First Steps in Basic Biological Process That Could be Harnessed to Make Therapeutic Cells

First Steps in Basic Biological Process That Could be Harnessed to Make Therapeutic Cells

Friday, 17 April 2015

Pioneer factor binding DNA on nucleosome is

shown. Credit: Ken Zaret, Ph.D., Perelman

School of Medicine, University of Pennsylvania.

Understanding the molecular signals that guide early cells in the embryo to develop into different types of organs provides insight into how tissues regenerate and repair themselves. By knowing the principles that underlie the intricate steps in this transformation, researchers will be able to make new cells at will for transplantation and tissue repair in such situations as liver or heart disease.

Now, investigators at the Perelman School of Medicine at the University of Pennsylvania are able to explain how cell identity changes occur at the very beginning of the process.

"During my scientific life, I've been fascinated by how early cells make 'decisions' to turn on one genetic program and exclude others," says Kenneth S. Zaret, PhD, director of the Institute for Regenerative Medicine and a professor of Cell and Developmental Biology. Zaret and postdoctoral fellow Abdenour Soufi, PhD, led a team that describes this research, which appeared online this week ahead of print in Cell. Soufi is now at the MRC Centre for Regenerative Medicine, University of Edinburgh.

What they found could be applied to guiding cells to fates proposed by scientists for a wide variety of biomedical contexts, for example, to better understand molecular changes in the early embryo after fertilization, when one cell type morphs into another. Another application could be to directly change one cell type into another for therapeutic purposes, for example transforming a skin cell directly into a liver, blood, or heart cell.

Pioneer factor binding chromosomes is shown.

Credit: Kenneth S. Zaret, Ph.D., Perelman

School of Medicine, University of Pennsylvania.

Tightly Packed

DNA in each cell is two meters long and 20 atoms wide. All of this genetic material needs to be wound into the nucleus in each of the 14 trillion cells in the human body. This is done by coiling DNA around chromosomal proteins to make repeating units of nucleosomes. These units are further compacted into a structure called chromatin, to make the entire DNA fit into the nucleus of the cell. How proteins that regulate gene expression search through the nucleosomes to find their sites of action on DNA has been a mystery.

Nobel Prize winner Shinya Yamanaka from Kyoto University found that turning on four gene regulatory proteins in mouse skin cells can convert these into embryonic-like stem cells called induced pluripotent stem cells, or iPS cells. The special gene regulatory proteins that make iPS cells, called Oct4, Sox2, KIf4, and c-Myc, are normally active in the early embryo and are collectively known as the Yamanaka factors.

Building on this knowledge, the Zaret lab compared the nucleosome and chromatin targeting activities of the Yamanaka factors. To elicit cell programming or reprogramming, the gene regulatory factors must be able to engage genes that are silenced and not meant for expression in the original cell type. These silenced genes are typically embedded in tightly coiled, "closed" chromatin that is covered by nucleosomes. Transcription factors with the highest reprogramming activity have the necessary ability to interact with their target sites on closed nucleosome DNA. These transcription proteins are called "pioneer factors" because they initiate molecular changes in closed chromatin.

"We found that pioneer protein activity relates simply to the ability of a transcription factor to adapt to particular areas of DNA building blocks on the nucleosome surface. This was an 'aha moment' of simplicity," recalls Zaret.

The Wiggle Factor

The DNA-binding domain (DBD) of pioneer factors allows the protein to recognize its target site on a segment of nucleosome DNA, where part of the DNA structure is occluded by proteins associated with chromosomes. The initial targeting of this DNA by pioneers in closed, silent chromatin allows the pioneer factor to initiate expression of silent genes in a given cell, enabling conversion of one cell type to another.

Zaret and Soufi found that the pioneer factors have an adaptable DBD that wiggles in a special fashion. The Yamanaka factors – Oct4, Sox2, and Klf4 all have the wiggle, and thus act as pioneers, while c-Myc is more rigid and is aided by a pioneer. The wiggle allows the pioneer factor to adapt physically to the shape of the DNA molecule that is in a complex with chromosomal proteins.

"We showed that Oct4, Sox2, and Klf4, but not c-Myc, could function as pioneers during reprogramming by virtue of their ability to target 'closed' chromatin sites that are 'naïve' in that they lack chemical modifications that active parts of the DNA might have," explains Zaret.

To check for the generality of the principles they found, the team looked to other studies and found that the same mechanisms applied to other examples where gene regulatory proteins act as pioneers during cellular reprograming, such as in the creation of neurons from skin cells.

"We all want to know how to transform one cell into another," says Zaret.

"Now we understand how that happens at the first step. We are working on how the pioneer gene regulatory proteins physically open up the chromatin to loosen it, in preparation for gene activity. These points are fundamentally important for understanding tissue development, cell regeneration, and for making designer cells."

Source: Penn Medicine

Contact: Karen Kreeger

Reference:

Pioneer Transcription Factors Target Partial DNA Motifs on Nucleosomes to Initiate Reprogramming

Abdenour Soufi, Meilin Fernandez Garcia, Artur Jaroszewicz, Nebiyu Osman, Matteo Pellegrini, Kenneth S. Zaret

Cell, published online: April 16, 2015, DOI: http://dx.doi.org/10.1016/j.cell.2015.03.017

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Tumour Suppressor Also Inhibits Key Property of Stem Cells

Tumour Suppressor Also Inhibits Key Property of Stem Cells

Friday, 14 November 2014

A protein that plays a critical role in preventing the development of many types of human cancers has been shown also to inhibit a vital stem cell property called pluripotency, according to a study by researchers at the Stanford University School of Medicine.

Blocking expression of the protein, called retinoblastoma, in mouse cells allowed the researchers to more easily transform them into what are known as induced pluripotent stem cells, or iPS cells. Pluripotent is a term used to describe a cell that is similar to an embryonic stem cell and can become any tissue in the body.

The study provides a direct and unexpected molecular link between cancer and stem cell science through retinoblastoma, or Rb, one of the best known of a class of proteins called tumour suppressors. Although Rb has long been known to control the rate of cell division, the researchers found that it also directly binds and inhibits the expression of genes involved in pluripotency.

