New Clues into How Stem Cells Get Their Identity

New Clues into How Stem Cells Get Their Identity

Wednesday, 06 May 2015

Scientists at the University of Copenhagen have identified one mechanism that explains how some stem cells choose to become a given cell type: the cells combine specific sets of proteins at precise positions along the DNA. When these particular groups of proteins are combined, the gates are opened so that certain groups of genes can now be used, giving the cells a new identity.

Scientists have now identified one of these combinations, which drive the cells along the path that allow them to become organs such as liver and pancreas. This latest research could lead scientists to a better understanding on how to generate insulin-producing cells in the laboratory to use as therapy for Type I diabetes. The work has just been published in the journal Cell Stem Cell.

Specificity – choosing the combinations

Scientists working under the leadership of Henrik Semb from the DanStem Center at the University of Copenhagen have explained how the acquisition of a new cell identity is achieved; cells respond to information from their surroundings, in turn activating a specific combination of proteins at certain places on the DNA, to turn on a genetic programme.

Stem cells. 

“We added one particular chemical compound to the culture media to promote the generation of new cell types. The information transmitted by this compound is deciphered only by a small number of proteins. We then looked all along the cell’s DNA to find the positions of the proteins that were activated by the compound. We repeated the experiment using additional compounds, to get an idea of how specific the responses were and to categorize the genes that the cells decided to use when being directed toward different cellular fates,” says Assistant Professor Karen Schachter.

Getting the identity right

The work in the field of human pluripotent stem cell research has concentrated on finding the correct combination of drugs or chemical compounds that can be used to drive the cells into specific cell types in the culture dish.

“There is however a lack of understanding of how these compounds activates the genes that give the cells unique identities, which has resulted in a lack of reproducibility of the methods used by different labs. As a comparison; if you use a pre-mixed powder to bake a cake you will face problems if you run out on an important ingredient and do not know how to replace its action. We believe that our study provides useful information that will help us to understand the recipe better, so that we can generate functional cells in a more controlled manner,” adds Post doc Nina Funa.

There is already a lot of focus in the stem cell community to generate cells in the laboratory to use as therapy, so the scientists at DanStem want to emphasize the importance of continuing doing this important basic research work.

“Our ultimate aim is to understand how stem cells make choices, which will also help improve the quality of the work that will put stem cells into therapeutic use,” concludes Funa.

Source: University of Copenhagen

Contact: Assistant Professor Karen Schachter

Reference:

β-Catenin Regulates Primitive Streak Induction through Collaborative Interactions with SMAD2/SMAD3 and OCT4

Nina S. Funa, Karen A. Schachter, Mads Lerdrup, Jenny Ekberg, Katja Hess, Nikolaj Dietrich, Christian Honoré, Klaus Hansen, Henrik Semb

Cell Stem Cell, April 23, 2015, DOI: http://dx.doi.org/10.1016/j.stem.2015.03.008

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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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Stem Cell Advance May Increase Efficiency of Tissue Regeneration

Stem Cell Advance May Increase Efficiency of Tissue Regeneration

Wednesday, 30 July 2014 

A new stem-cell discovery might one day lead to a more streamlined process for obtaining stem cells, which in turn could be used in the development of replacement tissue for failing body parts, according to UC San Francisco scientists who reported the findings in the current edition of Cell.

Miguel Ramalho-Santos, PhD. .

Credit: Gladstone Institutes. 

The work builds on a strategy that involves reprogramming adult cells back to an embryonic state in which they again have the potential to become any type of cell.

The efficiency of this process may soon increase thanks to the scientists' identification of biochemical pathways that can inhibit the necessary reprogramming of gene activity in adult human cells. Removing these barriers increased the efficiency of stem-cell production, the researchers found.

"Our new work has important implications for both regenerative medicine and cancer research," said Miguel Ramalho-Santos, PhD, associate professor of obstetrics, gynecology and reproductive sciences and a member of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF, who led the research, funded in part by a prestigious NIH Director's New Innovator Award.

