New Technology Directly Reprograms Skin Fibroblasts for a New Role

Penn study has implications for new skin disease treatments

Tuesday, 16 December 2014

Dermal fibroblasts are directly reprogrammed to

pigmented melanocytes by three transcription

factors (SOX10, MITF and PAX3).

Credit: Ruifeng Yang, Perelman School of

Medicine, University of Pennsylvania. 

As the main component of connective tissue in the body, fibroblasts are the most common type of cell. Taking advantage of that ready availability, scientists from the Perelman School of Medicine at the University of Pennsylvania, the Wistar Institute, Boston University School of Medicine, and New Jersey Institute of Technology have discovered a way to repurpose fibroblasts into functional melanocytes, the body's pigment-producing cells. The technique has immediate and important implications for developing new cell-based treatments for skin diseases such as vitiligo, as well as new screening strategies for melanoma. The work was published this week in Nature Communications.

The new technique cuts out a cellular middleman. Study senior author Xiaowei "George" Xu, MD, PhD, an associate professor of Pathology and Laboratory Medicine, explains.

"Through direct reprogramming, we do not have to go through the pluripotent stem cell stage, but directly convert fibroblasts to melanocytes. So these cells do not have tumourigenicity."

Changing a cell from one type to another is hardly unusual. Nature does it all the time, most notably as cells divide and differentiate themselves into various types as an organism grows from an embryo into a fully-functional being. With stem cell therapies, medicine is learning how to tap into such cell specialization for new clinical treatments. But controlling and directing the process is challenging. It is difficult to identify the specific transcription factors needed to create a desired cell type. Also, the necessary process of first changing a cell into an induced pluripotent stem cell (iPSC) capable of differentiation, and then into the desired type, can inadvertently create tumours.

Xu and his colleagues began by conducting an extensive literature search to identify 10 specific cell transcription factors important for melanocyte development. They then performed a transcription factor screening assay and found three transcription factors out of those 10 that are required for melanocytes: SOX10, MITF, and PAX3, a combination dubbed SMP3.

"We did a huge amount of work," says Xu.

"We eliminated all the combinations of the other transcription factors and found that these three are essential."

The researchers first tested the SMP3 combination in mouse embryonic fibroblasts, which then quickly displayed melanocytic markers. Their next step used a human-derived SMP3 combination in human foetal dermal cells, and again melanocytes (human-induced melanocytes, or hiMels) rapidly appeared. Further testing confirmed that these hiMels indeed functioned as normal melanocytes, not only in cell culture but also in whole animals, using a hair-patch assay, in which the hiMels generated melanin pigment. The hiMels proved to be functionally identical in every respect to normal melanocytes.

Xu and his colleagues anticipate using their new technique in the treatment of a wide variety of skin diseases, particularly those such as vitiligo for which cell-based therapies are the best and most efficient approach.

The method could also provide a new way to study melanoma.

By generating melanocytes from the fibroblasts of melanoma patients, Xu explains: "we can screen not only to find why these patients easily develop melanoma, but possibly use their cells to screen for small compounds that can prevent melanoma from happening."

Perhaps most significantly, say the researchers, is the far greater number of fibroblasts available in the body for reprogramming compared to tissue-specific adult stem cells, which makes this new technique well-suited for other cell-based treatments.

Source: University of Pennsylvania School of Medicine

Contact: Karen Kreeger

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Reprogramming Cells, Long Term

Positive finding could have major implications for diabetes treatment

Tuesday, 18 November 2014

Harvard Stem Cell Institute (HSCI) researchers, representing five Harvard departments and affiliated institutions as well as the Massachusetts Institute of Technology (MIT), have demonstrated that adult cells, reprogrammed into another cell type in a living animal, can remain functional over a long period.

Joe Zhou is a Harvard Stem Cell Institute

Principal Faculty Member and an Associate

Professor in Harvard's Department of Stem Cell

and Regenerative Biology. Zhou and colleagues

have demonstrated that cells reprogrammed in

vivo can survive, thrive, and be therapeutically

useful. Credit: B. D. Colen/Harvard University.

