Tuesday, 15 May 2012

New York Stem Cell Foundation Scientist Grows Bone from Human Embryonic Stem Cells

New York Stem Cell Foundation Scientist Grows Bone from Human Embryonic Stem Cells

Monday, 14 May 2012

Dr. Darja Marolt, an Investigator at The New York Stem Cell Foundation (NYSCF) Laboratory, is lead author on a study showing that human embryonic stem cells can be used to grow bone tissue grafts for use in research and potential therapeutic application. Dr. Marolt conducted this research as a post-doctoral NYSCF – Druckenmiller Fellow at Columbia University in the laboratory of Dr. Gordana Vunjak-Novakovic.

The study is the first example of using bone cell progenitors derived from human embryonic stem cells to grow compact bone tissue in quantities large enough to repair centimetre-sized defects. When implanted in mice and studied over time, the implanted bone tissue supported blood vessel ingrowth, and continued development of normal bone structure, without demonstrating any incidence of tumour growth.

Dr. Marolt's work is a significant step forward in using pluripotent stem cells to repair and replace bone tissue in patients. Bone replacement therapies are relevant in treating patients with a variety of conditions, including wounded military personnel, patients with birth defects, or patients who have suffered other traumatic injury.

Since conducting this work as proof of principle at Columbia University, Dr. Marolt has continued to build upon this research as an Investigator in the NYSCF Laboratory, developing bone grafts from induced pluripotent stem (iPS) cells. iPS cells are similar to embryonic stem cells in that they can also give rise to nearly any type of cell in the body, but iPS cells are produced from adult cells and as such are individualized to each patient. By using iPS cells rather than embryonic stem cells to engineer tissue, Dr. Marolt hopes to develop personalized bone grafts that will avoid immune rejection and other implant complications.

Contact: David McKeon

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Friday, 11 May 2012

Successful Stem Cell Differentiation Requires DNA Compaction

Successful Stem Cell Differentiation Requires DNA Compaction

Friday, 11 May 2012

New research findings show that embryonic stem cells unable to fully compact the DNA inside them cannot complete their primary task: differentiation into specific cell types that give rise to the various types of tissues and structures in the body.

This image shows hematoxylin and eosin 
staining of sections of wild-type (top row) 
and H1 triple-knockout (bottom row) embryoid 
bodies. After 14 days in rotary suspension 
culture, the wild-type embryoid bodies showed 
more differentiated morphologies with cysts 
forming (black arrows) and the knockout 
embryoid bodies failed to form cavities (far 
right). Credit: Yuhong Fan. 
Researchers from the Georgia Institute of Technology and Emory University found that chromatin compaction is required for proper embryonic stem cell differentiation to occur. Chromatin, which is composed of histone proteins and DNA, packages DNA into a smaller volume so that it fits inside a cell.

A study published on May 10, 2012 in the journal PLoS Genetics found that embryonic stem cells lacking several histone H1 subtypes and exhibiting reduced chromatin compaction suffered from impaired differentiation under multiple scenarios and demonstrated inefficiency in silencing genes that must be suppressed to induce differentiation.

"While researchers have observed that embryonic stem cells exhibit a relaxed, open chromatin structure and differentiated cells exhibit a compact chromatin structure, our study is the first to show that this compaction is not a mere consequence of the differentiation process but is instead a necessity for differentiation to proceed normally," said Yuhong Fan, an assistant professor in the Georgia Tech School of Biology.

Fan and Todd McDevitt, an associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, led the study with assistance from Georgia Tech graduate students Yunzhe Zhang and Kaixiang Cao, research technician Marissa Cooke, and postdoctoral fellow Shiraj Panjwani.

The work was supported by the National Institutes of Health's National Institute of General Medical Sciences (NIGMS), the National Science Foundation, a Georgia Cancer Coalition Distinguished Scholar Award, and a Johnson & Johnson/Georgia Tech Healthcare Innovation Award.