"We were very surprised to see that retinoblastoma directly connects control of the cell cycle with pluripotency," said Julien Sage, PhD, associate professor of paediatrics and of genetics.

"This is a completely new idea as to how retinoblastoma functions. It physically prevents the reacquisition of stem cell-ness and pluripotency by inhibiting gene expression."

"The loss of Rb appears to directly change a cell's identity. Without the protein, the cell is much more developmentally fluid and is easier to reprogram into an iPS cell," said Marius Wernig, MD, associate professor of pathology.

Wernig and Sage, both members of the Stanford Cancer Institute, share senior authorship of the study, which will be published online Nov. 13 in Cell Stem Cell. Postdoctoral scholar Michael Kareta, PhD, is the lead author.

Tumour Suppressor

Pluripotent stem cells are able to become any tissue in the body. In 2006, researchers in Shinya Yamanaka's laboratory in Kyoto University found that it's possible to push a fully specialized adult cell, such as a skin cell, backward along the developmental pathway to assume a pluripotent state. They did so by adding four proteins – Sox2, Oct4, c-Myc and Klf4 – that are normally found in cells only very early in embryonic development. The resulting cells were called induced pluripotent stem cells.

Rb was first identified as a tumour suppressor because of its role in a rare but rapidly developing childhood cancer of the retina. It has since been shown to be missing or functionally inactive in nearly all human cancers. Intact Rb prevents cancer by acting as a natural brake on the cell cycle, the process by which cells divide to make daughter cells. Loss of Rb allows a cell to divide more quickly and potentially accumulate more cancer-causing mutations. However, the new research shows that Rb's effect on pluripotency is independent of its role in cell cycle control.

Cancerous cells often appear less mature than their noncancerous peers. They persist in dividing in the face of external cues that curb the proliferation of normal cells, and they often seem to regress developmentally, assuming the form and mimicking the behaviour of their more developmentally flexible ancestors. A similar cascade of events occurs when researchers create iPS cells from specialized adult cells.

"The process of creating iPS cells from fully differentiated, or specialized, cells is in many ways very similar to what happens when a cell becomes cancerous," said Sage, who holds the Harriet and Mary Zelencik Endowed Professorship in Pediatrics.

"We wondered if we could learn more about both processes by investigating whether the loss of Rb affects reprogramming efficiency."

Previous studies in other laboratories have suggested that Rb may also be involved in promoting cellular differentiation – a cell's developmental progression toward a more specialized state.

Link between Rb and Pluripotency

The researchers found that embryonic mouse cells unable to express functional Rb were much more efficiently and quickly converted to iPS cells than were cells in which Rb was present. Conversely, cells with higher-than-normal levels of the Rb protein were more difficult to reprogram into iPS cells. When the researchers compared the rate of division of the control cells with those in which Rb expression was lost, they found no significant differences.

"It didn't change the cell proliferation rates at all," said Wernig.

"This indicated that Rb's mechanism of action on reprogramming was something completely different than what we had expected."

Further investigation showed that Rb directly binds to many genes involved in the acquisition of pluripotency, including those encoding two of the proteins often used by researchers to create iPS cells: Sox2 and Oct4. Loss of Rb increased the expression of the proteins, thereby affecting a large "pluripotency network."

"We saw a global effect on a network of genes involved in pluripotency," said Sage.

The net effect, according to the researchers, is an overall reduction in the natural barrier that exists to prevent specialized adult cells from dedifferentiating – that is, spontaneously becoming pluripotent, an occurrence that could easily wreak havoc on a multicellular organism that depends on an orderly arrangement of tissues.

The researchers also showed that Rb's effect on the pluripotency network is an important driver of cancer in a mouse model. Animals in which Rb expression is blocked typically develop pituitary tumours within a few months. However, the researchers found the cancers didn't occur when Sox2 was also removed.

"It's clear that Sox2 expression is also required for the development of cancers in the animals," said Wernig.

"This implies that Rb's effect on Sox2 expression is critical for cancer development."

The researchers plan to continue their investigations into the relationship between Rb and pluripotency. In particular, Wernig is interested in learning whether Rb expression plays a role in a phenomenon he discovered called direct conversion, in which one cell type, such as a skin cell, can be directly converted into another, such as a neuron, without first entering a pluripotent state.

Source: Stanford University Medical Center

Contact: Krista Conger

Reference:

Inhibition of Pluripotency Networks by the Rb Tumor Suppressor Restricts Reprogramming and Tumorigenesis

Michael S. Kareta, Laura L. Gorges, Sana Hafeez, Bérénice A. Benayoun, Samuele Marro, Anne-Flore Zmoos, Matthew J. Cecchini, Damek Spacek, Luis F.Z. Batista, Megan O’Brien, Yi-Han Ng, Cheen Euong Ang, Dedeepya Vaka, Steven E. Artandi, Frederick A. Dick, Anne Brunet, Julien Sage, Marius Wernig

Cell Stem Cell, November 13, 2014, DOI: http://dx.doi.org/10.1016/j.stem.2014.10.019

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New Efficiency to Stem Cell Reprogramming

Biologists reveal genes key to development of pluripotency, in single cells

Friday, 14 September 2012

Several years ago, biologists discovered that regular body cells can be reprogrammed into pluripotent stem cells — cells with the ability to become any other type of cell. Such cells hold great promise for treating many human diseases.

These induced pluripotent stem cells (iPSCs) are usually created by genetically modifying cells to overexpress four genes that make them revert to an immature, embryonic state. However, the procedure works in only a small percentage of cells.

Now, new genetic markers identified by researchers at Whitehead Institute and MIT could help make that process more efficient, allowing scientists to predict which treated cells will successfully become pluripotent.

The new paper, published in the Sept. 13 online edition of Cell, also identifies new combinations of reprogramming factors that produce iPSCs, according to the researchers.

Led by Rudolf Jaenisch, a Whitehead Founding Member and an MIT professor of biology, the study is the first to examine genetic changes that occur in individual cells as they become pluripotent. Previous studies have only looked at gene-expression changes in large populations of cells — not all of which will actually reprogram — making it harder to pick out genes involved in the process.

