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Acid bath offers easy path to stem cells
Just squeezing or bathing cells in acidic conditions can readily reprogram them into an embryonic state.
29 January 2014
In 2006, Japanese researchers reported1 a technique for creating cells that have the embryonic ability to turn into almost any cell type in the mammalian body — the now-famous induced pluripotent stem (iPS) cells. In papers published this week in Nature2, 3, another Japanese team says that it has come up with a surprisingly simple method — exposure to stress, including a low pH — that can make cells that are even more malleable than iPS cells, and do it faster and more efficiently.
”It’s amazing. I would have never thought external stress could have this effect,” says Yoshiki Sasai, a stem-cell researcher at the RIKEN Center for Developmental Biology in Kobe, Japan, and a co-author of the latest studies. It took Haruko Obokata, a young stem-cell biologist at the same centre, five years to develop the method and persuade Sasai and others that it works. “Everyone said it was an artefact — there were some really hard days,” says Obokata.
Obokata says that the idea that stressing cells might make them pluripotent came to her when she was culturing cells and noticed that some, after being squeezed through a capillary tube, would shrink to a size similar to that of stem cells. She decided to try applying different kinds of stress, including heat, starvation and a high-calcium environment. Three stressors — a bacterial toxin that perforates the cell membrane, exposure to low pH and physical squeezing — were each able to coax the cells to show markers of pluripotency.
But to earn the name pluripotent, the cells had to show that they could turn into all cell types — demonstrated by injecting fluorescently tagged cells into a mouse embryo. If the introduced cells are pluripotent, the glowing cells show up in every tissue of the resultant mouse. This test proved tricky and required a change in strategy. Hundreds of mice made with help from mouse-cloning pioneer Teruhiko Wakayama at the University of Yamanashi, Japan, were only faintly fluorescent. Wakayama, who had initially thought that the project would probably be a “huge effort in vain”, suggested stressing fully differentiated cells from newborn mice instead of those from adult mice. This worked to produce a fully green mouse embryo.
Still, the whole idea was radical, and Obokata’s hope that glowing mice would be enough to win acceptance was optimistic. Her manuscript was rejected multiple times, she says.
To convince sceptics, Obokata had to prove that the pluripotent cells were converted mature cells and not pre-existing pluripotent cells. So she made pluripotent cells by stressing T cells, a type of white blood cell whose maturity is clear from a rearrangement that its genes undergo during development. She also caught the conversion of T cells to pluripotent cells on video. Obokata called the phenomenon stimulus-triggered acquisition of pluripotency (STAP).
The results could fuel a long-running debate. For years, various groups of scientists have reported finding pluripotent cells in the mammalian body, such as the multipotent adult progenitor cells described4 by Catherine Verfaillie, a molecular biologist then at the University of Minnesota in Minneapolis. But others have had difficulty reproducing such findings. Obokata started the current project in the laboratory of tissue engineer Charles Vacanti at Harvard University in Cambridge, Massachusetts, by looking at cells that Vacanti’s group thought to be pluripotent cells isolated from the body5. But her results suggested a different explanation: that pluripotent cells are created when the body’s cells endure physical stress. “The generation of these cells is essentially Mother Nature’s way of responding to injury,” says Vacanti, a co-author of the latest papers2, 3.
One of the most surprising findings is that the STAP cells can also form placental tissue, something that neither iPS cells nor embryonic stem cells can do. That could make cloning dramatically easier, says Wakayama. Currently, cloning requires extraction of unfertilized eggs, transfer of a donor nucleus into the egg, in vitro cultivation of an embryo and then transfer of the embryo to a surrogate. If STAP cells can create their own placenta, they could be transferred directly to the surrogate. Wakayama is cautious, however, saying that the idea is currently at “dream stage”.