These are phase contrast images showing that 
H1 triple-knockout (bottom) embryonic stem 
cells were unable to adequately form neurites 
and neural networks compared to wild-type 
embryonic stem cells (top). Credit: Yuhong 
To investigate the impact of linker histones and chromatin folding on stem cell differentiation, the researchers used embryonic stem cells that lacked three subtypes of linker histone H1 – H1c, H1d and H1e – which is the structural protein that facilitates the folding of chromatin into a higher-order structure. They found that the expression levels of these H1 subtypes increased during embryonic stem cell differentiation, and embryonic stem cells lacking these H1s resisted spontaneous differentiation for a prolonged time, showed impairment during embryoid body differentiation and were unsuccessful in forming a high-quality network of neural cells.

"This study has uncovered a new, regulatory function for histone H1, a protein known mostly for its role as a structural component of chromosomes," said Anthony Carter, who oversees epigenetics grants at NIGMS.

"By showing that H1 plays a part in controlling genes that direct embryonic stem cell differentiation, the study expands our understanding of H1's function and offers valuable new insights into the cellular processes that induce stem cells to change into specific cell types."

During spontaneous differentiation, the majority of the H1 triple-knockout embryonic stem cells studied by the researchers retained a tightly packed colony structure typical of undifferentiated cells and expressed high levels of Oct4 for a prolonged time. Oct4 is a pluripotency gene that maintains an embryonic stem cell's ability to self-renew and must be suppressed to induce differentiation.

"H1 depletion impaired the suppression of the Oct4 and Nanog pluripotency genes, suggesting a novel mechanistic link by which H1 and chromatin compaction may mediate pluripotent stem cell differentiation by contributing to the epigenetic silencing of pluripotency genes," explained Fan.

"While a significant reduction in H1 levels does not interfere with embryonic stem cell self-renewal, it appears to impair differentiation."

This image shows immunostaining of wild-type 
(top) and H1 triple-knockout (bottom) cultures 
under a neural differentiation protocol. The H1 
triple-knockout cells were defective in forming 
neuronal and glial cells and a neural network, 
which is essential for nervous system 
development. Credit: Yuhong Fan. 
The researchers also used a rotary suspension culture method developed by McDevitt to produce with high efficiency homogenous 3D clumps of embryonic stem cells called embryoid bodies. Embryoid bodies typically contain cell types from all three germ layers – the ectoderm, mesoderm and endoderm – that give rise to the various types of tissues and structures in the body. However, the majority of the H1 triple-knockout embryoid bodies formed in rotary suspension culture lacked differentiated structures and displayed gene expression signatures characteristic of undifferentiated stem cells.

"H1 triple-knockout embryoid bodies displayed a reduced level of activation of many developmental genes and markers in rotary culture, suggesting that differentiation to all three germ layers was affected." noted McDevitt.

The embryoid bodies also lacked the epigenetic changes at the pluripotency genes necessary for differentiation, according to Fan.

"When we added one of the deleted H1 subtypes to the embryoid bodies, Oct4 was suppressed normally and embryoid body differentiation continued," explained Fan.

"The epigenetic regulation of Oct4 expression by H1 was also evident in mouse embryos."

In another experiment, the researchers provided an environment that would encourage embryonic stem cells to differentiate into neural cells. However, the H1 triple-knockout cells were defective in forming neuronal and glial cells and a neural network, which is essential for nervous system development. Only 10 percent of the H1 triple-knockout embryoid bodies formed neurites and they produced on average eight neurites each. In contrast, half of the normal embryoid bodies produced, on average, 18 neurites.

In future work, the researchers plan to investigate whether controlling H1 histone levels can be used to influence the reprogramming of adult cells to obtain induced pluripotent stem cells, which are capable of differentiating into tissues in a way similar to embryonic stem cells.

Contact: Abby Robinson

Histone H1 Depletion Impairs Embryonic Stem Cell Differentiation
Yunzhe Zhang, Marissa Cooke, Shiraj Panjwani,Kaixiang Cao, Beth Krauth, Po-Yi Ho, Magdalena Medrzycki, Dawit T. Berhe, Chenyi Pan, Todd C. McDevitt, Yuhong Fan
PLoS Genet 8(5): e1002691. doi:10.1371/journal.pgen.1002691

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Sunday, 6 May 2012

New York’s Investment in Stem Cell Research

Program generate new medical treatments and innovative research

Sunday, 06 May 2012

The Associated Medical Schools of New York (AMSNY) today released a 2012 report showing how New York’s stem cell program has enabled it to emerge as a leader in stem cell research, and strengthened the state’s economy through job creation.