"In previous studies, you weren't able to detect the few cells that expressed predictive pluripotency markers. The really cool part of this study is that you can detect two or three cells that express these important genes early, which has never been done before," says Dina Faddah, a graduate student in Jaenisch's lab and one of the paper's lead authors.

The other lead author is Yosef Buganim, a postdoc at Whitehead Institute.

Single-cell analysis

In 2007, scientists discovered that adult human cells could be reprogrammed by overexpressing four genes — Oct4, Sox2, c-Myc and Klf4. However, in a population of cells in which those genes are overexpressed, only about 0.1 to 1 percent will become pluripotent.

In this image of mouse embryonic fibroblasts

undergoing reprogramming, each coloured dot

represents messenger RNA associated with a

specific gene that is active in cells being

reprogrammed. Red dots represent mRNA for

the gene Sall4, green is Sox2, and blue is

Fbxo15. The researchers determined that Sox2

activates Sall4 and then activates the

downstream gene Fbxo15, creating a gene

hierarchy in the later phase of reprogramming.

Credit: Dina Faddah/Whitehead Institute. 

In the new study, Jaenisch's team reprogrammed mouse embryonic fibroblast cells and then measured their expression of 48 genes known or suspected to be involved in pluripotency at several points during the process. This allowed them to compare gene-expression profiles in cells that became pluripotent, those that did not, and those that were only partially reprogrammed.

Once the reprogramming, which took between 32 and 94 days, was complete, the researchers looked for genes expressed only in the cells that ended up becoming pluripotent.

The team identified four genes that were turned on very early — around six days after the reprogramming genes were delivered — in cells that ended up becoming pluripotent: Esrrb, Utf1, Lin28 and Dppa2, which control the transcription of other genes involved in pluripotency.

The researchers also found that several previously proposed markers for pluripotency were active in cells that became only partially programmed, suggesting those markers would not be useful. With their newly discovered markers, "you can eliminate all the colonies that are not completely reprogrammed," Buganim says.

"You don't want to use partially reprogrammed iPSCs for patient-specific therapies."

To read cells' genetic profiles so precisely, the researchers screened for genes using a microfluidic system called Fluidigm, then confirmed their results with a fluorescence imaging technique that can detect single strands of messenger RNA.

Not totally random

The findings also allowed the researchers to develop a new model for how genes interact with each other to steer cells toward pluripotency. Previously, it had been thought that reprogramming was a random process — that is, once the four reprogramming genes were overexpressed, it was a matter of chance whether they would activate the correct genes to make a particular cell pluripotent.

However, the new study reveals that only the earliest phase of the process is random. Once those chance events awaken the cell's own dormant copy of the Sox2 gene, that gene launches a deterministic pathway that leads to pluripotency.

During the early, random stage, there are probably many ways that Sox2 can be activated, Buganim says.

"Different cells will activate Sox2 in different ways," he says.

"As soon as you have a specific combination that allows the activation of Sox2, you are on the way toward full reprogramming."

The new model also predicted six combinations of factors that could activate Sox2. The researchers tested these combinations in reprogrammed cells and found that they were successful, with varying rates of efficiency.

Interestingly, they found combinations that do not include any of the original reprogramming factors. The researchers are now testing their new combinations to see if they produce healthier iPSCs. The most stringent test involves injecting iPSCs into an embryo that cannot give rise to normal cells because it has four sets of chromosomes instead of two. If a healthy animal develops from those cells, it is entirely the product of the iPSCs, demonstrating that the iPSCs were equivalent to embryonic stem cells. Most iPSCs injected into embryos do not pass this test.

Source: Whitehead Institute for Biomedical Research, written by Anne Trafton, MIT News.

Contact: Nicole Rura

Reference:

Single-cell gene expression analyses of cellular reprogramming reveal a stochastic early and hierarchic late phase

Yosef Buganim, Dina A. Faddah, Albert W. Cheng, Elena Itskovich, Styliani Markoulaki, Kibibi Ganz, Sandy L. Klemm, Alexander van Oudenaarden, and Rudolf Jaenisch

Cell, September 14, 2012, in print

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Cancer-causing Gene Crucial in Stem Cell Development

Cancer-causing Gene Crucial in Stem Cell Development
Friday, 03 September 2010

Stem cells might be thought of as trunks in the tree of life. All multi-cellular organisms have them, and they can turn into a dazzling variety other cells — kidney, brain, heart or skin, for example. One class, pluripotent stem cells, has the capacity to turn into virtually any cell type in the body, making them a focal point in the development of cell therapies, the conquering of age-old diseases or even re-growing defective body parts.

Now, a research team at the University of Georgia has shown for the first time that a gene called Myc may be far more important in the development and persistence of stem cells than was known before. Myc is traditionally thought of as a cancer-causing gene, or oncogene, but recent studies from the UGA team have established critical roles for it in stem cell biology. The discovery has important implications for the basic understanding of developmental processes and how stem cells can be used for therapeutic purposes.

"This new research has uncovered a really unexpected role for Myc," said Stephen Dalton, GRA Eminent Scholar of Molecular Cell Biology and Georgia Cancer Coalition Distinguished Scientist at UGA.

"Our work here represents the first mechanistic characterization of how Myc controls the pluripotent stem cell state."

The research was published today in the journal Cell Stem Cell. Other authors of the paper include Keriayn Smith and Amar Singh of the Dalton lab at UGA. Smith left recently to begin a postdoc at the University of North Carolina. Dalton also is a member of the department of biochemistry and molecular biology in the Franklin College of Arts and Sciences and is affiliated with the UGA Cancer Center and the Biomedical and Health Sciences Institute.

In previous work, Dalton and his colleagues showed that Myc is critical for stem cell maintenance and that it affects widespread changes in gene expression. This latter function is crucial when stem cells differentiate into more specific cell types. In the new research, Dalton's team showed that Myc sustains the important pluripotency process by repressing a "master regulator" gene called GATA6.

"Pluripotency is the inherent property of a cell to create all cell types, from an embryo to an adult organism," said Dalton.