Obokata has already reprogrammed a dozen cell types, including those from the brain, skin, lung and liver, hinting that the method will work with most, if not all, cell types. On average, she says, 25% of the cells survive the stress and 30% of those convert to pluripotent cells — already a higher proportion than the roughly 1% conversion rate of iPS cells, which take several weeks to become pluripotent. She now wants to use these results to examine how reprogramming in the body is related to the activity of stem cells. Obokata is also trying to make the method work with cells from adult mice and humans.
“The findings are important to understand nuclear reprogramming,” says Shinya Yamanaka, who pioneered iPS cell research. “From a practical point of view toward clinical applications, I see this as a new approach to generate iPS-like cells.”
(30 January 2014)
Nature 505, 641–647 (30 January 2014) | doi:10.1038/nature12968
Stimulus-triggered fate conversion of somatic cells into pluripotency
Haruko Obokata , Teruhiko Wakayama , Yoshiki Sasai , Koji Kojima , Martin P. Vacanti , Hitoshi Niwa , Masayuki Yamato & Charles A. Vacanti
Here we report a unique cellular reprogramming phenomenon, called stimulus-triggered acquisition of pluripotency (STAP), which requires neither nuclear transfer nor the introduction of transcription factors. In STAP, strong external stimuli such as a transient low-pH stressor reprogrammed mammalian somatic cells, resulting in the generation of pluripotent cells. Through real-time imaging of STAP cells derived from purified lymphocytes, as well as gene rearrangement analysis, we found that committed somatic cells give rise to STAP cells by reprogramming rather than selection. STAP cells showed a substantial decrease in DNA methylation in the regulatory regions of pluripotency marker genes. Blastocyst injection showed that STAP cells efficiently contribute to chimaeric embryos and to offspring via germline transmission. We also demonstrate the derivation of robustly expandable pluripotent cell lines from STAP cells. Thus, our findings indicate that epigenetic fate determination of mammalian cells can be markedly converted in a context-dependent manner by strong environmental cues.
Letters to Editor
Nature 505, 676–680 (30 January 2014) | doi:10.1038/nature12969
Bidirectional developmental potential in reprogrammed cells with acquired pluripotency
Haruko Obokata , Yoshiki Sasai , Hitoshi Niwa , Mitsutaka Kadota , Munazah Andrabi , Nozomu Takata , Mikiko Tokoro , Yukari Terashita , Shigenobu Yonemura , Charles A. Vacanti & Teruhiko Wakayama
We recently discovered an unexpected phenomenon of somatic cell reprogramming into pluripotent cells by exposure to sublethal stimuli, which we call stimulus-triggered acquisition of pluripotency (STAP). This reprogramming does not require nuclear transfer or genetic manipulation. Here we report that reprogrammed STAP cells, unlike embryonic stem (ES) cells, can contribute to both embryonic and placental tissues, as seen in a blastocyst injection assay. Mouse STAP cells lose the ability to contribute to the placenta as well as trophoblast marker expression on converting into ES-like stem cells by treatment with adrenocorticotropic hormone (ACTH) and leukaemia inhibitory factor (LIF).
Stem cell breakthrough may be simple, fast, cheap
By Elizabeth Landau, CNN
January 30, 2014 -- Updated 1320 GMT (2120 HKT)
(CNN) -- We run too hard, we fall down, we're sick -- all of this puts stress on the cells in our bodies. But in what's being called a breakthrough in regenerative medicine, researchers have found a way to make stem cells by purposely putting mature cells under stress.
Two new studies published Wednesday in the journal Nature describe a method of taking mature cells from mice and turning them into embryonic-like stem cells, which can be coaxed into becoming any other kind of cell possible. One method effectively boils down to this: Put the cells in an acidic environment.
"I think the process we've described mimics Mother Nature," said Dr. Charles Vacanti, director of the laboratory for Tissue Engineering and Regenerative Medicine at Brigham & Women's Hospital in Boston and senior author on one of the studies. "It's a natural process that cells normally respond to."
Both studies represent a new step in the thriving science of stem cell research, which seeks to develop therapies to repair bodily damage and cure disease by being able to insert cells that can grow into whatever tissues or organs are needed. If you take an organ that's functioning at 10% of normal and bring it up to 25% functionality, that could greatly reduce the likelihood of fatality in that particular disease, Vacanti said.