“This report demonstrates the foresight of New York’s leaders in funding stem cell research. Not only are scientists across the state making progress towards understanding how to treat or prevent debilitating diseases, New York’s stem cell program generates jobs, attracts promising young women and men into medical and scientific careers, and enhances our state’s leadership in biomedical research,” said Dr. Lee Goldman, AMSNY’s chair, and executive vice president and dean of the Columbia University College of Physicians & Surgeons.

In 2007, New York State allocated $600 million over 11 years to the Empire State Stem Cell Program (NYSTEM), making it the second largest publically-financed stem cell program in the country. To date, New York has awarded nearly $223 million of the $600 million to support stem cell research for the purpose of exploring innovative cures and treatment to life threatening and chronic illnesses, such as Alzheimer’s, and Arterial Lateral Sclerosis (ALS).

In addition to supporting ground-breaking stem cell research projects, the state’s investment has been a tool for economic development by creating or maintaining more than 400 jobs at AMSNY institutions since the program’s inception, and is attracting world-renowned researchers and scientists to New York.

Dr. Ronald G. Crystal, chairman and professor of Genetic Medicine, Weill Cornell Medical College, said:
“Stem cell research holds the promise of tomorrow’s medical breakthroughs to improve human health. Continuing to advance stem cell research in New York is vital since we are one of the strongest and largest centres of stem cell science in the world and home to some of the most talented medical researchers. Funding for stem cell research in New York is critical and any reduction in support will slow our progress toward securing the important discoveries and cures for the devastating diseases that affect New Yorkers.”

Dr. Allen M. Spiegel, dean of Albert Einstein College of Medicine said:
“The Albert Einstein College of Medicine has made a major commitment to the stem cell research field because it offers tremendous potential for understanding the causes of and developing better treatments for diseases like cancer, type 1 diabetes, and Parkinson's. The NYSTEM program has been critical in helping Einstein support innovative and technically advanced work in this vital field.”

Dennis S. Charney, MD, Anne and Joel Ehrenkranz Dean, Mount Sinai School of Medicine, and executive vice president for Academic Affairs at the Mount Sinai Medical Center said:
“Stem cell research has the potential to transform the way medicine is practiced and it is an area that has been one of Mount Sinai’s top priorities. Our researchers at the Black Family Stem Cell Institute, with funding from The Empire State Stem Cell Program, were the first to develop abnormal heart cells from human stem cells, allowing them to study potential treatments for cardiomyopathy. Maintaining funding for stem cell research is essential to the continued success of our research programs which, in addition to studying heart disease, include researching potential treatments for schizophrenia, autism, Alzheimer’s disease and diabetes.”

Ruth Lehmann, PhD, Director of the Kimmel Center for Stem Cell Biology and the Skirball Institute of Biomolecular Medicine at NYU School of Medicine, part of the NYU Langone Medical Center, said:
“In the current political and economic climate, where the government’s funding choices are being scrutinized, it is important to realize the impact of continuing to support early stage research and development, particularly in stem cell biology. The majority of scientific and medical discoveries originate in early stage laboratory research. By focusing its support on early stage research, NYSTEM has attracted new researchers to the field of stem cell biology who are bringing creative ideas and new approaches to this important field. At NYU Langone, this support has contributed to understanding the underlying causes of leukaemia and to developing new approaches for cancer stem cell therapies. Without this funding, academic medical centres cannot thrive, and our leadership as innovators in health and science is threatened.”

According to the report, New York’s funding commitment is critical to the state’s stem cell research and patient communities given its unique nature. NYSTEM funds early stage projects that have not been able to access other funding sources such as those granted by the National Institutes of Health (NIH). NYSTEM also is distinct among other research grants in that it provides funding for capital projects and equipment, allowing institutions to develop or expand their stem cell research infrastructure.

“NYSTEM has made it possible for cutting-edge stem cell research to thrive in New York,” said Jo Wiederhorn, AMSNY’s president and CEO.