"It's an extremely important biological process, and knowing how it is controlled is crucial not only from a basic developmental perspective but also so that we can harness the potential of stem cells for the development of therapies, including those for diabetes, cardiovascular disease and a range of neurological disorders. Through a detailed understanding of early development, we hope to apply this information so that pluripotent stem cells can be differentiated into therapeutically useful cell types."

"These cells can then be used in a clinical setting to cure degenerative diseases and treat acute injury."

The finding that Myc inhibits GATA6 came as a big surprise to the Dalton team and points out that researchers have only seen the tip of the "molecular iceberg" in terms of what Myc does in stem cells. It now seems likely that understanding Myc's role in further detail will reshape current ideas about the basic biology of stem cells.

Dalton's new work addressed the uncertainty about how Myc maintains the pluripotency of stem cells by examining what happens when two forms of Myc — c-Myc and N-Myc — are inactivated in pluripotent stem cells. What he found was that either c- or N-Myc is sufficient to maintain pluripotency, but that the absence of both triggers the differentiation of pluripotent stem cells. Myc is therefore acting as a "brake" to restrain differentiation. When the "differentiation brake" is removed, cells lose their stem cell properties, and, potentially, they can become any one of over a hundred different cell types.

Pluripotent stem cells can now be made from skin fibroblasts and even from blood samples. (Fibroblasts are cells common in connective tissues of animals and play an important role in the healing of wounds, among many functions.) The conversion of mature fibroblast or blood cells back to pluripotent stem cells is called "reprogramming." Myc also has a critical role in this process. The ability to make stem cells from a patient's blood or skin is going to revolutionize medicine as it opens the way for patient-specific stem cells that would circumvent problems associated with immune rejection, said Dalton.

"During the reprogramming of cells, Myc represses genes associated with the differentiated state and primes them for the expression of stem cell genes," he said.

"We now speculate that during the early reprogramming stage, Myc serves to change the cell cycle so that stem cells can divide for long periods of time without aging. This is also what Myc does in cancer cells."

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

"This is clearly going to be a major area of research for many years to come," Dalton said.

Source: University of Georgia
Contact: Stephen Dalton

Reference:
Myc Represses Primitive Endoderm Differentiation in Pluripotent Stem Cells
Keriayn N. Smith, Amar M. Singh, Stephen Dalton
Cell Stem Cell, Volume 7, Issue 3, 343-354, 3 September 2010, 10.1016/j.stem.2010.06.023
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Our Genes Can be Set on Pause

Embryonic stem cells reveal oncogenes secret growth formula Friday, 30 April 2010

A comprehensive new gene expression study in embryonic stem cells has uncovered a transcription control mechanism that is not only more pervasive than once thought but is also heavily regulated by the cancer-causing gene c-Myc.

In research published in the April 30th edition of Cell, a team of Whitehead Institute for Biomedical Research researchers describes a pausing step in the transcription process that serves to regulate expression of as many as 80% of the genes in mammalian cells.

Scientists have long known that DNA-binding transcription factors recruit the RNA polymerase Pol II (which prompts copying of DNA into mRNA protein codes) to promoters in order to kick off the transcription process. Now researchers in the lab of Whitehead Member Richard Young have found that additional factors recruited to the promoters serve to stop transcription in its tracks shortly after it has begun.

"It's like the engine's running, but the transmission is not engaged on that transcription apparatus," says Young, who is also a professor of biology at MIT.

"You need something to engage that transmission."

It turns out that for a surprisingly large number of genes in embryonic stem cells, that "something" is the transcription factor c-Myc. This so-called pause release role for c-Myc is significant, as many of c-Myc's targets are genes in highly proliferative cells. Over-expression of c-Myc is a hallmark of a number of tumours, and it now appears that c-Myc's ability to release transcriptional pausing is linked with the hyper-proliferation that is characteristic of cancer cells.

"Our findings provide the molecular basis for loss of proliferation control in some cancers," says Peter Rahl, a postdoctoral researcher in Young's lab and first author of the Cell paper.

Armed with this new understanding of c-Myc's role in controlling proliferation genes, Young and his colleagues have embarked on a search for drugs that could interrupt c-Myc's pause-release activity in tumours where it is over-expressed.

"Clearly, cancer cells are able to exploit mechanisms that normally operate in embryonic stem cells, so I expect further understanding of embryonic stem cell control mechanisms will give us additional insights into human disease mechanisms." says Young.

Reference:
c-Myc regulates transcriptional pause release
Peter B. Rahl, Charles Y. Lin, Amy C. Seila, Ryan A. Flynn, Scott McCuine, Christopher B. Burge, Phillip A. Sharp, Richard A. Young
Cell, Volume 141, Issue 3, 432-445, 30 April 2010, 10.1016/j.cell.2010.03.030
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For more on stem cells and cloning, go to CellNEWS at
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Amniotic Fluid Cells More Efficiently Reprogrammed to Pluripotency than Adult Cells