The earlier discovery that it was possible to take specialized adult cells and reverse the developmental clock to strip the mature cells of their distinctive identities and characteristics — and to make them immortal, reprogrammable cells that theoretically can be used to replace any tissue type — led to a share of the Nobel Prize in Physiology or Medicine being awarded to UCSF, Gladstone Institutes and Kyoto University researcher Shinya Yamanaka, MD, in 2012.

Turning Back the Clock on Cellular Maturation

Induced pluripotent stem cells — known as iPS

cells, and which act very much like embryonic

stem cells—are here growing into heart cells

(blue) and nerve cells (green). Credit: Gladstone

Institutes/Chris Goodfellow. 

These induced pluripotent stem (iPS) cells are regarded as an alternative experimental approach to ongoing efforts to develop tissue from stem cells obtained from early-stage human embryos. However despite the promise of iPS cells and the excitement surrounding iPS research, the percentage of adult cells successfully converted to iPS cells is typically low, and the resultant cells often retain traces of their earlier lives as specialized cells.

Researchers generate stem cells by forcing the activation within adult cells of pluripotency-inducing genes — starting with the so-called "Yamanaka factors" — a process that turns back the clock on cellular maturation.

Yet, as Ramalho-Santos notes:

"From the time of the discovery of iPS cells, it was appreciated that the specialized cells from which they are derived are not a blank slate. They express their own genes that may resist or counter reprogramming."

But the nature of what exactly was getting in the way of reprogramming remained poorly understood.

"Now, by genetically removing multiple barriers to reprogramming, we have found that the efficiency of generation of iPS cells can be greatly increased," he said.

The discovery will contribute to accelerating the safe and efficient use of iPS cells and other reprogrammed cells, according to Ramalho-Santos.

Removing Roadblocks to Reprogramming

Barriers to human cell reprogramming.
Cell, Volume 158, Issue 2, p449–461. 

The researchers found not merely isolated genes acting as barriers, but rather sets of genes acting in concert through different mechanisms to throw up roadblocks to reprogramming.

"At practically every level of a cell's functions there are genes that act in an intricately coordinated fashion to antagonize reprogramming," Ramalho-Santos said.

These mechanisms are likely to help adult cells maintain their identities and functional roles.

"Much like the Red Queen running constantly to remain in the same place in Lewis Carroll's 'Through the Looking-Glass,' adult cells appear to put a lot of effort into remaining in the same state," he said.

To uncover this previously unidentified bustling biochemical matrix of inhibitory gene activity, the scientists had to simultaneously master a few different technical feats in the lab. They combined cutting-edge genetic, cellular and bioinformatics technologies to comprehensively identify genes that act as barriers to the generation of human iPS cells, and probed how these novel barriers work.

Apart from maintaining the integrity of our adult tissues, the barrier genes probably serve important roles in other diseases – including in the prevention of certain cancers, according to Ramalho-Santos.

Source: University of California - San Francisco 

Contact: Jeffrey Norris

Reference:

Systematic Identification of Barriers to Human iPSC Generation

Han Qin, Aaron Diaz, Laure Blouin, Robert Jan Lebbink, Weronika Patena, Priscilia Tanbun, Emily M. LeProust, Michael T. McManus, Jun S. Song, Miguel Ramalho-Santos

Cell, Volume 158, Issue 2, p449–461, 17 July 2014

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Researchers Create 'Naïve' Pluripotent Human Embryonic Stem Cells

Researchers Create 'Naïve' Pluripotent Human Embryonic Stem Cells

Saturday, 26 July 2014

Phase and fluorescence images of conventional

(primed) human embryonic stem cells (ESCs)

and naïve human ESCs generated in the

presence of 5 small molecule inhibitors.

The naïve human ESCs exhibit activation of a

fluorescent reporter linked to an enhancer of

theOCT4 gene that is specifically used in

the naïve state. 40X magnification. Credit:

Courtesy of Thorold Theunissen. 