The work by Joe Zhou, an associate professor in Harvard's Department of Stem Cell and Regenerative Biology, and his collaborators is an important advance in the effort to develop cell-based therapies for tissue repair, and specifically in the effort to develop improved treatment for diabetes.

The researchers used a combination of genes to change pancreatic exocrine cells – one of the main forms of cells in the pancreas – in adult mice that have diabetes into insulin-producing beta cells that appeared to cure about a third of the mice of the metabolic disease, and improved insulin production in most of the other mice. A report on the work was published today in the journal Nature Biotechnology.

The new findings are a major advance in work by HSCI co-director Doug Melton and Zhou, who in 2008 reported having converted exocrine cells into functional beta cells in mice. At that time, however, it was not known how long, and how well, the repurposed cells would function.

"The efficiency of reprogramming has always been an issue," Zhou said.

"Until now, the new cells have either dropped dramatically in number or disappeared completely," he said, noting that since his work with Melton in 2008 there have been reports published in other programing systems that question whether the reprogrammed cells could be stable enough ultimately to be useful.

"What we have demonstrated is that yes, the reprogrammed cells can be useful, and for that to happen you have to create a niche environment in which the cells can survive," Zhou continued.

"We have improved the reprogramming efficiency to a point where one can create a large enough number of the new cells that the new cells create their own niche environment."

Zhou said that the researchers studied the mice for up to about 13 months, approximately half their normal life span, and found that "the cells are still there, and fairly robust. These are diabetic animals, and we were able to, I wouldn't use the word 'cure' because that's a very freighted word for me to use, but they became highly glycaemic animals – though not every animal became normal. That may be because to completely control the glucose level of the animal, you not only need beta cells, you need about a quarter of a million functional beta cells. If you are short of this number, even if the beta cells are perfectly normal," they can't completely control blood sugar levels, Zhou said.

When discussing the implications of the study for the field of cellular reprogramming, Zhou cautioned that the pancreas has a particularly simple cellular organization and structure, and thus findings in the pancreas might not necessarily apply to other organs.

Diabetes is a metabolic disease that is seen in two basic forms.

Type 1 diabetes is an autoimmune disease affecting about 3 million Americans, in which the patient's immune system ultimate destroys all the insulin-producing beta cells in the pancreas, and the patient has to inject insulin in order to regulate blood glucose levels.

Type 2 diabetes, which is now at epidemic prevalence rates in the United States and around the world, is usually caused by being overweight, lack of proper exercise, and improper diet, and can make a patient insulin-resistant, so the insulin the body produces is not sufficient to control blood glucose levels.

If the kind of treatment approach suggested by the new study were to succeed in humans – and that is a question to be answered with further animal, and eventually human, studies – it could be useful in treating both forms of diabetes.

One drawback to the current form of the new approach is that the cellular reprogramming is done with genes, and there might ultimately be unwanted effects on the cells. Zhou said the goal would be to replace the genes with chemicals or, perhaps, RNAs.

"I've talked to many clinicians about whether our approach could be used in humans," Zhou said.

"And the opinion is divided. Some say this could be developed into a human treatment, and some say it should be improved. But there seems to be general agreement that it could potentially be useful."

Source: Harvard University

Contact: B. D. Colen

Reference:

Long-term persistence and development of induced pancreatic beta cells generated by lineage conversion of acinar cells

Weida Li, Claudia Cavelti-Weder, Yinying Zhang, Kendell Clement, Scott Donovan, Gabriel Gonzalez, Jiang Zhu, Marianne Stemann, Ke Xu, Tatsu Hashimoto, Takatsugu Yamada, Mio Nakanishi, Yuemei Zhang, Samuel Zeng, David Gifford, Alexander Meissner, Gordon Weir & Qiao Zhou

Nature Biotechnology (2014), doi:10.1038/nbt.3082

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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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Some Stem Cell Methods Closer to "Gold Standard" than Others

Nuclear transfer appears superior for creating embryonic stem cells

Thursday, 03 July 2014

Researchers around the world have turned to stem cells, which have the potential to develop into any cell type in the body, for potential regenerative and disease therapeutics.