Amniotic Fluid Cells More Efficiently Reprogrammed to Pluripotency than Adult Cells Tuesday, 16 March 2010 Researchers at Mount Sinai School of Medicine have demonstrated that skin cells found in human amniotic fluid can be efficiently "reprogrammed" to pluripotency, where they have characteristics similar to human embryonic stem cells that can develop into almost any type of cell in the human body. The study is online now and will appear in print in the next issue of the journal Cellular Reprogramming, to be published next month. The Mount Sinai researchers found that when compared to cultured adult skin cells, the amniotic fluid skin cells formed stem cell colonies in about half the time and yielded nearly a 200 percent increase in number. Reprogramming foetal skin cells also cuts significantly the cost of generating patient-specific induced pluripotent stem cells when compared to reprogramming other cell types. "There remains today a need in stem cell research for an easily reprogrammable cell type," said the study's lead author, Dr. Katalin Polgar, Assistant Professor of Medicine, Cardiology and Obstetrics, Gynecology and Reproductive Science, Mount Sinai School of Medicine. "Our study shows that reprogramming of cultured, terminally differentiated amniotic fluid cells results in pluripotent stem cells that are identical to human embryonic stem cells, and that it is much easier, faster and more efficient than reprogramming neonatal and adult cells." Amniotic fluid skin cells can be safely obtained from pregnant women undergoing amniocentesis at about 15 weeks of pregnancy as part of a diagnostic workup for chromosome aberrations and other genetic diseases. About 99 percent of cells found in amniotic fluid are terminally differentiated cells mostly from foetal skin, which are shed into the amniotic fluid as a foetus develops. Since these cells can be reprogrammed to pluripotency more efficiently than other cell types, they could be an important source for generating stem cells for basic research and future therapies and may be used to study and potentially cure fatal embryonic diseases with prenatal, perinatal gene therapy. "We induced amniotic fluid skin cells to return from their final differentiated stage back to an undifferentiated stem cell stage from where they can develop into any cell type of the body," said Dr. Polgar. Amniotic fluid cells work much better than any other cell types when turning back their 'internal clock.' These cells can potentially be used as a model system in studying different regenerative therapies for diseases of the heart, liver, kidney, lung, pancreas, as well as for replacement of lost neurons in Alzheimer's, Parkinson's, even for cancer vaccines. They may also be used for future personalized stem cell banks. As the pluripotent stem cells induced from amniotic fluid skin cells are the patient's own cells, there is no risk of immuno-rejection or teratocarcinoma formation. "Additionally, stem cells reprogrammed from amniotic fluid skin cells could be used for drug discovery in disease models," added Dr. Polgar. "Their potential use in toxicology models could reduce the need for experimental animals. Developing cell lines from individual amniotic fluid samples can accelerate the development of existing targets for different diseases. This all will bring new opportunities to explore innovative therapeutic models or targets in regenerative personalized medicine." The scientists were able to genetically reprogram the amniotic fluid skin cells using the four transcription factors (proteins that regulate the transcription of genes) Oct3/4, Sox2, Klf4, and c-Myc. After reprogramming, the cells were found to be identical to human embryonic stem cells in numerous ways, including for morphological and growth characteristics, antigenic stem cell markers, stem cell gene expression, and telomerase activity, in vitro and in vivo differentiation. "These reprogrammed amniotic fluid cells are able to form, as embryonic stem cells can, three dimensional spheroid structures called 'embryoid bodies.' They also have the ability to self-renew themselves indefinitely. Pluripotent stem cells created from amniotic fluid cells shed from the foetal skin maintain all the potential of embryonic stem cells without using embryos, thereby eliminating ethical concerns associated with human embryonic stem cells obtained from preimplantation embryos," Dr. Polgar said. ......... ZenMaster

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p53 Immortality Improves Cell Reprogramming

Tumour Suppressor Pulls Double Shift as Reprogramming Watchdog Monday, 10 August 2009 A collaborative study by researchers at the Salk Institute for Biological Studies uncovered that the tumour suppressor p53, which made its name as “guardian of the genome,” not only stops cells that could become cancerous in their tracks but also controls somatic cell reprogramming. Although scientists have learned how to reprogram adult human cells such as skin cells into so-called induced pluripotent stem cells (iPSCs), the reprogramming efficiency is still woefully low. The Salk study, published in the Aug. 9 advance online edition of Nature, gives new insight into why only a few cells out of many can be persuaded to turn back the clock. “Although we have been able to reprogram specialized cells for a while now, there had been nothing known about the control mechanisms that prevent it from happening spontaneously in the body and why it has been so hard to change their fate in a Petri dish,” says Juan-Carlos Izpisúa Belmonte, Ph.D., a professors in the Gene Expression Laboratory, who worked closely with Geoffrey M. Wahl, Ph.D., also a professor in the Gene Expression Laboratory. Their findings bring iPSCs technology a step closer to fulfilling its promise as source of patient-specific stem cells but also force scientists to rethink the development of cancer. “There’s been a decade-old idea that cancer arises through the de-differentiation of fully committed and specialized cells but eventually it was discarded in favour of the currently fashionable cancer stem cell theory,” says Wahl. “Now, that we know that p53 prevents de-differentiation, I believe it is time to reconsider the possibility that reprogramming plays a role in the development of cancer since virtually all cancer cells lose p53 function in one way or another.” As mammalian embryos transition through a series of developmental stages, the choices of embryonic stem cells, which enjoy almost limitless prospects, are progressively limited till they eventually give rise to the roughly 200 cell types that make up our body and generally lack the ability to revert back to a less specialized stage. Although differentiation is generally irreversible, scientists have developed several methods to overcome the cells’ reluctance to be reprogrammed. The most widely used technology involves the forced expression of four transcription factors — Oct4, Sox2, Klf4, and c-Myc — in fully committed adult cells. “Unfortunately, Klf4 and c-Myc are oncogenes and adding them carries the risk of inducing cancer,” says Belmonte. Yet, despite the extra push provided by those powerful oncogenes, only a tiny fraction transmogrifies into iPSCs that look and act like embryonic stem cells, leading Belmonte to question whether what they were doing to get the cells to reprogram induced a response that stopped the cells from growing? A conversation with his next-door neighbour, cancer expert Wahl provided some fresh ideas that could be tested in the lab. “Normally, cells don’t reprogram so there must be a mechanism in place that prevents it,” says Wahl. “We knew that c-Myc and some of the other genes that are required for reprogramming activate the tumour suppressor p53 and we wondered whether it had any part in it.” And sure enough, experiments by postdoctoral researchers and co-first authors Teruhisa Kawamura, Ph.D., and Jotaro Suzuki, Ph.D., revealed that adding the reprogramming factors c-Myc and Klf4, alone or in various combinations activated the p53 pathway. As a first-responder, the tumour suppressor p53 is called to action when cells experience stressful conditions. Depending on the situation, p53 then turns on genes that halt cell division to allow time for repairs or, when all rescue attempts prove futile, order the cell to stop dividing forever or to commit suicide. In cells genetically engineered to lack p53, reprogramming efficiency was at least 10-fold increased compared to control cells, demonstrating that p53 clearly played an important role in reigning in cells trying to revert back into a stem-like state. Because iPSCs generated with the full complement of reprogramming factors run the risk to turn malignant, Belmonte and his team wanted to know whether mouse cells lacking p53 could be reprogrammed using only two factors, Oct4 and Sox2. The cells readily converted into iPSCs and gave rise to healthy, full term mice that were able to reproduce passing the ultimate test for pluripotent embryonic stem cells. “This very successful collaboration is a prime example of what makes the Salk such a special place,” says Wahl. “Juan Carlos and I talk every day and we approach the same question from very different perspectives. He comes from a developmental biology perspective, while I come from the cancer side but when put together they can make for a great story.” About the Salk Institute for Biological Studies The Salk Institute for Biological Studies is one of the world's preeminent basic research institutions, where internationally renowned faculty probe fundamental life science questions in a unique, collaborative, and creative environment. Focused both on discovery and on mentoring future generations of researchers, Salk scientists make groundbreaking contributions to our understanding of cancer, aging, Alzheimer's, diabetes, and cardiovascular disorders by studying neuroscience, genetics, cell and plant biology, and related disciplines. Faculty achievements have been recognized with numerous honours, including Nobel Prizes and memberships in the National Academy of Sciences. Founded in 1960 by polio vaccine pioneer Jonas Salk, M.D., the Institute is an independent non-profit organization and architectural landmark. Comment: Now, five research teams, including Shinya Yamanaka's, have boosted their success rates by around a 100-fold by silencing the p53 pathway, which prevents mutations and preserves the sequence of the genome (see references below). Reference: Suppression of induced pluripotent stem cell generation by the p53–p21 pathway Hyenjong Hong, Kazutoshi Takahashi, Tomoko Ichisaka, Takashi Aoi, Osami Kanagawa, Masato Nakagawa, Keisuke Okita & Shinya Yamanaka Nature advance online publication 9 August 2009, doi:10.1038/nature08235 Immortalization eliminates a roadblock during cellular reprogramming into iPS cells Jochen Utikal, Jose M. Polo, Matthias Stadtfeld, Nimet Maherali, Warakorn Kulalert, Ryan M. Walsh, Adam Khalil, James G. Rheinwald & Konrad Hochedlinger Nature advance online publication 9 August 2009, doi:10.1038/nature08285 A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity Rosa M. Marión, Katerina Strati, Han Li, Matilde Murga, Raquel Blanco, Sagrario Ortega, Oscar Fernandez-Capetillo, Manuel Serrano & Maria A. Blasco Nature advance online publication 9 August 2009, doi:10.1038/nature08287 The Ink4/Arf locus is a barrier for iPS cell reprogramming Han Li, Manuel Collado, Aranzazu Villasante, Katerina Strati, Sagrario Ortega, Marta Cañamero, Maria A. Blasco & Manuel Serrano Nature advance online publication 9 August 2009, doi:10.1038/nature08290 Linking the p53 tumour suppressor pathway to somatic cell reprogramming Teruhisa Kawamura, Jotaro Suzuki, Yunyuan V. Wang, Sergio Menendez, Laura Batlle Morera, Angel Raya, Geoffrey M. Wahl & Juan Carlos Izpisúa Belmonte Nature advance online publication 9 August 2009, doi:10.1038/nature08311 See also: Immortality improves cell reprogramming Nature News Published online 9 August 2009, doi:10.1038/news.2009.809 ......... ZenMaster