“Across the state, medical schools and research institutions have been renovating laboratories and building state-of-the-art stem cell centers – spurring economic development and fostering medical innovation. None of this would have been possible without NYSTEM.”

The program also has stimulated state research institutions to make their own investments in stem cell research, which in turn has improved their ability to win additional NIH grants and attract private sector and philanthropic funding.

Source: AMSNY 2012 STEM Cell Report

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Saturday, 5 May 2012

A Single Stem Cell Mutation Triggers Fibroid Tumours

Mutated stem cell 'goes wild' in frenzied tumour expansion

Saturday, 05 May 2012

Fibroid uterine tumours affect an estimated 15 million women in the United States, causing irregular bleeding, anaemia, pain and infertility. Despite the high prevalence of the tumours, which occur in 60 percent of women by age 45, the molecular cause has been unknown.

New Northwestern Medicine preclinical research has for the first time identified the molecular trigger of the tumour – a single stem cell that develops a mutation, starts to grow uncontrollably and activates other cells to join its frenzied expansion.

"It loses its way and goes wild," said Serdar Bulun, M.D., the chair of obstetrics and gynaecology at Northwestern University Feinberg School of Medicine and Northwestern Memorial Hospital.

"No one knew how these came about before. The stem cells make up only 1 ½ percent of the cells in the tumour, yet they are the essential drivers of its growth."

The paper is published in the journal PLoS ONE. Masanori Ono, M.D., a post-doctoral student in Bulun's lab, is the lead author.

The stem cell initiating the tumour carries a mutation called MED12. Recently, mutations in the MED12 gene have been reported in the majority of uterine fibroid tissues. Once the mutation kicks off the abnormal expansion, the tumours grow in response to steroid hormones, particularly progesterone.

For the study, researchers examined the behaviour of human fibroid stem cells when grafted into a mouse, a novel model initiated by Northwestern scientist Takeshi Kurita, a research associate professor of obstetrics and gynaecology. The most important characteristic of fibroid stem cells is their ability to generate tumours. Tumours originating from the fibroid stem cell population grew 10 times larger compared to tumours initiated with the main cell population, suggesting a key role of these tumour stem cells is to initiate and sustain tumour growth.

"Understanding how this mutation directs the tumour growth gives us a new direction to develop therapies," said Bulun, also the George H. Gardner Professor of Clinical Gynecology.

Contact: Marla Paul

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Researchers Develop New Muscular Dystrophy Treatment Approach Using Human Stem Cells

Researchers Develop New Muscular Dystrophy Treatment Approach Using Human Stem Cells

Saturday, 05 May 2012

Researchers from the University of Minnesota's Lillehei Heart Institute have effectively treated muscular dystrophy in mice using human stem cells derived from a new process that – for the first time – makes the production of human muscle cells from stem cells efficient and effective.

The research, published today in Cell Stem Cell, outlines the strategy for the development of a rapidly dividing population of skeletal myogenic progenitor cells (muscle-forming cells) derived from induced pluripotent (iPS) cells. iPS cells have all of the potential of embryonic stem (ES) cells, but are derived by reprogramming skin cells. They can be patient-specific, which renders them unlikely to be rejected, and do not involve the destruction of embryos.

This is the first time that human stem cells have been shown to be effective in the treatment of muscular dystrophy.

According to U of M researchers – who were also the first to use ES cells from mice to treat muscular dystrophy – there has been a significant lag in translating studies using mouse stem cells into therapeutically relevant studies involving human stem cells. This lag has dramatically limited the development of cell therapies or clinical trials for human patients.

The latest research from the U of M provides the proof-of-principle for treating muscular dystrophy with human iPS cells, setting the stage for future human clinical trials.

"One of the biggest barriers to the development of cell-based therapies for neuromuscular disorders like muscular dystrophy has been obtaining sufficient muscle progenitor cells to produce a therapeutically effective response," said principal investigator Rita Perlingeiro, Ph.D., associate professor of medicine in the Medical School's Division of Cardiology.

"Up until now, deriving engraftable skeletal muscle stem cells from human pluripotent stem cells hasn't been possible. Our results demonstrate that it is indeed possible and sets the stage for the development of a clinically meaningful treatment approach."