For years, researchers and patients have hoped that embryonic stem cells (ESCs) — capable of forming nearly any cell type in the body — could provide insight into numerous diseases perhaps even be used to treat them. Yet progress has been hampered by the inability to transfer research and tools from mouse ESC studies to their human counterparts, in part because human ESCs are "primed" and slightly less plastic than the mouse cells.

Now Thorold Theunissen, Benjamin Powell, and Haoyi Wang, who are scientists in the lab of Whitehead Institute Founding Member Rudolf Jaenisch, have discovered how to manipulate and maintain human ESCs in a "naïve" or base pluripotent state similar to that of mouse ESCs without the use of any reprogramming factors. Their work is described in this week's issue of the journal Cell Stem Cell.

Naïve mice ESCs are well-studied, and scientists have a strong understanding of how they function and mature into more specialized cells. But this understanding is of limited use in human ESC research, as the human cells look different, grow differently, and rely on different genes than mouse ESCs. According to Theunissen, the disparities between mouse and human ESCs are attributable not to species-specific differences but rather to differences of cell state.

In naïve mouse ESCs, a particular enhancer of the gene OCT4 is active, prompting the researchers to look for the presence of this marker as a means to identify rare naïve human ESCs. With this unbiased reporter system in hand, the Jaenisch team determined that a cocktail of five small molecules with a few additional growth factors can induce and support the conversion of primed human ESCs to a naïve state with or without using reprogramming factors to jumpstart the process.

By applying this cocktail to human blastocysts, the scientists could also isolate naïve human stem cells.

"This is important because if this cocktail only works in existing lines of human ESCs, you might wonder, does this really capture a distinct state or is this artificial?" says Theunissen.

"Since the cocktail works directly on human blastocysts, I think it suggests that we're really capturing a cell state that is already present in the early human embryo."

Although other labs have recently reported creating naïve human ESCs, Theunissen, Powell, and Wang question these results as the cells produced through these techniques lack the gene expression and epigenetic profiles of naïve human ESCs. Yet, the Jaenisch lab believes they have now finally unlocked a way to create and maintain this important type of cell and are looking forward to exploring its potential.

"We have discovered a new pathway to generate something we believe is a totally different state of pluripotency in human ESCs that is very close to the mouse naïve state," says Jaenisch, who is also a professor of biology at MIT.

"These cells may be essential for ESC technology, and that is an area we're looking forward to investigating. Now the big question for us is, does this state exist in vivo in embryos? Right now, we don't know, and that is a very interesting line of research."

Source: Whitehead Institute for Biomedical Research 

Contact: Nicole Giese Rura

Reference:

Systematic Identification of Culture Conditions for Induction and Maintenance of Naive Human Pluripotency

Thorold W. Theunissen, Benjamin E. Powell, Haoyi Wang, Maya Mitalipova, Dina A. Faddah, Jessica Reddy, Zi Peng Fan, Dorothea Maetzel, Kibibi Ganz, Linyu Shi, Tenzin Lungjangwa, Sumeth Imsoonthornruksa, Yonatan Stelzer, Sudharshan Rangarajan, Ana D'Alessio, Jianming Zhang, Qing Gao, Meelad M. Dawlaty, Richard A. Young, Nathanael S. Gray, and Rudolf Jaenisch

Cell Stem Cell, July 24, 2014, http://www.cell.com/cell-stem-cell/abstract/S1934-5909(14)00298-7

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Why Stem Cells Need to Stick With Their Friends

Why Stem Cells Need to Stick With Their Friends

Thursday, 07 November 2013

Embryonic stem cells stick together (green, left

image) while they express Oct4 protein in their

nuclei (magenta, left). When Oct4 is removed

their shape changes and their adhesion to each

other is reduced (right panel, nuclei in blue).

Credit: DanStem. 