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Scripps Team Also Make Mice from iPS Skin Cells

Scripps Team Also Make Mice from iPS Skin Cells Monday, 03 August 2009 A team at The Scripps Research Institute in La Jolla says it bred live mice from mouse skin cells, advancing a technique that could offer an alternative to the controversial use of embryonic stem cells. The work, reported online yesterday by the journal Nature, involves reprogramming normal cells to create what are known as induced pluripotent stem cells. In recent years, scientists have been studying these cells to see if they might be as useful as embryonic stem cells, the master cells that differentiate into more than 200 cell types in the body. The Scripps team, led by Assistant Professor Kristin Baldwin, is not the first to achieve the feat. Two teams of Chinese researchers, who published their findings online in Cell Stem Cell and Nature, reported success in similar experiments two weeks ago, creating mice that were as much as 95 percent genetically matched to the adult mouse whose cells were used. The first part of the team's new study involved gathering cells that were already "differentiated," i.e. developed into a particular cell type, such as skin, nerve, or muscle. In this case, the scientists worked with skin cells from foetal mice, though other cell types may also work. Viruses were then used to insert genes coding for four proteins, called reprogramming factors, into these cells' DNA. These reprogramming factors shifted the cells out of their normal differentiated state to a "pluripotent" state resembling that of embryonic stem cells, which allows the cells to produce a wide variety of cell types. This cellular rewiring caused the cells to change their size and shape so that after only 7 to 10 days they could not be visually distinguished from embryonic stem cells. The ultimate test of this developmental pluripotency was to generate live mice entirely from iPS cells. This week, the Baldwin team and two Chinese groups all reported that they had independently grown live mice from iPS cells all the way to fertile adulthood. The team’s best cell line produced live pups 13 percent of the time, compared with a 3.5 percent and 1 percent success rate reported by the Chinese teams. The Scripps team said it generated live mice in four of 15 cell lines generated in one experiment, compared with success rates of three in 37 and one in five for the Chinese teams. “We can’t say for sure yet, but it is possible that we may have identified a protocol more likely to produce mice that survive until birth than current methods in the field,” Baldwin said. Reference: Adult mice generated from induced pluripotent stem cells Michael J. Boland, Jennifer L. Hazen, Kristopher L. Nazor, Alberto R. Rodriguez, Wesley Gifford, Greg Martin, Sergey Kupriyanov & Kristin K. Baldwin Nature advance online publication 2 August 2009, doi:10.1038/nature08310 See also: Second Chinese Group Produce Mice from iPS Cells CellNEWS - Thursday, 23 July 2009 Mice Grown from iPS Cells by Chinese Researchers CellNEWS - Thursday, 23 July 2009 ......... ZenMaster

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Second Chinese Group Produce Mice from iPS Cells