Upon transplantation into mice suffering from muscular dystrophy, human skeletal myogenic progenitor cells provided both extensive and long-term muscle regeneration which resulted in improved muscle function.

To achieve their results, U of M researchers genetically modified two well-characterized human iPS cell lines and an existing human ES cell line with the PAX7 gene. This allowed them to regulate levels of the Pax7 protein, which is essential for the regeneration of skeletal muscle tissue after damage. The researchers found this regulation could prompt naïve ES and iPS cells to differentiate into muscle-forming cells.

Up until this point, researchers had struggled to make muscle efficiently from ES and iPS cells. PAX7 – induced at exactly the right time – helped determine the fate of human ES and iPS cells, pushing them into becoming human muscle progenitor cells.

Once Dr. Perlingeiro's team was able to pinpoint the optimal timing of differentiation, the cells were well suited to the regrowth needed to treat conditions such as muscular dystrophy. In fact, Pax7-induced muscle progenitors were far more effective than human myoblasts at improving muscle function. Myoblasts, which are cell cultures derived from adult muscle biopsies, had previously been tested in clinical trials for muscular dystrophy, however the myoblasts did not persist after transplantation.

"Seeing long-term maintenance of these cells without major adverse side effects is exciting," said Perlingeiro.

"Our research proves that these differentiated stem cells have real staying power in the fight against muscular dystrophy."

According to John Wagner, M.D., scientific director of clinical research at the University's Stem Cell Institute and renowned blood and marrow transplant expert,
"This research is a phenomenal breakthrough. Dr. Perlingeiro and her collaborators have overcome one of the most significant obstacles to moving stem cell therapies into the treatment of children with devastating and life threatening muscular dystrophies."

The U of M researchers say alternative methods of Pax7 induction will need to be investigated before this study can be turned into a human clinical trial. Their method of delivering the Pax7 protein involved genetic modification of cells with viruses and because viruses sometimes cause mutations, they add risk to a clinical trial. But the U of M researchers are committed to developing a safe and effective clinical protocol, and are actively testing alternate methods of delivering Pax7.

Contact: Caroline Marin

Human ES- and iPS-Derived Myogenic Progenitors Restore DYSTROPHIN and Improve Contractility upon Transplantation in Dystrophic Mice
Radbod Darabi, Robert W. Arpke, Stefan Irion, John T. Dimos, Marica Grskovic, Michael Kyba, Rita C.R. Perlingeiro
Cell Stem Cell, Volume 10, Issue 5, 610-619, 4 May 2012, 10.1016/j.stem.2012.02.015

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Friday, 4 May 2012

Stem Cells Poised to Self-destruct for the Good of the Embryo

Stem Cells Poised to Self-destruct for the Good of the Embryo

Friday, 04 May 2012

Embryonic stem cells — those revered cells that give rise to every cell type in the body — just got another badge of honour. If they suffer damage that makes them a threat to the developing embryo, they swiftly fall on their swords for the greater good, according to a study published online May 3, 2012 in the journal Molecular Cell.

This is an image depicting active Bax 
(red) located at Golgi of human embryonic 
stem cells. Nuclei are stained in blue.
Credit: Deshmukh Lab, UNC-Chapel Hill. 
The finding offers a new glimpse into the private lives of stem cells that could help scientists use them to grow new neurons or other cells to replace those that have been lost in patients with Parkinson's and other diseases.

"Despite the huge potential of stem cells for therapeutic use, very few people have actually investigated their basic biology," said study senior researcher Mohanish Deshmukh, PhD, professor of cell and developmental biology at the University of North Carolina at Chapel Hill.

"These results could have significant implications from a therapeutic perspective."

Of all the important things our bodies' cells do, staying alive is clearly key. But a cell's ability to die when something goes wrong is equally critical. For example, a faulty self-destruct button is one factor that allows cancer cells to proliferate unchecked and cause tumours.

Deshmukh and his colleagues discovered stem cells are extremely sensitive to DNA damage, which can be caused by factors like chemicals, radiation or viruses. The experiment showed that virtually 100 percent of human embryonic stem cells treated with a DNA-damaging drug killed themselves within 5 hours, as compared to 24 hours for other types of cells.

