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Thread: Human embryonic stem cell lines accumulate changes in their genetic material over time

  1. #1
    Senior Member
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    Jun 2005

    Human embryonic stem cell lines accumulate changes in their genetic material over time

    <H2>An international team of researchers has discovered that human embryonic stem cell lines accumulate changes in their genetic material over time.

    The findings do not limit the utility of the cells for some types of research or for some future clinical applications, the researchers say, but draw attention to the need to closely monitor stem cell lines for genetic changes and to study how these alterations affect the cells' behavior. The researchers' work is described in the Sept. 4 online edition of Nature Genetics.

    "This is just the first step," says Aravinda Chakravarti, Ph.D., one of the research team's leaders and professor and director of the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins. "While this is a snapshot of the genomic changes that can happen, it's certainly not everything going on. We still need comprehensive analyses of the changes and what they mean for the functions of embryonic stem cells."

    "Embryonic stem cells are actually far more genetically stable than other stem cells, but our work shows that even they can accumulate potentially deleterious changes over time," adds Anirban Maitra, M.B.B.S., an assistant professor of pathology at Johns Hopkins who shares first authorship of the paper with Dan Arking, Ph.D., an instructor at Hopkins. Both are members of the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins. "Now it will be important to figure out why these changes occur, how they affect the cells' behavior and how time affects other human embryonic stem cell lines."

    The researchers in the United States, Singapore, Canada and Sweden compared "early" and "late" batches of each of nine federally approved human embryonic stem cell lines. Twenty-nine human embryonic stem cell lines from seven different companies are approved by the United States National Institutes of Health under President George W. Bush's policy restricting federal funding of this research to cell lines in existence before his announcement of the policy at 9 p.m. ET, Aug. 9, 2001. The dozens of human embryonic stem cell lines developed since that announcement cannot be used in federally funded research.

    Most of the "late" batches of stem cells -- those grown in the lab a year to three years longer than their early counterparts -- displayed gross changes in the number of copies of chromosomes or parts of chromosomes, in the marks that control whether a gene is used by the cell, or in the sequence of DNA found in the cell's mitochondria.

    "The majority of the lines we tested had genetic changes over time," says Chakravarti. "Whenever you have something in a culture dish, it can change, and it will be important to identify, keep track of and understand these changes."

    At this point, the precise effects of these changes on the cells aren't known, but some of the changes resemble those seen in cancerous cells. At any rate, the changes presumably became entrenched in a particular cell line because they conferred some advantage as the cells were grown in laboratory dishes. Whether the changes affect the stem cells' abilities to become other cell types is also unknown.

    Although research with human embryonic stem cells is still in the lab -- not the clinic -- focusing on what the cells can do and how they are controlled, the hope is that in the future these cells might help replace or repair tissues lost to disease or injury. Because embryonic stem cells can become any type of cell found in the body, in theory they could replace certain pancreas cells in people with type I diabetes, or regenerate brain cells lost in a person with Parkinson's disease, for example.

    The analyses of the embryonic stem cell lines and the computer comparisons of the mounds of resulting data required the efforts of scientists at four academic centers, two federal laboratories and three companies. Critical to the team's success was prescient support of cutting-edge technology development by the National Institutes of Health, support that enabled development of the technological infrastructure necessary for large-scale comparative research, particularly the Human Genome Project, says study co-author Mahendra Rao, M.B.B.S., Ph.D., of the Laboratory of Neurosciences at the National Institute on Aging.

    The scientists used so-called GeneChip microarrays, or oligonucleotide arrays, to determine whether there were genetic differences between the early and the late batch of each of the stem cell lines, including whether any genes were present in extra copies. Depending on the gene affected, extra copies could lead to accelerated cell growth, increased cell death, or no measurable effect at all.

    In addition to probing changes in the nuclear and mitochondrial DNA sequences and copy numbers, the researchers examined whether the cells' genetic material had shifts in marks that sit on genes and are passed from cell to cell during cell division. These so-called epigenetic marks -- in this case methyl groups on a gene region known as the promoter -- help control whether a gene is used by a cell to make proteins. The researchers determined the methylation status of 14 genes in each of the batches of stem cells; three of the genes did show different methylation patterns in late batches compared to early batches.

    The scientists' analysis revealed that five of the nine cell lines had extra or fewer copies of at least one section of their genetic material in the late batch compared to the same cell line's early batch. Two of the nine lines had changes in their mitochondrial DNA over time, and all nine stem cell lines exhibited some shift in methylation of at least one of three genes. One of these genes, called RASSF1A, is also methylated in many cancers, but what effect the methylation has on the stem cells is unknown.

    The team is already planning to conduct similar analyses of the remaining NIH-approved cell lines, but analysis of stem cell lines not available for use with federal funds will also be needed, the team members say.

    The Johns Hopkins researchers were funded by the Henry J. Knott Professorship in Genetic Medicine, the Sol Goldman Pancreatic Cancer Research Center at Johns Hopkins, the National Cancer Institute, the Maryland Cigarette Restitution Fund and the Donald W. Reynolds Foundation Clinical Cardiovascular Research Center at Johns Hopkins.