Now, for the first time, researchers at the Salk Institute, with collaborators from Oregon Health & Science University and the University of California, San Diego, have shown that stem cells created using two different methods are far from identical. The finding could lead to improved avenues for developing stem cell therapies as well as a better understanding of the basic biology of stem cells.

The researchers discovered that stem cells created by moving genetic material from a skin cell into an empty egg cell — rather than coaxing adult cells back to their embryonic state by artificially turning on a small number of genes — more closely resemble human embryonic stem cells, which are considered the gold standard in the field.

Joseph R. Ecker, Professor, Genomic Analysis

Laboratory. Credit: Courtesy of the Salk

Institute for Biological Studies. 

"These cells created using eggs' cytoplasm have fewer reprogramming issues, fewer alterations in gene expression levels and are closer to real embryonic stem cells," says co-senior author Joseph R. Ecker, professor and director of Salk's Genomic Analysis Laboratory and co-director of the Center of Excellence for Stem Cell Genomics. The results of the study were published today in Nature.

Human embryonic stem cells (hESCs) are directly pulled from unused embryos discarded from in-vitro fertilization, but ethical and logistical quandaries have restricted their access. In the United States, federal funds have limited the use of hESCs so researchers have turned to other methods to create stem cells. Most commonly, scientists create induced pluripotent stem (iPS) cells by starting with adult cells (often from the skin) and adding a mixture of genes that, when expressed, regress the cells to a pluripotent stem-cell state. Researchers can then coax the new stem cells to develop into cells that resemble those in the brain or in the heart, giving scientists a valuable model for studying human disease in the lab.

Over the past year, a team at OHSU built upon a technique called somatic cell nuclear transfer (the same that is used for cloning an organism, such as Dolly the sheep) to transplant the DNA-containing nucleus of a skin cell into an empty human egg, which then naturally matures into a group of stem cells.

Shoukhrat Mitalipov, Ph.D., Oregon Health &

Science University, led a team that found that a

process called "somatic cell nuclear transfer" is

much better and more accurate at

reprogramming human skin cells to become

embryonic stem cells. Credit: Oregon Health &

Science University.

Ecker, holder of the Salk International Council Chair in Genetics, teamed up with Shoukhrat Mitalipov, developer of the new technique and director of the Center for Embryonic Cell and Gene Therapy at OHSU, and UCSD assistant professor Louise Laurent to carry out the first direct comparison of the two approaches. The scientists created four lines of nuclear transfer stem cells all using eggs from a single donor, along with seven lines of iPS cells and two lines of the gold standard hESCs. All cell lines were shown to be able to develop into multiple cell types and had nearly identical DNA content contained within them.

But when they looked closer at the cells, the researchers spotted some differences: the patterns of methylation — chemical flags that are added to genes to control their expression — varied between the cell lines. This indicates a difference in how and when genes, despite having identical sequences, might be expressed. The methylation of nuclear transfer cells more closely resembled hESCs than the iPS cells did. And when the investigators looked at patterns of actual gene expression — by measuring the levels of particular RNA strands produced by each cell — the differences continued. Once again, nuclear transfer cells had RNA levels closer to embryonic cells, making them more accurate for basic research and therapeutic studies.

"Both the DNA methylation and gene expression data show that nuclear transfer does a better job at erasing the signature of the original skin cell," says Laurent, who is a co-senior author of the paper.

"If you believe that gene expression is important, which we do, then the closer you get to the gene expression patterns of embryonic stem cells, the better," Ecker says.

"Right now, nuclear transfer cells look closer to the embryonic stem cells than do the iPS cells."

Ecker doesn't expect labs to race to make the switch to nuclear transfer protocols — after all, the method falls within those restricted for federal funding. But he thinks the new observation likely holds lessons that could help improve the protocols for making iPS cells.

"What this is telling us is that you can use the standard mix of genes and they do a pretty good job of creating iPS cells," Ecker says.

"But they're not perfect. The material in an egg does a better job than just those four genes alone."