"That's an incredibly rapid rate of death," said Deshmukh, who also is a member of the UNC Neuroscience Center and Lineberger Comprehensive Cancer Center.

The hair-trigger suicidal response is an important adaptation for embryonic stem cells, said the UNC School of Medicine researcher, because a slower response could allow DNA damage to proliferate and harm the embryo.

"Mutations that develop in these cells could be catastrophic for the developing organism, so it would make sense for these cells to be rapidly eliminated."

The key to the stem cells' quick response is that they pre-activate a critical protein called Bax, the researchers found. In most cells, Bax is kept in an inactive form, waiting for a long chain of events to rouse it into action if the cell becomes damaged enough to kill itself. In human embryonic stem cells, the team found Bax standing at attention in its active form in the Golgi apparatus, a part of the cell that processes and modifies proteins.

"What these cells do is very clever," said Deshmukh.

"They have activated Bax, but they've also parked it in a safe little compartment — the Golgi."

If the cell detects DNA damage, Bax zips over to the mitochondrion (the cell's power plant), where it signals other proteins to shut the cell down.

It's like starting a 100-yard race at the 80-yard line, said Deshmukh. You're guaranteed to get to the finish line first because you did most of the work before the race began. However, there are built-in safeguards against a hair trigger activation of death. Pre-activated Bax is housed in the Golgi keeping the protein from accidentally triggering cell death when it's not warranted.

This extreme sensitivity to DNA damage lasts only a few days during early development. After the embryonic stem cells begin differentiating into early progenitors that give rise to specific cell types (like heart cells or skin cells), Bax reverts to its inactive state.

Contact: Les Lang

Human Embryonic Stem Cells Have Constitutively Active Bax at the Golgi and Are Primed to Undergo Rapid Apoptosis
Raluca Dumitru, Vivian Gama, B. Matthew Fagan, Jacquelyn J. Bower, Vijay Swahari, Larysa H. Pevny, and Mohanish Deshmukh
Molecular Cell, 03 May 2012, 10.1016/j.molcel.2012.04.002

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Study Reveals Dynamic Changes in Gene Regulation in Human Stem Cells

Results suggest researchers implement careful quality control

Friday, 04 May 2012

A team led by scientists at The Scripps Research Institute and the University of California (UC) San Diego has discovered a new type of dynamic change in human stem cells.

Last year, this team reported recurrent changes in the genomes of human pluripotent stem cells as they are expanded in culture. The current report, which appears in the May 4, 2012 issue of the journal Cell Stem Cell, shows that these cells can also change their epigenomes, the patterns of DNA modifications that regulate the activity of specific genes—sometimes radically. These changes may influence the cells' abilities to serve as models of human disease and development.

"Our results show that human pluripotent stem cells change during expansion and differentiation in ways that are not easily detected, but that have important implications in using these cells for basic and clinical research," said team leader Louise Laurent, assistant professor in the UC San Diego School of Medicine.

Human pluripotent stem cells can give rise to virtually every type of cell in the body. Because of this remarkable quality, they hold huge potential for cell replacement therapies and drug development.

Many avenues of stem cell research focus on determining how genes are turned on and off during the course of normal development or at the onset of a disease transformation. It is widely accepted that gene activation and silencing play important roles in transforming all-purpose stem cells into the specific adult cell types that make up the specialized tissues of organs such as the heart and brain.

In the new study, Laurent and her collaborator, Professor Jeanne Loring of Scripps Research, and their colleagues focused on understanding gene silencing via DNA methylation, a process whereby bits of DNA are chemically marked with tags that prevent the genes from being expressed, effectively switching them off. Errors in gene silencing via DNA methylation are known contributors to serious developmental defects and cancer.

Specifically, the team assessed the state of both DNA methylation and gene expression in the most comprehensive set of human stem cell samples to date, comprised of more than 200 human pluripotent stem cell samples from more than 100 cell lines, along with 80 adult cell samples representing 17 distinct tissue types. The researchers used a new global DNA methylation array, developed in collaboration with Illumina, Inc, which detects the methylation state of 450,000 sites in the human genome. The results showed surprising changes in patterns of DNA methylation in the stem cells. Because of the unprecedented breadth of the study, the researchers were able to determine the frequency of different types of changes.

