iPS Cells Used to Correct Genetic Mutations that Cause Muscular Dystrophy

iPS Cells Used to Correct Genetic Mutations that Cause Muscular Dystrophy

Thursday, 27 November 2014

This image shows immunofluorescence staining

of skeletal cells differentiated from DMD-iPS

cells. Untreated DMD skeletal cells do not

express dystrophin (green) due to the deletion of

exon 44. However, after any of the three

correction strategies are applied to iPS cells,

differentiation into skeletal cells results in normal

dystrophin expression. Scale bar, 50 μm.

Credit: Dr. Akitsu Hotta, Kyoto University.

Researchers at the Center for iPS Cell Research and Application (CiRA), Kyoto University, show that induced pluripotent stem (iPS) cells can be used to correct genetic mutations that cause Duchenne muscular dystrophy (DMD). The research, published in Stem Cell Reports, demonstrates how engineered nucleases, such as TALEN and CRISPR, can be used to edit the genome of iPS cells generated from the skin cells of a DMD patient. The cells were then differentiated into skeletal muscles, in which the mutation responsible for DMD had disappeared.

DMD is a severe muscular degenerative disease caused by a loss-of-function mutation in the dystrophin gene. It inflicts 1 in 3500 boys and normally leads to death by early adulthood. Currently, very little is available in terms of treatment for patients outside palliative care. One option gaining interest is genomic editing by TALEN and CRISPR, which have quickly become invaluable tools in molecular biology. These enzymes allow scientists to cleave genes at specific locations and then modify the remnants to produce a genomic sequence to their liking. However, programmable nucleases are not pristine and often mistakenly edit similar sequences that vary a few base pairs from the target sequence, making them unreliable for clinical use because of the potential for undesired mutations.

For this reason, induced pluripotent stem cells (iPS cells) are ideal models, because they provide researchers an abundance of patient cells on which to test the programmable nucleases and find optimal conditions that minimize off-target modifications. CiRA scientists took advantage of this feature by generating iPS cells from a DMD patient. They used several different TALEN and CRISPR to modify the genome of the iPS cells, which were then differentiated into skeletal muscle cells. In all cases, dystrophin protein expression was convalesced, and in some cases, the dystrophin gene was fully corrected.

One key to the success was the development of a computational protocol that minimized the risk of off-target editing. The team built a database that all possible permutations of sequences up to 16 base pairs long. Among these, they extracted those that only appear once in the human genome, i.e. unique sequences. DMD can be caused by several different mutations; in the case of the patient used in this study, it was the result of the deletion of exon 44. The researchers therefore built a histogram of unique sequences that appeared in a genomic region that contained this exon. They found a stack of unique sequences in exon 45.

to Akitsu Hotta, who headed the project and holds joint positions at CiRA and the Institute for Integrated Cell-Materials Sciences at Kyoto University:

"Nearly half the human genome consists of repeated sequences. So even if we found one unique sequence, a change of one or two base pairs may result in these other repeated sequences, which risks the TALEN or CRISPR editing an incorrect region. To avoid this problem, we sought a region that hit high in the histogram".

With this target, the team considered three strategies to modify the frame-shift mutation of the dystrophin gene: exon skipping by connecting exons 43 and 46 to restore the reading frame, frame shifting by incorporating insertion or deletion (indel) mutations, and exon knock-in by inserting exon 44 before exon 45. All three strategies effectively increased dystrophin synthesis in differentiated skeletal cells, but only the exon knock-in approach recovered the gene to its natural state. Importantly, editing showed very high specificity, suggesting that their computational approach can be used to minimize off-target editing by the programming nucleases.

Moreover, the paper provides a proof-of-principle for using iPS cell technology to treat DMD in combination with TALEN or CRISPR. The group now aims to expand this protocol to other diseases.

First author Lisa Li explains: "We show that TALEN and CRISPR can be used to correct the mutation of the DMD gene. I want to apply the nucleases to correct mutations for other genetic-based diseases like point mutations".