    Authors on the paper are Anirban Maitra, Dan Arking, Morna Ikeda, Keyaunoosh Kassauei, Guoping Sui, David Cutler and Aravinda Chakravarti of Johns Hopkins; Narayan Shivapurkar, Victor Stastny and Adi Gazdar of the University of Texas Southwestern Medical Center; Ying Liu and Mahendra Rao of the National Institute on Aging; Sandii Brimble and Thomas Schulz of BresaGen Inc., Athens, Ga.; Karin Noaksson, Johan Hyllner and Peter Sartipy of Cellartis AB, Goteburg, Sweden; Xianmin Zeng and William Freed of the National Institute on Drug Abuse; Alan Coleman of ES Cell International, Singapore; Sei-Ichi Matsui of Roswell Park Cancer Institute and the State University of New York at Buffalo; and Melissa Carpenter of Robarts Research Institute, Ontario, Canada.

    The microarrays used in this work are the product of Affymetrix (Santa Clara, Calif.). Chakravarti is a paid member of the Affymetrix scientific advisory board. The terms of this arrangement are being managed by The Johns Hopkins University in accordance with its conflict of interest policies.

  2. #2
    Senior Member
    Join Date
    Jun 2005
    This should be in favour of removal of the US ban on embryonic stem cell research?

  3. #3
    Quote Originally Posted by Leif
    Leif, thanks for posting this. There were earlier rumors that this paper was having difficulties being approved for publication by NIH. Mahendra Rao had reported that all the cell lines approved for NIH funding are aneuploid, i.e. have missing genes or even chromosomes because they are so old.

    Scientists have long known that when cells are grown for long periods of time, through many thousands of generations, they will show genetic changes that are deleterious. There are technologies that can and must be developed to screen the cells for such changes. For example, it may be possible to genetically modify the cells so that they will commit suicide. More sensitive methods of detecting aneuploidy will also allow the cells to be screened and abnormal cells can be discarded.


  4. #4

    Stem Cell Instability Further Down The Line

    September, 7 2005 4:42

    A study in the October issue of Nature Genetics reports that human embryonic stem cell lines cultured for a long period of time develop changes in their genomes that may make them unusable for therapeutic purposes.
    While all cultured cells develop small mutations over time, some previous work had suggested that large-scale genetic alterations in embryonic stem cells were rare. Using a more sensitive technique, Aravinda Chakravarti and colleagues now show that stem cell lines that are cultured at length tend to accumulate more significant changes in certain regions of the genome, including large deletions and amplifications. Some of these regions are known to be involved in human cancers.

    The authors also found mutations in the mitochondrial DNA, which could affect the metabolic functions of the cells, as well as changes in the patterns of DNA methylation--a chemical mark that typically regulates the expression of genes--in most of the extensively cultured cell lines. This latter finding stands in contrast to a recent study reporting stable methylation patterns at select genes.

    The authors note that embryonic stem cell lines cultured for only a short time do not exhibit these structural changes in their genomes, making them suitable for therapeutic applications. For those cell lines that have been cultured for longer periods, such as those currently approved for research funding by the US government, the authors recommend monitoring them for various types of mutations using standardized and sensitive techniques. Additional embryonic stem cell lines that have not been extensively cultured will likely be needed, they conclude.

    Author contact:

    Aravinda Chakravarti (Johns Hopkins University School of Medicine, Baltimore, MD, USA)

    The abstract of the paper:


    Published online: 4 September 2005; | doi:10.1038/ng1631
    Genomic alterations in cultured human embryonic stem cells

    Anirban Maitra1, 2, 3, 12, Dan E Arking1, 12, Narayan Shivapurkar4, Morna Ikeda1, Victor Stastny4, Keyaunoosh Kassauei2, Guoping Sui2, David J Cutler1, Ying Liu5, Sandii N Brimble6, Karin Noaksson7, Johan Hyllner7, Thomas C Schulz6, Xianmin Zeng8, William J Freed8, Jeremy Crook9, Suman Abraham9, Alan Colman9, Peter Sartipy7, Sei-Ichi Matsui10, Melissa Carpenter11, Adi F Gazdar4, Mahendra Rao5 & Aravinda Chakravarti1

    1 McKusick-Nathans Institute of Genetic Medicine, The Sol Goldman Pancreatic Cancer Research Center, 733 N. Broadway Research Bldg., Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    2 Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, 733 N. Broadway Research Bldg., Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    3 Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center, 733 N. Broadway Research Bldg., Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    4 Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

    5 Laboratory of Neurosciences, National Institute on Aging, 333 Cassell Drive, Triad Bldg., Baltimore, Maryland 21224, USA.

    6 BresaGen, Inc., Athens, Georgia, USA.

    7 Cellartis AB, Goteborg, Sweden.

    8 Cellular Neurobiology Branch, National Institute on Drug Abuse, Baltimore, Maryland, USA.

    9 ES Cell International, Singapore.

    10 Roswell Park Cancer Institute, State University of New York at Buffalo, Buffalo, New York, USA.

    11 Robarts Research Institute, Ontario, Canada.

    12 These authors contributed equally to this work.

    Correspondence should be addressed to Aravinda Chakravarti or Mahendra Rao

    Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10-9 per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.

  5. #5
    Quote Originally Posted by Leif
    This should be in favour of removal of the US ban on embryonic stem cell research?
    One would think so..but our expert spinmasters will use this in a WHOLE nother way. Count on it.
    Life isn't about getting thru the storm but learning to dance in the rain.

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