One of the anomalies highlighted by the study centres on X chromosomes. Since female cells contain two X chromosomes and males only one, one of the X chromosomes in females is normally silenced by DNA methylation through a process called X-chromosome inactivation (XCI). The new study demonstrated that a majority of female human pluripotent stem cells cultured in the lab lost their X chromosome inactivation over time, resulting in cells with two active X chromosomes.

This phenomenon could affect stem cell-based models of diseases caused by mutations of the X chromosome, such as Lesch-Nyhan disease, the researchers note. These cell-based models require that only the diseased copy of an X-linked gene be expressed, with the normal copy of the gene in females silenced via XCI. As the originally inactive X chromosome becomes active, the normal copy of the gene is expressed, changing the phenotype of the cells from diseased to normal.

"If an X chromosome that was assumed to be inactive is actually active, scientists may find that their cells perplexingly change from mutant to normal over time in culture," Loring said.

Another epigenomic aberration noted in pluripotent cells was in imprinted genes. Human cells contain two copies of most genes: one inherited from the mother and one from the father. In most cases, both the maternal and paternal copies of a gene are expressed equally. This is not the case, however, for imprinted genes, some of which are only expressed from the paternal chromosomes and others expressed only from the maternal chromosomes. This parent-of-origin specific gene expression involves silencing of one of the copies of the gene. Abnormalities in this selective silencing of genes can lead to serious developmental diseases.

The study found that, while the patterns of DNA methylation required to maintain imprinted gene silencing were stable in all of the somatic tissues, surprisingly, frequent aberrations in the patterns of DNA methylation existed in imprinted genes in the stem cells. Some of these aberrations arose very early in the establishment of the cell lines, while others crept in with the passage of time.

Interestingly, the team was able to link at least some of these aberrations to the conditions under which the stem cells were cultured in the lab. This suggests that researchers who use stem cells to study diseases linked to genomic imprinting will need to use conditions that best maintain imprinted gene silencing.

The researchers found another surprise — this one having to do with the basic process by which stem cells become specialized adult cells. Scientists have assumed that most genes are active at the earliest stages of human development, and that unnecessary ones are switched off as the cells developed specialized functions.

"For example, during the process of differentiation from a stem cell into a neuron, you might expect to observe silencing of all the genes that are important for the kidney, the pancreas, and the liver," said Kristopher Nazor, a Scripps Research Kellogg School of Science and Technology graduate student who is lead author of the study.

"But we found something quite different."

When the team compared stem cells with adult cells taken from tissue samples, rather than seeing mostly active genes in the stem cells and selectively silenced genes in the adult ones, they saw the opposite: in the stem cells, the researchers found that genes linked to the development of specialized tissue cells were silent and methylated, while in the adult cells regions of DNA involved in cell type specification were active and unmethylated. The scientists could reproduce some aspects of the developmental changes in culture: when stem cells were differentiated into neural cells in the culture dish, the patterns of DNA methylation became similar to those seen in human brain tissue.

This implies that, contrary to conventional wisdom, the genes responsible for transforming stem cells into tissue cells were initially silent, and were switched on during the process of differentiation.

Contact: Mika Ono

Recurrent Variations in DNA Methylation in Human Pluripotent Stem Cells and Their Differentiated Derivatives
Kristopher L. Nazor, Gulsah Altun, Candace Lynch, Ha Tran, Julie V. Harness, Ileana Slavin, Ibon Garitaonandia, Franz-Josef Müller, Yu-Chieh Wang, Francesca S. Boscolo, Eyitayo Fakunle, Biljana Dumevska, Sunray Lee, Hyun Sook Park, Tsaiwei Olee, Darryl D. D'Lima, Ruslan Semechkin, Mana M. Parast, Vasiliy Galat, Andrew L. Laslett, Uli Schmidt, Hans S. Keirstead, Jeanne F. Loring, Louise C. Laurent
Cell Stem Cell, 2012; 10 (5): 620 DOI: 10.1016/j.stem.2012.02.013

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Wednesday, 2 May 2012

Improved Adult-derived Human Stem Cells have Fewer Genetic Changes than Expected

Study lends support to safe use for therapy
Wednesday, 02 May 2012

A team of researchers from Johns Hopkins University and the National Human Genome Research Institute has evaluated the whole genomic sequence of stem cells derived from human bone marrow cells — so-called induced pluripotent stem (iPS) cells — and found that relatively few genetic changes occur during stem cell conversion by an improved method. The findings, reported in the March issue of Cell Stem Cell, the official journal of the International Society for Stem Cell Research (ISSCR), will be presented at the annual ISSCR meeting in June.