If researchers can pin down what it is within an egg that drives the production of pluripotent stem cells, they may be able to integrate that knowledge into iPS methods to improve stem cell therapy for disease.

"At this point, nuclear transfer stem cells combine the key advantages of both hESCs and iPS cells and, as such, are ideal for clinical applications in regenerative therapy," adds Mitalipov.

Other researchers on the study were Ryan C. O'Neil, Yupeng He, Matthew D. Schultz, Manoj Heriharan, Joseph R. Nery, and Rosa Castanon of the Salk Institute for Biological Studies; Hong Ma, Brittany Daughtry, Masahito Tachibana, Eunju Kang, Rebecca Tippner-Hedges, Riffat Ahmed, Nuria Marti Gutierrez, Crystal Van Dyken, Alimujiang Fulati, Atsushi Sugawara, Michelle Sparman, Paula Amato and Don P. Wolf of Oregon Health & Science University; Robert Morey, Karen Sabatini and Rathi D. Thiagarajan of the University of California, San Diego; and Sumita Gokhale of the Boston University School of Medicine.

Source: Salk Institute for Biological Studies

Contact: Kristina Grifantini

Reference:

Abnormalities in human pluripotent cells due to reprogramming mechanisms

Hong Ma, Robert Morey, Ryan C. O'Neil, Yupeng He, Brittany Daughtry, Matthew D. Schultz, Manoj Hariharan, Joseph R. Nery, Rosa Castanon, Karen Sabatini, Rathi D. Thiagarajan, Masahito Tachibana, Eunju Kang, Rebecca Tippner-Hedges, Riffat Ahmed, Nuria Marti Gutierrez, Crystal Van Dyken, Alim Polat, Atsushi Sugawara, Michelle Sparman, Sumita Gokhale, Paula Amato, Don P.Wolf, Joseph R. Ecker, Louise C. Laurent & Shoukhrat Mitalipov

Nature (2014), doi:10.1038/nature13551

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Functional Nerve Cells from Skin Cells

Functional Nerve Cells from Skin Cells

Wednesday, 21 May 2014

A new method of generating mature nerve cells from skin cells could greatly enhance understanding of neurodegenerative diseases, and could accelerate the development of new drugs and stem cell-based regenerative medicine.

These are mature nerve cells generated from

human cells using enhanced transcription 
factors. Credit: Fahad Ali.

The nerve cells generated by this new method show the same functional characteristics as the mature cells found in the body, making them much better models for the study of age-related diseases such as Parkinson's and Alzheimer's, and for the testing of new drugs.

Eventually, the technique could also be used to generate mature nerve cells for transplantation into patients with a range of neurodegenerative diseases.

By studying how nerves form in developing tadpoles, researchers from the University of Cambridge were able to identify ways to speed up the cellular processes by which human nerve cells mature. The findings are reported in the May 27th edition of the journal Development.

Stem cells are our master cells, which can develop into almost any cell type within the body. Within a stem cell, there are mechanisms that tell it when to divide, and when to stop dividing and transform into another cell type, a process known as cell differentiation. Several years ago, researchers determined that a group of proteins known as transcription factors, which are found in many tissues throughout the body, regulate both mechanisms.

More recently, it was found that by adding these proteins to skin cells, they can be reprogrammed to form other cell types, including nerve cells. These cells are known as induced neurons, or iN cells. However, this method generates a low number of cells, and those that are produced are not fully functional, which is a requirement in order to be useful models of disease: for example, cortical neurons for stroke, or motor neurons for motor neuron disease.

In addition, for age-related diseases such as Parkinson's and Alzheimer's, both of which affect millions worldwide, mature nerve cells which show the same characteristics as those found in the body are crucial in order to enhance understanding of the disease and ultimately determine the best way to treat it.

"When you reprogram cells, you're essentially converting them from one form to another but often the cells you end up with look like they come from embryos rather than looking and acting like more mature adult cells," said Dr Anna Philpott of the Department of Oncology, who led the research.