Scientists at University of Copenhagen and University of Edinburgh have identified a core set of functionally relevant factors which regulates embryonic stem cells' ability for self-renewal. A key aspect is the protein Oct4 and how it makes stem cells stick together. The identification of these factors will be an important tool in devising better and safer ways of making specialised cells for future regenerative cell therapies for treatment of diseases like diabetes and Parkinson's disease. The results have just been published in the scientific journal Current Biology.

Scientists have known that the protein Oct4 plays a key role in maintaining the embryonic stem cells in pure form by turning on stem cell genes, however up until now it has not been know which of the 8.000 or more possible genes that Oct4 can choose from actually support self-renewal.

By comparing the evolution of stem cells in frogs, mice and humans, scientists at the Danish Stem Cell Center (DanStem) and the MRC Centre for Regenerative Medicine in Edinburgh have now been able to link the protein Oct4 with the ability of cells to stick together. They found that for embryonic stem cells to thrive they need to stick together and Oct4's role is to make sure they stay that way.

"Embryonic stem cells can stay forever young unless they become grown-up cells with a specialised job in a process called differentiation. Our study shows that Oct4 prevents this process by pushing stem cells to stick to each other," says Dr Alessandra Livigni, Research Fellow at the University of Edinburgh.

Identification of specific genes

The research teams in Edinburgh and Copenhagen successfully identified 53 genes, out of more than 8.000 possible candidates that together with Oct4, functionally regulate cell adhesion. Almost like finding needles in a haystack the scientists have paved the way for a more efficient way of maintaining stem cells as stem cells.

"Embryonic stem cells are characterized, among other things, by their ability to perpetuate themselves indefinitely and differentiate into all the cell types in the body – a trait called pluripotency. Though to be able to use them medically, we need to be able to maintain them as stem cells, until they're needed. When we want to turn a stem cell into a specific cell for example; an insulin producing beta cell, or a nerve cell like those in the brain, we'd like this process to occur accurately and efficiently. We cannot do this if we don't understand how to maintain stem cells as stem cells," says Professor Joshua Brickman from DanStem, University of Copenhagen.

Future potential

As well as maintaining embryonic stem cells in their pure state more effectively, this new insight will also enable scientists to more efficiently manipulate adult cells to revert to a stem cell like stage known as induced pluripotent stem cells (iPS cells). These cells have many of the same traits and characteristics as embryonic stem cells but can be derived from the patients to both help study degenerative disease and eventually treat them.

"This research knowledge has the potential for us to change the way we grow stem cells, enabling us to use them in a less costly and more efficient way. It will help us devise better and safer ways to create specialised cells for future regenerative medicine therapies," concludes Professor Joshua

Source: University of Copenhagen 

Contact: Joshua Brickman

Reference:

A Conserved Oct4/POUV-Dependent Network Links Adhesion and Migration to Progenitor Maintenance

Alessandra Livigni, Hanna Peradziryi, Alexei A. Sharov, Gloryn Chia, Fella Hammachi, Rosa Portero Migueles, Woranop Sukparangsi, Salvatore Pernagallo, Mark Bradley, Jennifer Nichols, Minoru S.H. Ko, Joshua M. Brickman

Current Biology, 07 November 2013, 10.1016/j.cub.2013.09.048

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For more on stem cells and cloning, go to CellNEWS at

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Researchers Develop Efficient Model for Generating Human iPSCs

Approach has potential to simplify generation of iPSCs for use in human stem cell therapies

Thursday, 01 August 2013

Researchers at the University of California, San Diego School of Medicine report a simple, easily reproducible RNA-based method of generating human induced pluripotent stem cells (iPSCs) in the August 1 edition of Cell Stem Cell. Their approach has broad applicability for the successful production of iPSCs for use in human stem cell studies and eventual cell therapies.

This graphic depicts single RNA generation

of human iPS cells. Credit: Peter Allen, UC

Santa Barbara. 