This method by Vacanti and his colleagues "is truly the simplest, cheapest, fastest method ever achieved for reprogramming [cells]," said Jeff Karp, associate professor of medicine at the Brigham & Women's Hospital and principal faculty member at the Harvard Stem Cell Institute. He was not involved in the study.
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Before the technique described in Nature, the leading candidates for creating stem cells artificially were those derived from embryos and stem cells from adult cells that require the insertion of DNA to become reprogrammable.
Stem cells are created the natural way every time an egg that is fertilized begins to divide. During the first four to five days of cell division, so-called pluripotent stem cells develop. They have the ability to turn into any cell in the body. Removing stem cells from the embryo destroys it, which is why this type of research is controversial.
Researchers have also developed a method of producing embryonic-like stem cells by taking a skin cell from a patient, for example, and adding a few bits of foreign DNA to reprogram the skin cell to become like an embryo and produce pluripotent cells, too. However, these cells are usually used for research because researchers do not want to give patients cells with extra DNA.
The new method does not involve the destruction of embryos or inserting new genetic material into cells, Vacanti said. It also avoids the problem of rejection: The body may reject stem cells that came from other people, but this method uses an individual's own mature cells.
"It was really surprising to see that such a remarkable transformation could be triggered simply by stimuli from outside of the cell," said Haruko Obokata of the Riken Center for Developmental Biology in Japan in a news conference this week.
The process is called STAP, which stands for "stimulus-triggered acquisition of pluripotency." Karp estimates that the method is five to 10 times faster than other means of reprogramming cells.
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Researchers used mice to study the STAP cell phenomenon. They genetically altered the mice donating stem cells to "label" those cells with the color green. For instance, they modified mice such that their cells would light up green in response to a particular wavelength of light.
The scientists exposed blood cells from these genetically altered mice to an acidic environment. A few days later, they saw that these cells turned into the embryonic-like state and grew in spherical clusters.
Scientists put the cell clusters into a mouse embryo that had not been genetically modified. It turned out, the implanted clusters could form tissues in all of the organs that the researchers tested. The scientists knew that the cells came from the original mouse because they turned green when exposed to a particular light.
Besides modifying acidity, researchers also stressed the cells in other ways, such as lowering the oxygen environment and disrupting the cell membrane. Increasing acidity was one of the most effective methods of turning mouse blood cells into STAP cells.
There are, of course, some caveats.
For now, the STAP cell procedure has only been demonstrated in cells from young mice. The effectiveness in humans, and the risks, are unknown.
Researchers have not yet shown how STAP embryonic-like stem cells compare with bona fide embryonic stem cells or induced pluripotent stem cells, Karp said.
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Also, although the study was "rigorous" and "well-controlled," it did not demonstrate exactly why the stress on the cells caused them to become STAP cells, Karp said.
As with everything in science, more research is required to confirm the findings and learn more about the implications.
Vacanti hopes the process could get tested clinically in humans within three years. He noted that induced pluripotent stem cells are already being explored in Japan in humans and the same "platforms" could be utilized for STAP cells.
STAP cells also have an additional property that embryonic stem cells and induced pluripotent stem cells do not: They can become placental cells. Scientists can manipulate them to contribute to tissues of either the embryo or the placenta.
What therapeutic purpose growing more placenta could serve, Vacanti isn't sure -- unless, that is, you wanted to create an embryo and bring it to term.
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But that's not the goal of this research. Vacanti and colleagues want to explore possible ties to cancer from the STAP cell process; it could potentially help to model the process by which cells become cancerous and explore if there is a way to reverse the process.
Stem cell research as a field has been growing at "lightning speed," Karp said.
New reprogramming approaches to stem cells are emerging all the time, he said, and this one in particular "looks incredibly promising."
Follow Elizabeth Landau on Twitter at @lizlandau