Source: Center for iPS Cell Research and Application - Kyoto University

Contact: Akemi Nakamura

Reference:

Precise correction of the DYSTROPHIN gene in Duchenne Muscular Dystrophy patient-derived iPS cells by TALEN and CRISPR-Cas9

Hongmei Lisa Li, Naoko Fujimoto, Noriko Sasakawa, Saya Shirai, Tokiko Ohkame, tetsushi Sakuma, Michihiro tanaka, Naoki Amano, Akira Watanabe, Hidetoshi Sakurai, Takashi Yamamoto, Shinya Yamanaka, and Akitsu Hotta

Stem Cell Reports, November 26, 2014, DOI: http://dx.doi.org/10.1016/j.stemcr.2014.10.013

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WNT Signalling Molecule Crucial to Stem Cell Reprogramming

Surprise finding has implications for wound repair therapies and inhibiting cancer

Friday, 21 November 2014

While investigating a rare genetic disorder, researchers at the University of California, San Diego School of Medicine have discovered that a ubiquitous signalling molecule is crucial to cellular reprogramming, a finding with significant implications for stem cell-based regenerative medicine, wound repair therapies and potential cancer treatments.

The findings are published in the Nov. 20 online issue of Cell Reports.

Karl Willert, PhD, assistant professor in the Department of Cellular and Molecular Medicine, and colleagues were attempting to use induced pluripotent stem cells (iPSC) to create a "disease-in-a-dish" model for focal dermal hypoplasia (FDH), a rare inherited disorder caused by mutations in a gene called PORCN. Study co-authors V. Reid Sutton and Ignatia Van den Veyver at Baylor College of Medicine had published the observation that PORCN mutations underlie FDH in humans in 2007.

FDH is characterized by skin abnormalities such as streaks of very thin skin or different shades, clusters of visible veins and wart like growths. Many individuals with FDH also suffer from hand and foot abnormalities and distinct facial features. The condition is also known as Goltz syndrome after Robert Goltz, who first described it in the 1960s. Goltz spent the last portion of his career as a professor at UC San Diego School of Medicine. He retired in 2004 and passed away earlier this year.

To their surprise, Willert and colleagues discovered that attempts to reprogram FDH fibroblasts or skin cells with the requisite PORCN mutation into iPSCs failed using standard methods, but succeeded when they added WNT proteins - a family of highly conserved signalling molecules that regulate cell-to-cell interactions during embryogenesis.

"WNT signalling is ubiquitous," said Willert.

"Every cell expresses one or more WNT genes and every cell is able to receive WNT signals. Individual cells in a dish can grow and divide without WNT, but in an organism, WNT is critical for cell-cell communication so that cells distinguish themselves from neighbours and thus generate distinct tissues, organs and body parts."

WNT signalling is also critical in limb regeneration (in some organisms) and tissue repair.

"We've shown that WNT signalling is required for cellular reprogramming," said Willert.

"Some of the processes that occur during cellular reprogramming resemble those that occur during regenerative processes and wound repair. For example, limb regeneration in organisms like axolotl and zebrafish require cells at the injury site to de-differentiate (change their function) and then rebuild the damaged tissue. WNT is essential for these amazing regenerative processes."

Willert cautioned that "it would be a stretch to say that activation of WNT signalling will allow us to regenerate limbs," but said WNT activation is likely valuable in assisting tissue repair.

A variety of efforts are already underway exploring how to leverage WNT signalling to promote wound healing, such as speeding bone fracture repairs, and even hair growth.

"That's not really a wound repair process, but WNT is required for hair growth," Willert said.

The caveat, he noted, is that "there's a fine line between repairing tissue and promoting cancer growth." Willert said there are efforts underway to create therapeutics that block WNT signalling as a means to block cancer growth. Earlier this year, for example, Willert and colleagues published findings describing the use of an antibody to disrupt WNT signalling in embryonic stem cells. In cancer cells with mutations in the WNT signalling pathway this antibody may inhibit their growth and development.

Source: University of California - San Diego

Contact: Scott LaFee

Reference:

A Rare Human Syndrome Provides Genetic Evidence that WNT Signaling Is Required for Reprogramming of Fibroblasts to Induced Pluripotent Stem Cells

Jason Ross, Julia Busch, Ellen Mintz, Damian Ng, Alexandra Stanley, David Brafman, V. Reid Sutton, Ignatia Van den Veyver, Karl Willert

Cell Reports, November 20, 2014, DOI: http://dx.doi.org/10.1016/j.celrep.2014.10.049

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Pluripotent Cells Created by Nuclear Transfer Can Prompt Immune Reaction

Pluripotent Cells Created by Nuclear Transfer Can Prompt Immune Reaction

Friday, 21 November 2014

Mouse cells and tissues created through nuclear transfer can be rejected by the body because of a previously unknown immune response to the cell's mitochondria, according to a study in mice by researchers at the Stanford University School of Medicine and colleagues in Germany, England and at MIT.