Partially funded by grants from the California Institute for Regenerative Medicine (CIRM) and the National Institutes of Health (NIH), the methods developed by the UC San Diego researchers dramatically improve upon existing DNA-based approaches – avoiding potential integration problems and providing what appears to be a safer and simpler method for future clinical applications.

The generation of human iPSCs has opened the potential for regenerative medicine therapies based on patient-specific, personalized stem cells. Pluripotent means that these cells have the ability to give rise to any of the body's cell types. The human iPSCs are typically artificially derived from a non-pluripotent adult cell, such as a skin cell. They retain the characteristics of the body's natural pluripotent stem cells, commonly known as embryonic stem cells. Because iPSCs are developed from a patient's own cells, it was first thought that treatment using them would avoid any immunogenic responses. However, depending on methods used to generate such iPSCs, they may pose significant risks that limit their use. For example, using viruses to alter the cell's genome could promote cancer in the host cell.

Methods previously developed to generate integration-free iPSCs were not easily and efficiently reproducible. Therefore, the UC San Diego researchers focused their approach on developing a self-replicating, RNA-based method (one that doesn't integrate into the DNA) with the ability to be retained and degraded in a controlled fashion, and that would only need to be introduced once into the cell.

Using a Venezuelan equine virus (VEE) with structural proteins deleted, but non-structural proteins still present, the scientists added four reprogramming factors (OCT4, KLF4, SOX2 with either c-MYC or GLIS1). They made a single transfection of the VEE replicative form (RF) RNA into new-born or adult human fibroblasts, connective tissue cells that provide a structural framework for many other tissues.

"This resulted in efficient generation of iPSCs with all the hallmarks of stem cells," said principal investigator Steven Dowdy, PhD, professor in the UC San Diego Department of Cellular & Molecular Medicine.

"The method is highly reproducible, efficient, non-integrative – and it works."

Dowdy added that it worked on both young and old human cells. He explained that this is important since – in order to be used therapeutically in fighting disease or to create disease models for research – iPSCs will need to be derived from the cells of middle-aged to old adults who are more prone to the diseases scientists are attempting to treat. In addition, reprogramming factors can be easily changed.

Source: University of California - San Diego 

Contact: Debra Kain

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For more on stem cells and cloning, go to CellNEWS at

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Scientists Discover More Versatile Approach to Creating Stem Cells

New method should hasten promise of regenerative medicine

Friday, 19 July 2013

Stem cells are key to the promise of regenerative medicine: the repair or replacement of injured tissues with custom grown substitutes. Essential to this process are induced pluripotent stem cells (iPSCs), which can be created from a patient's own tissues, thus eliminating the risk of immune rejection. However, Shinya Yamanaka's formula for iPSCs, for which he was awarded last year's Nobel Prize, uses a strict recipe that allows for limited variations in human cells, restricting their full potential for clinical application.

From left: Emmanuel Nivet Martinez and Juan

Carlos Belmonte. Seated: Ignacio Sancho

Martinez. Credit: Courtesy of the Salk Institute

for Biological Studies. 

Now, in this week's issue of Cell Stem Cell, the Salk Institute's Juan Carlos Izpisua Belmonte and his colleagues show that the recipe for iPSCs is far more versatile than originally thought. For the first time, they have replaced a gene once thought impossible to substitute, creating the potential for more flexible recipes that should speed the adoption of stem cells therapies.

Stem cells come in two types: embryonic stem cells (ESCs), which are immature cells that have never differentiated into specific cell types, and induced pluripotent stem cells, which are mature cells that have been reprogrammed back into an undifferentiated state. After the initial discovery in 2006 by Yamanaka that introducing four different genes into a mature cell could suffice for reprogramming the cell to pluripotency, most researchers adopted his recipe.

Izpisua Belmonte and his colleagues took a fresh approach and discovered that pluripotency (the stem cell's ability to differentiate into nearly any kind of adult cell) can also be accomplished by balancing the genes required for differentiation. These genes code for "lineage transcription factors," proteins that start a stem cell down the path to differentiate first into a particular cell lineage, or type, such as a blood cell versus a skin cell, and then finally into a specific cell, such as a white blood cell.

