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.

Source: University of North Carolina School of Medicine

Contact: Les Lang

Reference:

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.

Source: Scripps Research Institute 

Contact: Mika Ono

Reference:

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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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."

Source: Johns Hopkins Medical Institutions 

Contact: Vanessa McMains
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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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