"In order to increase our understanding of diseases like Alzheimer's, we need to be able to work with cells that look and behave like those you would see in older individuals who have developed the disease, so producing more 'adult' cells after reprogramming is really important."

By manipulating the signals which transcription factors send to the cells, Dr Philpott and her collaborators were able to promote cell differentiation and maturation, even in the presence of conflicting signals that were directing the cell to continue dividing.

When cells are dividing, transcription factors are modified by the addition of phosphate molecules, a process known as phosphorylation, but this can limit how well cells can convert to mature nerves. However, by engineering proteins which cannot be modified by phosphate and adding them to human cells, the researchers found they could produce nerve cells that were significantly more mature, and therefore more useful as models for disease such as Alzheimer's.

Additionally, very similar protein control mechanisms are at work to mature important cells in other tissues such as pancreatic islets, the cell type that fails to function effectively in type 2 diabetes. As well as making more mature nerves, Dr Philpott's lab is now using similar methods to improve the function of insulin-producing pancreas cells for future therapeutic applications.

"We've found that not only do you have to think about how you start the process of cell differentiation in stem cells, but you also have to think about what you need to do to make differentiation complete - we can learn a lot from how cells in developing embryos manage this," said Dr Philpott.

Source: University of Cambridge 

Contact: Sarah Collins

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One Step Closer to Cell Reprogramming

One Step Closer to Cell Reprogramming

Wednesday, 07 May 2014

In 2012, John B. Gurdon and Shinya Yamanaka were awarded the Nobel Prize in medicine for discovering that adult cells can be reprogrammed into pluripotent ones (iPS); the cells obtained are capable of behaving in a similar way to embryonic stem cells, and hence have enormous potential for regenerative medicine.

Cells with activated Wnt can no longer be

reprogrammed (in green) are located on the

periphery; cells that can be reprogrammed are

aggregated and can be seen in the centre of the

image (in red). Credit: CRG.

However, although there are many research groups around the world studying this process, it is still not completely understood, it is not totally efficient, and it is not safe enough to be used as the basis for a new cell therapy.

Now, researchers at the Centre for Genomic Regulation (CRG) in Barcelona have taken a very important step towards understanding cell reprogramming and its efficiency: they have discovered the key role of the Wnt signalling pathway in transforming adult cells into iPS cells.

"Generally, transcription factors are used to try to increase or decrease the cell reprogramming process. We have discovered that we can increase the efficiency of the process by inhibiting the Wnt route", explains Francesco Aulicino, a PhD student in the Reprogramming and Regeneration group, led by Maria Pia Cosma and co-author of the study that has just been published in Stem Cell Reports.

The Wnt signalling pathway is a series of biochemical reactions that are produced in cells. In frogs or lizards, for example, these reactions are those that allow their extremities to regenerate if the animal suffers an injury. Although in general, humans and mammals have lost this regenerative capacity, the Wnt pathway is involved in numerous processes during embryonic development and cell fusion, as it is in reprogramming.

The researchers have studied how the Wnt route behaves throughout the entire process of transforming cells into iPS cells, which usually lasts two weeks. It is a very dynamic process that produces oscillations from the pathway, which is not active all the time.

"We have seen that there are two phases and that in each one of them, Wnt fulfils a different function. And we have shown that by inhibiting it at the beginning of the process and activating it at the end we can increase the efficiency of reprogramming and obtain a larger number of pluripotent cells", indicates Ilda Theka, also a PhD student in Pia Cosma's group and a co-author of the article.

To artificially control the pathway, the group has employed a chemical molecule, Iwp2, which is a Wnt secretion inhibitor that does not permanently alter the cells, something which other research into reprogramming using different factors has still has not been able to achieve.

They have also seen that the exact moment when the Wnt pathway is activated is crucial. Doing it too early, makes the cells begin to differentiate, for example into neurones or endodermal cells, and they are not reprogrammed.

"It is a very important and an innovative advance in the field of cell reprogramming, because until now this was a very inefficient process. There are many groups trying to understand the mechanism by which adult cells become pluripotent, and what blocks that process and makes only a small percentage of cells end up being reprogrammed. We are providing information on why it happens", says Theka.