The findings reveal a likely, but surmountable, hurdle if such therapies are ever used in humans, the researchers said.

Stem cell therapies hold vast potential for repairing organs and treating disease. The greatest hope rests on the potential of pluripotent stem cells, which can become nearly any kind of cell in the body. One method of creating pluripotent stem cells is called somatic cell nuclear transfer, and involves taking the nucleus of an adult cell and injecting it into an egg cell from which the nucleus has been removed.

The promise of the SCNT method is that the nucleus of a patient's skin cell, for example, could be used to create pluripotent cells that might be able to repair a part of that patient's body.

"One attraction of SCNT has always been that the genetic identity of the new pluripotent cell would be the same as the patient's, since the transplanted nucleus carries the patient's DNA," said cardiothoracic surgeon Sonja Schrepfer, MD, PhD, a co-senior author of the study, which will be published online Nov. 20 in Cell Stem Cell.

"The hope has been that this would eliminate the problem of the patient's immune system attacking the pluripotent cells as foreign tissue, which is a problem with most organs and tissues when they are transplanted from one patient to another," added Schrepfer, who is a visiting scholar at Stanford's Cardiovascular Institute. She is also a Heisenberg Professor of the German Research Foundation at the University Heart Center in Hamburg, and at the German Center for Cardiovascular Research.

Possibility of rejection

A dozen years ago, when Irving Weissman, MD, professor of pathology and of developmental biology at Stanford, headed a National Academy of Sciences panel on stem cells, he raised the possibility that the immune system of a patient who received SCNT-derived cells might still react against the cells' mitochondria, which act as the energy factories for the cell and have their own DNA. This reaction could occur because cells created through SCNT contain mitochondria from the egg donor and not from the patient, and therefore could still look like foreign tissue to the recipient's immune system, said Weissman, the other co-senior author of the paper. Weissman is the Virginia and D.K. Ludwig Professor for Clinical Investigation in Cancer Research and the director of the Stanford Institute for Stem Cell Biology and Regenerative Medicine.

That hypothesis was never tested until Schrepfer and her colleagues took up the challenge.

"There was a thought that because the mitochondria were on the inside of the cell, they would not be exposed to the host's immune system," Schrepfer said.

"We found out that this was not the case."

Schrepfer, who heads the Transplant and Stem Cell Immunobiology Laboratory in Hamburg, used cells that were created by transferring the nuclei of adult mouse cells into enucleated eggs cells from genetically different mice. When transplanted back into the nucleus donor strain, the cells were rejected although there were only two single nucleotide substitutions in the mitochondrial DNA of these SCNT-derived cells compared to that of the nucleus donor.

"We were surprised to find that just two small differences in the mitochondrial DNA were enough to cause an immune reaction," she said.

"We didn't do the experiment in humans, but we assume the same sort of reaction could occur," Schrepfer added.

Until recently, researchers were able to perform SCNT in many species, but not in humans. When scientists at the Oregon Health and Science University announced success in performing SCNT with human cells last year, it reignited interest in eventually using the technique for human therapies. Although many stem cell researchers are focused on a different method of creating pluripotent stem cells, called induced pluripotent stem cells, there may be some applications for which SCNT-derived pluripotent cells are better suited.

Handling the reaction

The immunological reactions reported in the new paper will be a consideration if clinicians ever use SCNT-derived stem cells in human therapy, but such reactions should not prevent their use, Weissman said.

"This research informs us of the margin of safety that would be required if, in the distant future, we need to use SCNT to create pluripotent cells to treat someone," he said.

"In that case, clinicians would likely be able to handle the immunological reaction using the immunosuppression methods that are currently available."

In the future, scientists might also lessen the immune reaction by using eggs from someone who is genetically similar to the recipient, such as a mother or sister, Schrepfer added.