Second Chinese Group Produce Mice from iPS Cells Thursday, 23 July 2009 Two teams of Chinese researchers have created live mice from induced pluripotent stem (iPS) cells, answering a lingering question about the developmental potential of the cells. Animal cloners Qi Zhou of the Institute of Zoology in Beijing and Fanyi Zeng of Shanghai Jiao Tong University started by creating iPS cells the same way as Shinya Yamanaka of Kyoto University in Japan, by using viral vectors to introduce four genes into mouse fibroblast cells. The Chinese team tried hard, tweaking the culture medium and analysing 250 developing embryos before getting their first mouse. In the paper, the team reports 27 live births. With their best cell line and optimal recipe, they were able to get 22 live births from 624 injected embryos, a success rate of 3.5%. Some of their mice passed one of the most fundamental tests of health: all 12 mice that were mated produced offspring, and the offspring showed no abnormalities. The team says it now has hundreds of second-generation, and more than 100 third-generation, mice. The team found no tumours in the mice, although they have not systematically looked for them. The leader of the other team, Shaorong Gao of the National Institute of Biological Sciences in Beijing, also credits persistence for success. His group, which used the same basic technique as Zeng and Zhou, transferred iPS cells to 187 tetraploid complementation embryos to get just two live births (a 1.1% efficiency rate), although one died in infancy. "The chance for generating such a cell line is rare but we tried very hard," he says. Gao's team is now trying to mate its surviving mouse. Reference: iPS cells produce viable mice through tetraploid complementation Xiao-yang Zhao, Wei Li, Zhuo Lu, Lei Liu, Man Tong, Tang Hai, Jie Hao, Chang-long Guo, Qing-wen Ma, Liu Wang, Fanyi Zeng & Qi Zhou Nature advance online publication 23 July 2009, doi:10.1038/nature08267 See also: Mice made from induced stem cells David Cyranoski Nature News, Published online 23 July 2009, doi:10.1038/460560a Mice Grown from iPS Cells by Chinese Researchers Reprogrammed mouse fibroblasts can make a whole mouse CellNEWS - Thursday, 23 July 2009 ......... ZenMaster

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Mice Grown from iPS Cells by Chinese Researchers

Reprogrammed mouse fibroblasts can make a whole mouse Thursday, 23 July 2009 In a paper publishing online July 23 in Cell Stem Cell, a Cell Press journal, Dr. Shaorong Gao and colleagues from the National Institute of Biological Sciences in Beijing, China, report an important advance in the characterization of reprogrammed induced pluripotent stem cells, or iPSCs. Scientists working with iPSCs have been eager to find out if these cells are fully pluripotent, as this would tell us to what extent they have in fact been truly reprogrammed and resemble normal embryonic stem cells (ESCs). The generally accepted "gold standard" for determining whether a mouse iPSC line has been fully reprogrammed is to show that when injected into an early embryo (or blastocyst), the iPSCs can contribute to many different tissues in the resulting chimeric mouse, including the germline. However, unlike bona fide mouse ESCs, until now mouse iPSCs have not been able to pass a more stringent test of true pluripotency termed "tetraploid complementation," which uses a hybrid embryo method to generate full-term mice entirely comprised of ESC-derived cells. In their current report, Gao and his colleagues used established methods to reprogram mouse cells to isolate five new iPSC lines, and then found that, using one of these lines, they were able to make by tetraploid complementation embryos that survived until birth, and one embryo that also survived to adulthood. The authors decided to test this specific iPSC line in the tetraploid complementation experiment because it gave an unusually high level of chimerism when injected into blastocysts and thus might have unique characteristics not found in many other iPSC lines. As emphasized by Gao: "Although these findings are an important proof of principle, it would be premature to make claims about whether iPSCs in general are functionally equivalent to normal ESCs." As the authors remark in their paper, it will be interesting to determine if there are specific reasons why this particular line succeeded where others have failed. The demonstration that mouse iPSCs can, in fact, pass the most stringent test of pluripotency offers added hope that the process of reprogramming may indeed one day overcome the need for embryo destruction in order to derive pluripotent cells for research and potential therapies. However, it remains to be seen whether lessons obtained from these findings can be applied to human cells and thus whether human iPSCs will be a viable alternative to human ESCs in all circumstances. Reference: iPS Cells Can Support Full-Term Development of Tetraploid Blastocyst-Complemented Embryos Lan Kang, Jianle Wang, Yu Zhang, Zhaohui Kou and Shaorong Gao Cell Stem Cell, 23 July 2009, doi:10.1016/j.stem.2009.07.001 See also: Second Chinese Group Produce Mice from iPS Cells CellNEWS - Thursday, 23 July 2009 This press release is also available in Chinese. ......... ZenMaster

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Pigs' Connective Tissue Cells Converted into Stem Cells

