell rather than a neuron, for example?

The new results inevitably raise the question: Is there some way to make a cell dedifferentiate and allow all three genes to become expressed, so that the cell becomes pluripotent again? It can't be very easy at all, because as long as so many of the 25,000 genes in the genome are expressed that are silent in a pluripotent cell, the internal environment is very different. Will the genie go back in the bottle? On the other hand, the process of cloning via "somatic cell nuclear transfer", has precisely this effect of resetting the initial conditions simply by removing the nucleus of a fully differentiated cell (of skin, say) and inserting it into an egg cell whose own nucleus has been removed. Well, it's not quite that easy, but of course such cloning has been done.



References:

• September 9, 2005 - Researchers discover key to human embryonic stem-cell potential
  
• September 11, 2005 - Researchers Discover Key To Human Embryonic Stem-cell Potential
  
• September 17, 2005 - Forever Young: Digging for the roots of stem cells