Source: Stanford University Medical Center

Contact: Christopher Vaughan 

Reference:

SCNT-Derived ESCs with Mismatched Mitochondria Trigger an Immune Response in Allogeneic Hosts

Tobias Deuse, Dong Wang, Mandy Stubbendorff, Ryo Itagaki, Antje Grabosch, Laura C. Greaves, Malik Alawi, Anne Grünewald, Xiaomeng Hu, Xiaoqin Hua, Joachim Velden, Hermann Reichenspurner, Robert C. Robbins, Rudolf Jaenisch, Irving L. Weissman, Sonja Schrepfer

Cell Stem Cell, November 20, 2014, DOI: http://dx.doi.org/10.1016/j.stem.2014.11.003

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Establishment of Induced Pluripotent Stem Cells from Werner Syndrome Fibroblasts

Milestone for understanding diseases and for the development of new therapies

Tuesday, 18 November 2014

Scientists at Hiroshima University established induced pluripotent stem (iPS) cells from the fibroblasts of Werner Syndrome patients. These results were published in PLOS ONE in an article entitled "Reprogramming Suppresses Premature Senescence Phenotypes of Werner Syndrome Cells and Maintains Chromosomal Stability over Long-Term Culture."

Werner syndrome is characterized by the premature appearance of features associated with normal aging and cancer predisposition. This syndrome occurs frequently in Japan, affecting 1 in 20,000 to 1 in 40,000 people. The therapeutic methods for this disease are very limited and it is expected that iPS cells can be used for the development of innovative therapies.

Dr. Akira Shimamoto and his collaborators analysed patient-derived iPS cells and found that telomeric abnormalities in the fibroblasts of these patients, which were caused by the lack of WRN helicase encoded by the gene responsible for Werner syndrome, were recovered in the iPS cells generated from these patients. Furthermore, Dr. Shimamoto found that the expression levels of aging-related genes, including those encoding cell cycle inhibitors and inflammatory cytokines, in the patient-derived iPS cells were the same as those in normal iPS cells, even though the expression levels of these genes in the fibroblasts of the patients were higher than those in normal fibroblasts.

"So far, the use of patient cells was restricted to blood or dermal cells in basic research. The iPS cells that we have established will provide an opportunity for drug discovery for the treatment of Werner syndrome and also help with better understanding of the mechanism of this disease. In addition, the mutated WRN gene in patient-derived iPS cells can be corrected by genome editing. This advantage will be help in the development of new gene and cell therapies for Werner syndrome," Dr. Shimamoto said.

Associate Professor Akira Shimamoto and Professor Hidetoshi Tahara at the Graduate School of Biomedical & Health Science in Hiroshima University, Professor Koutaro Yokote at the Graduate School of Medicine in Chiba University, Visiting Professor Makoto Goto at the Medical Center East in Tokyo Women's Medical University, and collaborators including the staff at the Cancer Chemotherapy Center in the Japanese Foundation for Cancer Research, Tottori University, and Keio University also participated in the study.

Source: Hiroshima University

Contact: Norifumi Miyokawa

Reference:

Reprogramming Suppresses Premature Senescence Phenotypes of Werner Syndrome Cells and Maintains Chromosomal Stability over Long-Term Culture

Akira Shimamoto, Harunobu Kagawa, Kazumasa Zensho, Yukihiro Sera, Yasuhiro Kazuki, Mitsuhiko Osaki, Mitsuo Oshimura, Yasuhito Ishigaki, Kanya Hamasaki, Yoshiaki Kodama, Shinsuke Yuasa, Keiichi Fukuda, Kyotaro Hirashima, Hiroyuki Seimiya, Hirofumi Koyama, Takahiko Shimizu, Minoru Takemoto, Koutaro Yokote, Makoto Goto, Hidetoshi Tahara

PLOS ONE, published 12 Nov 2014, 10.1371/journal.pone.0112900

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Cardiac Stem Cell Therapy may Heal Heart Damage Caused by Duchenne Muscular Dystrophy

Late-breaking basic science research presented at American Heart Association Scientific Sessions shows stem cell treatment restores heart function damaged by muscular disease

Tuesday, 18 November 2014

Researchers at the Cedars-Sinai Heart Institute have found that injections of cardiac stem cells might help reverse heart damage caused by Duchenne muscular dystrophy, potentially resulting in a longer life expectancy for patients with the chronic muscle-wasting disease.

The study results were presented today at a Breaking Basic Science presentation during the American Heart Association Scientific Sessions in Chicago. After laboratory mice with Duchenne muscular dystrophy were infused with cardiac stem cells, the mice showed steady, marked improvement in heart function and increased exercise capacity.