"Prior to this series of experiments, most researchers in the field started from the premise that they were trying to impose an 'embryonic-like' state on mature cells," says Izpisua Belmonte, who holds the Institute's Roger Guillemin Chair.

"Accordingly, major efforts had focused on the identification of factors that are typical of naturally occurring embryonic stem cells, which would allow or further enhance reprogramming."

The picture shows newly reprogrammed cells

expressing marks of pluripotency as identified

by fluorescence (NANOG in green, TRA-1-81

in red). Credit: Courtesy of the Salk Institute

for Biological Studies.

Despite these efforts, there seemed to be no way to determine through genetic identity alone that cells were pluripotent. Instead, pluripotency was routinely evaluated by functional assays. In other words, if it acts like a stem cell, it must be a stem cell.

That condition led the team to their key insight.

"Pluripotency does not seem to represent a discrete cellular entity but rather a functional state elicited by a balance between opposite differentiation forces," says Izpisua Belmonte.

Once they understood this, they realized the four extra genes weren't necessary for pluripotency. Instead, it could be achieved by altering the balance of "lineage specifiers," genes that were already in the cell that specified what type of adult tissue a cell might become.

"One of the implications of our findings is that stem cell identity is actually not fixed but rather an equilibrium that can be achieved by multiple different combinations of factors that are not necessarily typical of ESCs," says Ignacio Sancho-Martinez, one of the first authors of the paper and a postdoctoral researcher in Izpisua Belmonte's laboratory.

The group was able to show that more than seven additional genes can facilitate reprogramming to iPSCs. Most importantly, for the first time in human cells, they were able to replace a gene from the original recipe called Oct4, which had been replaced in mouse cells, but was still thought indispensable for the reprogramming of human cells. Their ability to replace it, as well as SOX2, another gene once thought essential that had never been replaced in combination with Oct4, demonstrated that stem cell development must be viewed in an entirely new way.

"It was generally assumed that development led to cell/tissue specification by 'opening' certain differentiation doors," says Emmanuel Nivet, a post-doctoral researcher in Izpisua Belmonte's laboratory and co-first author of the paper, along with Sancho-Martinez and Nuria Montserrat of the Center for Regenerative Medicine in Barcelona, Spain.

Instead, the successful substitution of both Oct4 and SOX2 shows the opposite.

"Pluripotency is like a room with all doors open, in which differentiation is accomplished by 'closing' doors," Nivet says.

"Inversely, reprogramming to pluripotency is accomplished by opening doors."

The team believes their work should help to overcome one of the major hurdles to the widespread adoption of stem cell therapies: the original four genes used to reprogram stem cells had been implicated in cancer.

"Recent studies in cancer, many of them done by my Salk colleagues, have shown molecular similarities between the proliferation of stem cells and cancer cells, so it is not surprising that oncogenes [genes linked to cancer] would be part of the iPSC recipe," says Izpisua Belmonte.

With this new method, which allows for a customized recipe, the team hopes to push therapeutic research forward.

"Since we have shown that it is possible to replace genes thought essential for reprogramming with several different genes that have not been previously involved in tumorigenesis, it is our hope that this study will enable iPSC research to more quickly translate into the clinic," says Izpisua Belmonte.

Source: Salk Institute for Biological Studies

Contact: Kat Kearney 

Reference:

Reprogramming of human fibroblasts to pluripotency with lineage specifiers

Nuria Montserrat, Emmanuel Nivet, Ignacio Sancho-Martinez, Tomoaki Hishida, Sachin Kumar, Laia Miquel, Carme Cortina, Yuriko Hishida, Yun Xia, Concepcion Rodriguez Esteban and Juan Carlos Izpisua Belmonte

Cell Stem Cell, 18 July 2013, doi:10.1016/j.stem.2013.06.019

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