Human embryonic stem cells display a unique pattern of chemical modification to DNA

Study suggests additional hurdles to therapeutic cloning may lie ahead

San Diego, Calif. -- Scientists from the Burnham Institute for Medical Research (BIMR) and Illumina Inc., in collaboration with stem cell researchers around the world, have found that the DNA of human embryonic stem cells is chemically modified in a characteristic, predictable pattern. This pattern distinguishes human embryonic stem cells from normal adult cells and cell lines, including cancer cells. The study, which appears online today in Genome Research, should help researchers understand how epigenetic factors contribute to self-renewal and developmental pluripotence, unique characteristics of human embryonic stem cells that may one day allow them to be used to replace diseased or damaged cells with healthy ones in a process called therapeutic cloning.

Embryonic stem cells are derived from embryos that are undergoing a period of intense cellular activity, including the chemical addition of methyl groups to specific DNA sequences in a process known as DNA methylation. The methylation and demethylation of particular DNA sequences in the genome are known to have profound effects on cellular behavior and differentiation. For example, DNA methylation is one of the critical epigenetic events leading to the inactivation of one X chromosome in female cells. Failure to establish a normal pattern of DNA methylation during embryogenesis can cause immunological deficiencies, mental retardation and other abnormalities such as Rett, Prader-Willi, Angelman and Beckwith-Wiedemann syndromes.

Until recently, DNA methylation could only be studied one gene at a time. But a new microarray-based technique developed at Illumina enabled the scientists conducting this new study to simultaneously examine hundreds of potential methylation sites, thereby revealing global patterns. "Analyzing the DNA methylation pattern of hundreds of genes at a time opens a new window for epigenetic research," says Dr. Jian-Bing Fan, director of molecular biology at Illumina. "Exciting insights into development, aging, and cancer should come quickly from understanding global patterns of DNA methylation."

To examine global DNA methylation patterns in human embryonic stem cells, the researchers analyzed 14 human embryonic stem cell lines from diverse ethnic origins, derived in several different labs, and maintained for various times in culture. They tested over 1500 potential methylation sites in the DNA of these cells and in other cell types and found that the embryonic stem cells shared essentially identical methylation patterns in a large number of gene regions. Furthermore, these methylation patterns were distinct from those in adult stem cells, differentiated cells, and cancer cells.

"Our results suggest that therapeutic cloning of patient-specific human embryonic stem cells will be an enormous challenge, as nuclei from adult cells will have to be epigenetically reprogrammed to reflect the specific DNA methylation signature of normal human embryonic stem cells," explains Dr. Jeanne Loring, co-director of the stem cell center at BIMR. "This reinforces the need for basic research directed at understanding the fundamental biology of human embryonic stem cells before therapeutic uses can be considered."

Peter Thiel puts his weight behind Dr. Aubrey de Grey ?s engineering blueprint

San Francisco – Peter A. Thiel, co-founder and former CEO of online payments system PayPal, Founder and Managing Member of Clarium Capital Management, a San Francisco-based hedge fund, and angel investor in social networking site Facebook, has announced his pledge of $3.5 Million to support scientific research into the alleviation and eventual reversal of the debilities caused by aging, to be conducted under the auspices of the Methuselah Foundation, a charity co-founded and Chaired by Dr. Aubrey de Grey.
Mr. Thiel commented "Rapid advances in biological science foretell of a treasure trove of discoveries this century, including dramatically improved health and longevity for all. I’m backing Dr. de Grey, because I believe that his revolutionary approach to aging research will accelerate this process, allowing many people alive today to enjoy radically longer and healthier lives for themselves and their loved ones.
Mr. Thiel will donate a total of $500,000 over the next three years to fund pilot research projects intended to deliver early stage validation of the “SENS” approach to combating the debilitation caused by aging.
Additionally, from now until the end of 2009, Mr. Thiel promises to match every Dollar donated to the Methuselah Foundation for SENS research with a 50 cent matching contribution from himself, up to a maximum of $3 Million of matching funds.
Dr. de Grey said “I am extremely grateful to Peter for his bold and visionary initiative. I have been working with leading biologists and biochemists around the world in identifying promising research projects, and with this generous donation we will go to work straightaway.”

MitoSENS

About MitoSENS

Mitochondria are a component of cells that perform cellular respiration. That is to say, they react oxygen with nutrients from our free radicalfood to produce water, carbon dioxide, and useable energy (in the form of ATP). A side effect of this process is the production of reactive chemical byproducts that damage nearby components of the cell. Mitochondria are unique in that they have their own DNA (mtDNA), separate from the nucleus. Being at the site of cellular respiration, the mtDNA is vulnerable to its reactive byproducts. Worse yet, the mitochondria's capacity for repairing DNA damage is much more limited than that of the nucleus. Mutations to the mtDNA inevitably accumulate leading to dysfunction of mitochondria, and contributing to aging of the organism. The goal of MitoSENS is to obviate mtDNA mutations by expressing the mtDNA genes from the nucleus.

Fortunately, we would be completing a process that evolution has already started.

The mitochondrial genome originally had thousands of genes, but evolution has reduced it to a mere 13 (protein encoding) genes in humans. By studying how nature transfered expression of other genes from the mitochondria to the nucleus, we can identify the necessary steps to transfer the remaining 13 genes (in humans).