Duchenne muscular dystrophy, which affects 1 in 3,600 boys, is a neuromuscular disease caused by a shortage of a protein called dystrophin, leading to progressive muscle weakness. Most Duchenne patients lose their ability to walk by age 12. Average life expectancy is about 25. The cause of death often is heart failure because the dystrophin deficiency leads to cardiomyopathy, a weakness of the heart muscle that makes the heart less able to pump blood and maintain a regular rhythm.

"Most research into treatments for Duchenne muscular dystrophy patients has focused on the skeletal muscle aspects of the disease, but more often than not, the cause of death has been the heart failure that affects Duchenne patients," said Eduardo Marbán, MD, PhD, director of the Cedars-Sinai Heart Institute and study leader.

"Currently, there is no treatment to address the loss of functional heart muscle in these patients."

During the past five years, the Cedars-Sinai Heart Institute has become a world leader in studying the use of stem cells to regenerate heart muscle in patients who have had heart attacks. In 2009, Marbán and his team completed the world's first procedure in which a patient's own heart tissue was used to grow specialized heart stem cells. The specialized cells were then injected back into the patient's heart in an effort to repair and regrow healthy muscle in a heart that had been injured by a heart attack. Results, published in The Lancet in 2012, showed that one year after receiving the experimental stem cell treatment, heart attack patients demonstrated a significant reduction in the size of the scar left on the heart muscle.

Earlier this year, Heart Institute researchers began a new study, called ALLSTAR, in which heart attack patients are being infused with allogeneic stem cells, which are derived from donor-quality hearts.

Recently, the Heart Institute opened the nation's first Regenerative Medicine Clinic, designed to match heart and vascular disease patients with appropriate stem cell clinical trials being conducted at Cedars-Sinai and other institutions.

"We are committed to thoroughly investigating whether stem cells could repair heart damage caused by Duchenne muscular dystrophy," Marbán said.

In the study, 78 lab mice were injected with cardiac stem cells. Over the next three months, the lab mice demonstrated improved pumping ability and exercise capacity in addition to a reduction in heart inflammation. The researchers also discovered that the stem cells work indirectly, by secreting tiny fat droplets called exosomes. The exosomes, when purified and administered alone, reproduce the key benefits of the cardiac stem cells.

Marbán said the procedure could be ready for testing in human clinical studies as soon as next year. The process to grow cardiac-derived stem cells was developed by Marbán when he was on the faculty of Johns Hopkins University. Johns Hopkins has filed for a patent on that intellectual property and has licensed it to Capricor, a company in which Cedars-Sinai and Marbán have a financial interest. Capricor is providing funds for the ALLSTAR clinical trial at Cedars-Sinai.

The Cedars-Sinai Heart Institute has been at the forefront of developing investigational stem cell treatments for heart attack patients.

Source: Cedars-Sinai Medical Center

Contact: Sally Stewart

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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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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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Salk Scientists Discover a Key to Mending Broken Hearts

Researchers regenerate and heal mouse hearts by using the molecular machinery the animals had all along

Friday, 07 November 2014

Researchers at the Salk Institute have healed injured hearts of living mice by reactivating long dormant molecular machinery found in the animals’ cells, a finding that could help pave the way to new therapies for heart disorders in humans.

An injured zebrafish heart showing proliferating

cells in the wounded area of the heart (red) and

cardiac muscle cells (green). Credit: Courtesy of

the Salk Institute for Biological Studies. 

The new results, published November 6, 2014 in the journal Cell Stem Cell, suggest that although adult mammals don’t normally regenerate damaged tissue, they may retain a latent ability as a holdover, like their distant ancestors on the evolutionary tree. When the Salk researchers blocked four molecules thought to suppress these programs for regenerating organs, they saw a drastic improvement in heart regeneration and healing in the mice.

The findings provide proof-of-concept for a new type of clinical treatment in the fight against heart disease, which kills about 600,000 people each year in the United States – more than AIDS and all cancer types combined, according to the U.S. Centers for Disease Control and Prevention.

“Organ regeneration is a fascinating phenomenon that seemingly recapitulates the processes observed during development. However, despite our current understanding of how embryogenesis and development proceeds, the mechanisms preventing regeneration in adult mammals have remained elusive,” says the study’s senior author Juan Carlos Izpisua Belmonte, a professor in the Gene Expression Laboratory at Salk and holder of the Roger Guillemin Chair.

Within the genomes of every cell in our bodies, we have what information we need to generate an organ. Izpisua Belmonte’s group has for many years focused on elucidating the key molecules involved in embryonic development as well as those potentially underlying healing responses in regenerative organisms such as the zebrafish.