New finding could result in better tests for stem cell therapy, more accurate model Thursday, 25 June 2009 For years, proponents have touted the benefits of embryonic stem cell research, but the potential therapies still face hurdles. Side effects such as tumour development, a lack of an effective and long-term animal model to test new therapies, and genetic incompatibility between the host and donor cells are some of the problems faced by researchers. Now, scientists at the University of Missouri-Columbia have developed the ability to take regular cells from a pig's connective tissues, known as fibroblasts, and transform them into stem cells, eliminating several of these hurdles. The new study appeared in a recent issue of the Proceedings of the National Academy of Sciences (PNAS). "It's important to develop a good, accurate animal model to test these new therapies," said R. Michael Roberts, Curator's Professor of Animal Science and Biochemistry and a researcher in the Bond Life Sciences Center. "Cures with stem cells are not right around the corner, but the pig could be an excellent model for testing new therapies because it is so similar to humans in many ways." In their research, Roberts; Toshihiko Ezashi, a research assistant professor of animal sciences in the College of Agriculture, Food and Natural Resources and lead author on the study; and Bhanu Telugu, a post-doctoral fellow in animal sciences; cultured fibroblasts from a foetal pig. The scientists then inserted four specific genes into the cells. These genes have the ability to "re-program" the differentiated fibroblasts so that they "believe" they are stem cells, take on many of the properties of stem cells that would normally be derived from embryos, and, like embryonic stem cells, differentiate into many, possibly all, of the more than 250 cell types found in the body of an adult pig. Since these "induced pluripotent stem cells" were not derived from embryos and no cloning technique was used to obtain them, the approach eliminates some of the controversy that has accompanied stem cell research in the past. The next step is for Roberts and his team to remove the four genes that reprogrammed the original cells. Then the researchers will determine what needs to be done to direct the new stem cells to develop into specific cell types. "Right now, we researchers have not answered questions concerning how to make stem cells develop into just one type of cell, such as those of liver, kidney or blood cells, rather than a mixture," Roberts said. "Now that we have been able to turn regular cells into stem cells, we need to learn how to make the right type of tissue and then test putting that new tissue back into the animal." Roberts also noted that using the same animal for both the beginning and end of the research would eliminate any host rejection of the transplanted cells once scientists reach the point where they are putting the new tissue back into the animal. Using pigs rather than mice allows researchers to observe any long-term effects of the therapies. Because mice typically have a short life span and differ from humans more than pigs, it is less difficult to predict and/or study long-term effects using pigs, Telugu said. Reference: Derivation of induced pluripotent stem cells from pig somatic cells Toshihiko Ezashi, Bhanu Prakash V. L. Telugu, Andrei P. Alexenko, Shrikesh Sachdev, Sunilima Sinha and R. Michael Roberts PNAS June 18, 2009, doi: 10.1073/pnas.0905284106 ......... ZenMaster

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Combined Stem Cell and Gene Therapy Cures Fanconi’s Anaemia

Approach cures human genetic disease in vitro Sunday, 31 May 2009 A study led by researchers at the Salk Institute for Biological Studies, has catapulted the field of regenerative medicine significantly forward, proving in principle that a human genetic disease can be cured using a combination of gene therapy and induced pluripotent stem (iPS) cell technology. The study, published in the May 31, 2009 early online edition of Nature, is a major milestone on the path from the laboratory to the clinic. "It's been ten years since human stem cells were first cultured in a Petri dish," says the study's leader Juan-Carlos Izpisúa Belmonte, Ph.D., a professor in the Gene Expression Laboratory and director of the Center of Regenerative Medicine in Barcelona (CMRB), Spain. "The hope in the field has always been that we'll be able to correct a disease genetically and then make iPS cells that differentiate into the type of tissue where the disease is manifested and bring it to clinic." Although several studies have demonstrated the efficacy of the approach in mice, its feasibility in humans had not been established. The Salk study offers the first proof that this technology can work in human cells. Belmonte's team, working with Salk colleague Inder Verma, Ph.D., a professor in the Laboratory of Genetics, and colleagues at the CMRB, and the CIEMAT in Madrid, Spain, decided to focus on Fanconi anaemia (FA), a genetic disorder responsible for a series of haematological abnormalities that impair the body's ability to fight infection, deliver oxygen, and clot blood. Caused by mutations in one of 13 Fanconi anaemia (FA) genes, the disease often leads to bone marrow failure, leukaemia, and other cancers. Even after receiving bone marrow transplants to correct the haematological problems, patients remain at high risk of developing cancer and other serious health conditions.

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Shown in green are genetically-corrected fibroblasts from Fanconi anaemia patients are reprogrammed to generate induced pluripotent stem cells, which, in turn, can be differentiated into disease-free hematopoietic progenitors, capable of producing blood cells in vitro. Credit: Courtesy of Dr. Juan-Carlos Belmonte, Salk Institute for Biological Studies.

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After taking hair or skin cells from patients with Fanconi anaemia, the investigators corrected the defective gene in the patients' cells using gene therapy techniques pioneered in Verma's laboratory. They then successfully reprogrammed the repaired cells into induced pluripotent stem (iPS) cells using a combination of transcription factors, Oct4, Sox2, Klf4 and c-Myc. The resulting FA-iPS cells were indistinguishable from human embryonic stem cells and iPS cells generated from healthy donors. Since bone marrow failure as a result of the progressive decline in the numbers of functional hematopoietic stem cells is the most prominent feature of Fanconi anaemia, the researchers then tested whether patient-specific iPS cells could be used as a source for transplantable hematopoietic stem cells. They found that FA-iPS cells readily differentiated into hematopoietic progenitor cells primed to differentiate into healthy blood cells. "We haven't cured a human being, but we have cured a cell," Belmonte explains. "In theory we could transplant it into a human and cure the disease." Although hurdles still loom before that theory can become practice — in particular, preventing the reprogrammed cells from inducing tumours — in coming months Belmonte and Verma will be exploring ways to overcome that and other obstacles. In April 2009, they received a $6.6 million from the California Institute Regenerative Medicine (CIRM) to pursue research aimed at translating basic science into clinical cures. "If we can demonstrate that a combined iPS–gene therapy approach works in humans, then there is no limit to what we can do," says Verma. About the Salk Institute for Biological Studies: The Salk Institute for Biological Studies is one of the world's preeminent basic research institutions, where internationally renowned faculty probe fundamental life science questions in a unique, collaborative, and creative environment. Focused both on discovery and on mentoring future generations of researchers, Salk scientists make groundbreaking contributions to our understanding of cancer, aging, Alzheimer's, diabetes, and cardiovascular disorders by studying neuroscience, genetics, cell and plant biology, and related disciplines. Faculty achievements have been recognized with numerous honours, including Nobel Prizes and memberships in the National Academy of Sciences. Founded in 1960 by polio vaccine pioneer Jonas Salk, M.D., the Institute is an independent non-profit organization and architectural landmark. Reference: Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells Ángel Raya, Ignasi Rodríguez-Pizà, Guillermo Guenechea, Rita Vassena, Susana Navarro, María José Barrero, Antonella Consiglio, Maria Castellà, Paula Río, Eduard Sleep, Federico González, Gustavo Tiscornia, Elena Garreta, Trond Aasen, Anna Veiga, Inder M. Verma, Jordi Surrallés, Juan Bueren & Juan Carlos Izpisúa Belmonte Nature advance online publication 31 May 2009, doi:10.1038/nature08129 ......... ZenMaster

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