"Our results show that human iPS cells accrue genetic changes at about the same rate as any replicating cells, which we don't feel is a cause for concern," says Linzhao Cheng, Ph.D., a professor of medicine and oncology, and a member of the Johns Hopkins Institute for Cell Engineering.

Each time a cell divides, it has the chance to make errors and incorporate new genetic changes in its DNA, Cheng explains. Some genetic changes can be harmless, but others can lead to changes in cell behaviour that may lead to disease and, in the worst case, to cancer.

In the new study, the researchers showed that iPS cells derived from adult bone marrow cells contain random genetic changes that do not specifically predispose the cells to form cancer.

"Little research was done previously to determine the number of DNA changes in stem cells, but because whole genome sequencing is getting faster and cheaper, we can now more easily assess the genetic stability of these cells derived by various methods and from different tissues," Cheng says. Last year, a study published in Nature suggested higher than expected cancer gene mutation rates in iPS cells created from skin samples, which, according to Cheng, raised great concerns to many in the field pertaining to usefulness and safety of the cells. This study analysed both viral and the improved, non-viral methods to turn on stem cell genes making the iPS cells. 

To more thoroughly evaluate the number of genetic changes in iPS cells created by the improved, non-viral method, Cheng's team first converted human blood-forming cells or their support cells, so-called marrow stromal cells (MSCs) in adult bone marrow into iPS cells by turning on specific genes and giving them special nutrients. The researchers isolated DNA from  – and sequenced  – the genome of each type of iPS cells, in comparison with the original cells from which the iPS cells were derived.

Cheng says they then counted the number of small DNA differences in each cell line compared to the original bone marrow cells. A range of 1,000 to 1,800 changes in the nucleic acid "letters" A, C, T and G occurred across each genome, but only a few changes were found in actual genes – DNA sequences that act as blueprints for our body's proteins. Such genes make up two percent of the genome.

The blood-derived iPS cells contained six and the MSC-derived iPS cells contained 12 DNA letter changes in genes, which led the researchers to conclude that DNA changes in iPS cells are far more likely to occur in the spaces between genes, not in the genes themselves.

Next, the investigators examined the severity of the DNA changes – how likely each one would disrupt the function of each gene. They found that about half of the DNA changes were "silent," meaning these altered blueprints wouldn't change the nucleic acid building code for its corresponding protein or change its function.
For the remaining DNA changes, the researchers guessed these would, in fact, disrupt the function of the gene by either making the gene inactive or changing the way the gene works. Since each cell contains two copies of each gene, in many cases the other, normal copy of the gene could compensate for a disrupted gene, Cheng and the team reasoned.

Cheng cautions that disrupting a single gene copy could pose a problem though, for example, by shutting down a tumour suppressor gene that prevents cells from malignant growth. However, none of the disrupted genes his team found have been implicated in cancer.

He also noted the absence of overlap in the DNA changes found among the different stem cell lines examined, implying that the changes were random and unlikely to cluster.

Based on these findings, Cheng says, iPS cells don't seem to pose a heightened cancer risk, but the risk is not zero, the researchers say.

"We need to sequence more iPS cell lines, including those derived from different cell types and ones using different methods of stem cell conversion, before we have a better picture of mutation rates and spectrums in the iPS cell lines," says Paul Liu, M.D., Ph.D., co-senior author and the deputy scientific director at the National Human Genome Research Institute.

Just because these DNA changes in the stem cells don't specifically select for cancer formation, he adds, doesn't mean that cancer mutations can't arise in other iPS cells. Liu adds that to be on the safe side "it should become a routine procedure to sequence iPS cells before they are used in the clinic."

Contact: Vanessa McMains

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