From left: Alejandro Ocampo, Concepcion

Rodriguez Esteban, Juan Carlos Izpisua

Belmonte, Ignacio Sancho-Martinez, Tomoaki

Hishida and Eric Vazquez. Credit: Courtesy of

the Salk Institute for Biological Studies. 

Indeed, back in 2003, Izpisua Belmonte’s laboratory first identified the signals preceding zebrafish heart regeneration. And in a 2010 Nature paper, the researchers described how regeneration occurred in the zebrafish. Rather than stem cells invading injured heart tissue, the cardiac cells themselves were reverting to a precursor-like state (a process called ‘dedifferentiation’), which, in turn, allowed them to proliferate in tissue.

Although in theory it might have seemed like the next logical step to ask whether mammals had evolutionarily conserved any of the right molecular players for this kind of regenerative reprogramming, in practice it was a scientific risk, recalls Ignacio Sancho-Martinez, a postdoctoral researcher in Izpisua Belmonte’s lab.

“When you speak about these things, the first thing that comes to peoples’ minds is that you’re crazy,” he says.

“It’s a strange sounding idea, since we associate regeneration with salamanders and fish, but not mammals.”

Most other studies have looked to the hearts of neonatal mammals for molecular clues about proliferation, to no avail.

“Instead, we thought, ‘If fish know how to do it, there must be something they can teach us about it,’” says the study’s first author Aitor Aguirre, a postdoctoral researcher in Izpisua Belmonte’s group.

In a dish, heart muscle cells return to a precursor-

like state after pro-regenerative treatment with

microRNA inhibitors. Green shows a

disorganized cardiomyocyte cytoskeleton

indicative of cell dedifferentiation; red shows

mitochondrial organization. Credit: Courtesy of

the Salk Institute for Biological Studies. 

The team decided to focus on microRNAs, in part because these short strings of RNA control the expression of many genes. They performed a comprehensive screen for microRNAs that were changing in their expression levels during the healing of the zebrafish heart and that were also conserved in the mammalian genome.

Their studies uncovered four molecules in particular – MiR-99, MiR-100, Let-7a and Let-7c – that fit their criteria. All were heavily repressed during heart injury in zebrafish and they were also present in rats, mice and humans.

However, in studies of mammalian cells in a culture dish and studies of living mice with heart damage, the group saw that the levels of these molecules were high in adults and did not decline with injury. So the team used adeno-associated viruses specific for the heart to target each of those four microRNAs, suppressing their levels experimentally.

Injecting the inhibitors into the hearts of mice that had suffered a heart attack triggered the regeneration of cardiac cells, improving numerous physical and functional aspects of the heart, such as the thickness of its walls and its ability to pump blood. The scarring caused by the heart attack was much reduced with treatment compared to controls, the researchers found.

The improvements were still obvious three and six months after treatment – a long time in a mouse’s life.

“The good thing is that the success was not limited to the short term, which is quite common in cardiac regenerative biology,” Sancho-Martinez says.

The new study focused only on a handful of 70 some microRNA candidates that turned up in the group’s initial screen. These other molecules will likely also play a part in heart cell proliferation, healing scars and promoting the formation of new blood vessels – all processes critical for heart repair, Sancho-Martinez says. The data are available so that other research groups can focus on molecules that interest them.

The next step for Izpisua Belmonte’s team is to move into larger animals and see whether “regenerative reprogramming” can work in larger hearts, and for extended periods after treatment, says Sancho-Martinez. And, although the virus packaging disappeared from the animals’ bodies by 2 weeks after treatment, the scientists are working on a new way to deliver the inhibitors to avoid the need for viruses altogether.

Source: Salk Institute

Reference:

In vivo reprogramming with conserved regenerative effectors promotes robust mammalian heart regeneration

Authors: Aitor Aguirre, Nuria Montserrat, Serena Zachiggna, Emmanuel Nivet, Tomoaki Hishida, Marie Nicole Krause, Leo Kurian, Alejandro Ocampo, Eric Vazquez-Ferrer, Concepcion Rodriguez-Esteban, Sachin Kumar, James J. Moresco, John R. Yates III, Josep Maria Campistol, Ignacio Sancho-Martinez, Mauro Giacca and Juan Carlos Izpisua Belmonte

Cell Stem Cell, Volume 15, Issue 5, p589–604, 6 November 2014, DOI: http://dx.doi.org/10.1016/j.stem.2014.10.003