MitoSENS research is currently being conducted in the lab of Ian Holt at Cambridge University. To learn more about the MitoSENS strategy from its originator and Methuselah Foundation chairperson Aubrey de Grey, see the SENS website here (laymans terms) or here (technical).
The Research Team

Mark Hamalainen is a researcher for the Methuselah Foundation and a PhD Candidate at Cambridge University. He has previously worked on the LysoSENS project and is now focusing on MitoSENS.

Questions and Contact

Any questions or comments on MitoSENS research can be directed to mark.hamalainen@gmail.com

Switching off Aging in Stem Cells

From Howard Hughes Medical Institute
A single molecular switch plays a central role in inducing stem cells in the brain, pancreas, and blood to lose function as they age, researchers have found. Mice lacking that switch show considerably reduced aging-related decline in stem cell function and tissue regeneration.

“People tend to think that old tissues have less regenerative capacity because they are wearing out,” said Sean J. Morrison, a Howard Hughes Medical Institute investigator at the University of Michigan who led the study of the switch's role in the brain. “This work shows that they are not just wearing out; they are actively shutting themselves down.”

While the finding could ultimately lead to drugs to slow or reverse degeneration in the brain and other tissues, the researchers cautioned such treatments would have to be balanced against the chance of increasing cancer risk in patients. Stem cells are the immature progenitor cells that are the continuing self-renewing source of mature, differentiated cells in the body.

Morrison's study was published online September 6, 2006, in Nature, ahead of publication in the journal's print version, along with two other studies from independent research teams that studied how a protein called inhibitor of cyclin-dependent kinase 4A, or p16^INK4a, contributes to stem cell decline. The other papers reported studies of the gene's role in stem cell decline in insulin-producing pancreatic islet cells and hematopoietic stem cells, which generate blood cells. Those studies were headed, respectively, by Norman Sharpless of the University of North Carolina at Chapel Hill and David Scadden of Harvard University.

“Before this work, p16^INK4a was thought of only as a gene that inhibited cancer formation by inducing senescence in the cell,” said Morrison. “The idea was that it wasn't expressed in normal tissues, and therefore probably didn't have a physiological role but only came on when something went wrong in the cell.”

However, said Morrison, studies in Sharpless's laboratory found that the gene becomes increasingly active as tissues age. “That started us thinking that maybe this gene is part of why old tissues have less stem cell activity and less regenerative capacity, for example taking longer to heal,” said Morrison. “In our lab, for example, we've found that the brain makes fewer neurons with age, but the molecular mechanism for that effect was not known.”

Morrison and his colleagues followed stem cell activity in the brains of normal mice as they aged. The researchers analyzed a particular area of the forebrain, the subventricular zone, known to be an important center of neuronal production, called neurogenesis, in adults. The other two research teams studied pancreatic islet cells and hematopoietic stem cells for the same reason - that they are a constant source of new cells in the adult.

Morrison and his colleagues found that stem cell number and self-renewal function, as well as neurogenesis, declined with age in the mice. But they found that during aging, p16^INK4a gene activity increased.

However, in genetically engineered mice that were deficient in p16^INK4a, stem cell function and neuronal production were enhanced in old but not young mice as compared to normal mice. “We didn't turn an old brain into a young brain by deleting p16^INK4a, but the deficient mice did show significantly increased progenitor cell function and neurogenesis with age, compared to normal mice,” said Morrison. “This tells us that p16^INK4a is not the whole story, although it's an important part, and that other genes also regulate the aging process.”

Morrison and his colleagues also found evidence that the gene does not play the same role in other neural tissues. “There are different kinds of stem cells in different regions of the brain, and some of those stem cells are more sensitive to factors like p16^INK4a than others,” said Morrison. p16^INK4a deficiency did not prevent the atrophy of the cortex that normally occurs with aging, they found. Nor did the deficiency prevent loss of function in another brain region, the hippocampus, that is also a center for neurogenesis in adults. The researchers also analyzed peripheral nerve cells in the gut and found that p16^INK4a did not prevent loss of stem cell function there. “There are probably other factors that are important for aging of the hippocampus and the peripheral nervous system,” Morrison noted.

Nevertheless, he said, the discovery of the central role of p16^INK4a is highly significant. “I think if you asked before these studies whether you could delete a single gene and rescue stem cell function in multiple tissues, and neurogenesis in an old brain, many people would have said that aging is such a complex phenomenon that you would not get a significant effect,” he said.

Morrison theorized that p16^INK4a is a suppressor of stem cell function that evolved as part of the regulatory machinery that also includes proto-oncogenes that encourage cell proliferation. “We are all evolutionarily selected to, on the one hand, maintain regenerative capacity of our tissues through adult life so that we can repair our cells and survive injuries — while on the other hand, limit proliferation in our tissues with age, so cells don't divide out of control, causing cancers,” he said. “And the way that we achieve that balance is by having proto-oncogenes that promote proliferation come into balance with tumor suppressor genes that inhibit proliferation. This work shows one way that this balance changes with age.

“While these tumor suppressor mechanisms don't even exist during fetal development, where cells must divide rapidly, it makes sense that they become stronger in old age, when we are more at risk of getting a cancer,” said Morrison. “So, the benefit is that genes like p16 allow us to get older before we get cancer, but the bad news is that they make us lose function with age.”