The work opens the way to new advances in regenerative medicine and sheds light on certain types of tumours involving the Wnt pathway. Other labs are also working on ways to increase efficiency when inducing pluripotency in these cells. This is the case of the Haematopoietic Stem Cells, Transdifferentiation and Reprogramming laboratory, led by Thomas Graf, where they work on induced pluripotent stem cells (iPS).

Source: Center for Genomic Regulation 

Contact: Juan Manuel Sarasua

Reference:

Temporal Perturbation of the Wnt Signaling Pathway in the Control of Cell Reprogramming Is Modulated by TCF1

Francesco Aulicino, Ilda Theka, Luigi Ombrato, Frederic Lluis, Maria Pia Cosma

Stem Cell Reports, 6 May 2014, Volume 2, Issue 5, p707–720

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A Protein Required for Integrity of Induced Pluripotent Stem Cells

SIRT1 is necessary for telomere elongation and genome integrity during cell reprogramming

Monday, 21 April 2014

This image shows chromosome abnormalities in

reprogrammed cells in which SIRT1 protein has

been removed (in red). Credit: Centro Nacional

de Investigaciones Oncologicas. 

Cell reprogramming converts specialised cells such as nerve cells or skin cells towards an embryonic stem cell state. This reversal in the evolutionary development of cells also requires a reversal in the biology of telomeres, the structures that protect the ends of chromosomes; whilst under normal conditions telomeres shorten over time, during cell reprogramming they follow the opposite strategy and increase in length.

A study published today in the journal Stem Cell Reports, from the Cell Publishing Group, reveals that the SIRT1 protein is needed to lengthen and maintain telomeres during cell reprogramming. SIRT1 also guarantees the integrity of the genome of stem cells that come out of the cell reprogramming process; these cells are known as iPS cells (induced Pluripotent Stem cells).

The study has been carried out by the Spanish National Cancer Research Centre's Telomeres and Telomerase Group, in collaboration with the CNIO's Transgenic Mice Core Unit.

Since the Japanese scientist Shinya Yamanaka first obtained iPS cells from adult tissue in 2006, regenerative medicine has become one of the most exciting and rapidly developing fields in biomedicine. There is a very ambitious aim, given the ability to differentiate iPS cells into any type of cell; this would allow for the regeneration of organs damaged by diseases such as Alzheimer, diabetes or cardiovascular diseases.

The nature of iPS cells however is causing intense debate. The latest research shows that chromosome aberrations and DNA damage can accumulate in these cells.

"The problem is that we don't know if these cells are really safe", says María Luigia De Bonis, a postdoctoral researcher of the Telomeres and Telomerase Group who has done a large part of the work.

In 2009, the same CNIO laboratory discovered that telomeres increase in length during cell reprogramming (Marion et al., Cell Stem Cell, 2009); this increase is important as it allows stem cells to acquire the immortality that characterises them.

One year later, it was demonstrated that the levels of SIRT1 — a protein belonging to the sirtuin family and that is involved in the maintenance of telomeres, genomic stability and DNA damage response — are increased in embryonic stem cells. The question CNIO researchers asked was: is SIRT1 involved in cell reprogramming?

Safer Stem Cells

Employing mouse models and cell cultures as research tools in which SIRT1 had been removed, the team has discovered that this protein is necessary for reprogramming to occur correctly and safely.

"We observed cell reprogramming in the absence of SIRT1, but over time the produced iPS cells lengthen telomeres less efficiently and suffer from chromosome aberrations and DNA damage," says De Bonis.

"SIRT1 helps iPS cells to remain healthy," she concludes.

The authors describe how this protective effect on iPS cells is, in part, mediated by the cMYC regulator. SIRT1 slows the degradation of cMYC, which results in an increase in telomerase (the enzyme that increases telomere length) in cells.

The study sheds light on how cell reprogramming guarantees the healthy functioning of stem cells. This knowledge will help to overcome barriers that come out of the use of iPS cells so they may be used in regenerative medicine.