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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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Scientists find that SCNT Derived Cells and iPS Cells are Similar

Scientists find that SCNT Derived Cells and iPS Cells are Similar

Thursday, 06 November 2014

A team led by New York Stem Cell Foundation (NYSCF) Research Institute scientists conducted a study comparing induced pluripotent stem (iPS) cells and embryonic stem cells created using somatic cell nuclear transfer (SCNT). The scientists found that the cells derived from these two methods resulted in cells with highly similar gene expression and DNA methylation patterns. Both methods also resulted in stem cells with similar amounts of DNA mutations, showing that the process of turning an adult cell into a stem cell introduces mutations independent of the specific method used. This suggests that both methods of producing stem cells need to be further investigated before determining their suitability for the development of new therapies for chronic diseases.

The NYSCF Research Institute is one of the only laboratories in the world that currently pursues all forms of stem cell research including SCNT and iPS cell techniques for creating stem cells. The lack of laboratories attempting SCNT research was one of the reasons that the NYSCF Research Institute was established in 2006.

"We do not yet know which technique will allow scientists to create the best cells for new cellular therapies," said Susan L. Solomon, NYSCF CEO and co-founder.

"It is critical to pursue both SCNT and iPS cell techniques in order to accelerate research and bring new treatments to patients."

While both techniques result in pluripotent stem cells, or cells that can become any type of cell in the body, the two processes are different. SCNT consists of replacing the nucleus of a human egg cell or oocyte with the nucleus of an adult cell, resulting in human embryonic stem cells with the genetic material of the adult cell. In contrast, scientists create iPS cells by expressing a few key genes in adult cells, like a skin or blood cell, causing the cells to revert to an embryonic-like state. These differences in methods could, in principle, result in cells with different properties. Advances made earlier this year by NYSCF Research Institute scientists that showed that human embryonic stem cells could be derived using SCNT revived that debate.

"Our work shows that we now have two methods for the generation of a patient's personal stem cells, both with great potential for the development of treatments of chronic diseases. Our work will also be welcome news for the many scientists performing basic research on iPS cells. It shows that they are likely working with cells that are very similar to human embryonic stem cells, at least with regard to gene expression and DNA methylation. How the finding of mutations might affect clinical use of stem cells generated from adult cells is the subject of an ongoing debate," said Dr. Dieter Egli, NYSCF Senior Research Fellow, NYSCF - Robertson Investigator, Assistant Professor in Pediatrics & Molecular Genetics at Columbia University, and senior author on the paper.

The study, published today in Cell Stem Cell, compared cell lines derived from the same sources using the two differing techniques, specifically contrasting the frequency of genetic coding mutations seen and measuring how closely the stem cells matched the embryonic state through the analysis of DNA methylation and of gene expression patterns. The scientists showed that both methods resulted in cell types that were similar with regard to gene expression and DNA methylation patterns. This suggested that both methods were effective in turning a differentiated cell into a stem cell.

The scientists also showed that cells derived using both SCNT and iPS techniques showed similar numbers of genetic coding mutations, implying that neither technique is superior in that regard. A similar number of changes in DNA methylation at imprinted genes (genes that are methylated differentially at the maternal versus the paternal allele) were also found. It is important to note that both types of techniques led to cells that had more of these aberrations than embryonic stem cells derived from an unfertilized human oocyte, or than embryonic stem cells derived from leftover IVF embryos. These findings suggest that a small number of defects are inherent to the generation of stem cells from adult differentiated cells and occur regardless of the method used.

Source: New York Stem Cell Foundation

Contact: David McKeon

Reference:

Comparable Frequencies of Coding Mutations and Loss of Imprinting in Human Pluripotent Cells Derived by Nuclear Transfer and Defined Factors

Bjarki Johannesson, Ido Sagi, Athurva Gore, Daniel Paull, Mitsutoshi Yamada, Tamar Golan-Lev, Zhe Li, Charles LeDuc, Yufeng Shen, Samantha Stern, Nanfang Xu, Hong Ma, Eunju Kang, Shoukhrat Mitalipov, Mark V. Sauer, Kun Zhang, Nissim Benvenisty, Dieter Egli

Cell Stem Cell, Volume 15, Issue 5, p634–642, 6 November 2014, DOI: http://dx.doi.org/10.1016/j.stem.2014.10.002

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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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