Source: Centro Nacional de Investigaciones Oncologicas (CNIO) 

Contact: Nuria Noriega

Reference:

SIRT1 Is Necessary for Proficient Telomere Elongation and Genomic Stability of Induced Pluripotent Stem Cells

Maria Luigia De Bonis, Sagrario Ortega, Maria A. Blasco

Stem Cell Reports (2014), http://dx.doi.org/10.1016/j.stemcr.2014.03.002

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ZenMaster

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http://cellnews-blog.blogspot.com/

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Team Finds a Better Way to Grow Motor Neurons from Stem Cells

Team Finds a Better Way to Grow Motor Neurons from Stem Cells

Tuesday, 01 April 2014

University of Illinois cell and developmental

biology professor Fei Wang, left; visiting scholar

Qiuhao Qu, center; materials science and

engineering professor Jianjun Cheng; and their

colleagues improved the process of converting

stem cells into motor neurons. (Neurons are

green; motor neurons are red in the image on 

the screen.) Credit: L. Brian Stauffer.

Researchers report they can generate human motor neurons from stem cells much more quickly and efficiently than previous methods allowed. The finding, described in Nature Communications, will aid efforts to model human motor neuron development, and to understand and treat spinal cord injuries and motor neuron diseases such as amyotrophic lateral sclerosis (ALS).

The new method involves adding critical signalling molecules to precursor cells a few days earlier than previous methods specified. This increases the proportion of healthy motor neurons derived from stem cells (from 30 to 70 percent) and cuts in half the time required to do so.

"We would argue that whatever happens in the human body is going to be quite efficient, quite rapid," said University of Illinois cell and developmental biology professor Fei Wang, who led the study with visiting scholar Qiuhao Qu and materials science and engineering professor Jianjun Cheng.

"Previous approaches took 40 to 50 days, and then the efficiency was very low – 20 to 30 percent. So it's unlikely that those methods recreate human motor neuron development."

Qu's method produced a much larger population of mature, functional motor neurons in 20 days.

The new approach will allow scientists to induce mature human motor neuron development in cell culture, and to identify the factors that are vital to that process, Wang said.

Stem cells are unique in that they can adopt the shape and function of a variety of cell types. Generating neurons from stem cells (either embryonic stem cells or those "induced" to revert back to an embryo-like state) requires adding signalling molecules to the cells at critical moments in their development.

Wang and other colleagues previously discovered a molecule (called compound C) that converts stem cells into "neural progenitor cells," an early stage in the cells' development into neurons. But further coaxing these cells to become motor neurons presented unusual challenges.

Previous studies added two important signalling molecules at Day 6 (six days after exposure to compound C), but with limited success in generating motor neurons. In the new study, Qu discovered that adding the signalling molecules at Day 3 worked much better: The neural progenitor cells quickly and efficiently differentiated into motor neurons.

This indicates that Day 3 represents a previously unrecognized neural progenitor cell stage, Wang said.

The new approach has immediate applications in the lab. Watching how stem cells (derived from ALS patients' own skin cells, for example) develop into motor neurons will offer new insights into disease processes, and any method that improves the speed and efficiency of generating the motor neurons will aid scientists. The cells can also be used to screen for drugs to treat motor neuron diseases, and may one day be used therapeutically to restore lost function.

"To have a rapid, efficient way to generate motor neurons will undoubtedly be crucial to studying – and potentially also treating – spinal cord injuries and diseases like ALS," Wang said.

Source: University of Illinois at Urbana-Champaign 

Contact: Diana Yates

Reference:

High-efficiency motor neuron differentiation from human pluripotent stem cells and the function ofIslet-1

Qiuhao Qu, Dong Li, Kathleen R. Louis, Xiangzhen Li, Hong Yang, Qinyu Sun, Shane R. Crandall, Stephanie Tsang, Jiaxi Zhou, Charles L. Cox, Jianjun Cheng & Fei Wang

Nature Communications 5, 3449, doi:10.1038/ncomms4449

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ZenMaster

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

http://cellnews-blog.blogspot.com/

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