Wednesday, January 29, 2014

Stem Cell Reversal - WOW

Stem cell power unleashed after 30 minute dip in acid

A mouse embryo made with reprogrammed cells (Image: Haruko Obokata)

A LITTLE stress is all it took to make new life from old. Adult cells have been given the potential to turn into any type of body tissue just by tweaking their environment. This simple change alone promises to revolutionise stem cell medicine.

Yet New Scientist has also learned that this technique may have already been used to make a clone. "The implication is that you can very easily, from a drop of blood and simple techniques, create a perfect identical twin," says Charles Vacanti at Harvard Medical School, co-leader of the team involved.

Details were still emerging as New Scientist went to press, but the principles of the new technique were outlined in mice in work published this week. The implications are huge, and have far-reaching applications in regenerative medicine, cancer treatment and human cloning.

In the first few days after conception, an embryo consists of a bundle of cells that are pluripotent, which means they can develop into all cell types in the body. These embryonic stem cells have great potential for replacing tissue that is damaged or diseased but, as their use involves destroying an embryo, they have sparked much controversy.

To avoid this, in 2006 Shinya Yamanaka at Kyoto University, Japan, and colleagues worked out how to reprogram adult human cells into what they called induced pluripotent stem cells (iPSCs). They did this by introducing four genes that are normally found in pluripotent cells, using a harmless virus.
The breakthrough was hailed as a milestone of regenerative medicine – the ability to produce any cell type without destroying a human embryo. It won Yamanaka and his colleague John Gurdon at the University of Cambridge a Nobel prize in 2012. But turning these stem cells into therapies has been slow because there is a risk that the new genes can switch on others that cause cancer.

Now, Vacanti, along with Haruko Obokata at the Riken Center for Developmental Biology in Kobe, Japan, and colleagues have discovered a different way to rewind adult cells – without touching the DNA. The method is striking for its simplicity: all you need to do is place the cells in a stressful situation, such as an acidic environment.

The idea that this might work comes from a phenomenon seen in the plant kingdom, whereby drastic environmental stress can change an ordinary cell into an immature one from which a whole new plant can arise. For example, the presence of a specific hormone has been shown to transform a single adult carrot cell into a new plant. Some adult cells in reptiles and birds are also known to have the ability to do this.

To investigate whether the process could occur in mammals, Obokata and colleagues used mice that were bred to carry a gene that glows green in the presence of Oct-4, a protein that is only found in pluripotent cells. The team took a blood sample from the spleenof these mice when they were one week old, isolated white blood cells called lymphocytes, and exposed them to various strong but fleeting physical and chemical stresses.

One batch of cells was exposed to a "sub-lethal" acidic environment, with a pH of 5.7, for 30 minutes. The team then tried to grow the cells in the lab.

Not much happened at first – some cells died, and the rest still looked like white blood cells. But on day 2, a number of cells began to glow green, meaning they were producing Oct-4. By day 7, two-thirds of the surviving cells showed this pluripotent marker, together with other genetic markers of pluripotency – many of which are also seen in embryonic stem cells. In contrast, iPS cells can take four weeks to reach this stage.

The team call their new cells "stimulus-triggered acquisition of pluripotency", or STAP cells.
To make sure they really were pluripotent, the team injected the STAP cells from the spleen into an early-stage mouse embryo, or blastocyst. These are typically five or six days old with about eight cells already formed inside. The STAP cells seemed to integrate themselves into the structure, and the embryo went on to form the three "germ layers" that eventually give rise to all cell types in the body. The embryos developed into pups that incorporated STAP cells into every tissue in their body. These pups subsequently gave birth to offspring that also contained STAP cells – showing that the cells incorporated themselves into the animal's sperm or eggs, and were inherited.

In a second test, the team injected STAP cells into an adult mouse. They wanted to see if the cells formed a type of embryonic tumour called a teratoma – another gold standard test of pluripotency. They did.

The team wondered whether other adult cells might behave in a similar way. So they tried the acid-bath technique on brain, skin, muscle, fat, bone marrow, lung and liver tissues from one-week-old mice. Although the efficiency varied, the same thing happened in each case. In unpublished results, Vacanti says they have now found the procedure appears to work on cells from much older animals, including some from adult primates. He cautions though that these studies have yet to be completed.


"I don't think for one moment people thought this might be possible in humans," says Chris Mason, professor of regenerative medicine at University College London. "Who would have thought that to reprogram adult cells to a pluripotent state just required a small amount of acid for less than half an hour – it's an incredible discovery."

The researchers don't know whether the reprogramming they are seeing is initiated by the low pH or by some other type of stress, such as chemical changes happening further down the line. But they think they are tapping into a fundamental body-repair process. If you injure cells significantly enough, so that they almost die, certain genes get switched on or off, says Vacanti. This may result in a change in the cell's overall controls, meaning all genes have the potential to be switched on again. This could happen in all tissues throughout the body, Vacanti says. "Perhaps injuries like a bump on the arm or a burn cause mature cells to revert back to stem cells."

With the right environmental cues, these stem cells then specialise into healthy new cells to repair the damage. "We think that the more significant the injury, the further back down the tree these cells revert," says Vacanti. "It's mother nature's repair process."

That's not where the story ends, however. On their own, STAP cells do not readily multiply. But if they are placed alongside various growth factors, they undergo minor changes that allow them to multiply exponentially with no chromosomal abnormalities. The team call these slightly modified cells STAP stem cells (Nature, DOI: 10.1038/nature12968 and DOI: 10.1038/nature12969).
Depending on the medium they are in, STAP stem cells injected into a very early embryo and implanted into a mouse can incorporate themselves not only into the developing embryo, but also into the extra-embryonic tissue, which forms the placenta (see diagram).

"The team haven't just made pluripotent cells like embryonic stem cells," says Jos̩ Silva from the University of Cambridge, "they appear to have made totipotent cells." This means the cells have been rewound to a state with even more flexibility than pluripotent cells, which means they should be easier to manipulate. The only cells known to be totipotent Рable to form an embryo and a placenta Рin the body are those that have only undergone the first couple of cell divisions immediately after fertilisation. "They are like precursors to embryonic stem cells," says Silva.

"The word totipotent brings up all kinds of issues," says Robert Lanza of Advanced Cell Technology in Marlborough, Massachusetts. "If these cells are truly totipotent, and they are reproducible in humans then they can implant in a uterus and have the potential to be turned into a human being. At that point you're entering into a right-to-life quagmire" (see "Clone wars").

It may be closer than we think. Vacanti told us that he asked another collaborator to take the experiment further. Vacanti says the collaborator grew STAP cells from white blood cells and let them multiply into spherical clusters, then implanted one cluster directly into the uterus of an adult mouse. New Scientist has been unable to confirm this procedure: Vacanti says his understanding is that the cluster grew into a fetus, while Obokata says direct cloning has not yet been attempted. The collaborator named by Vacanti has not yet responded to our request for comment.

If this approach can be made to work, it would mean the creation of the world's first perfect cloned embryo. All animals cloned to date, including Dolly the sheep, were created using nuclear transfer, where the DNA from an unfertilised egg is replaced with DNA from an adult cell. The egg is stimulated to start dividing until it forms a blastocyst, and only then is it implanted in a uterus. However, the donor egg also contains other, mitochondrial DNA, which contributes to the resulting animal.

"Clones like Dolly are not actually a perfect copy," says Vacanti. "When you clone a cell using our technique, there is no egg, so there's no additional mitochondrial DNA. There's an embryo and a placenta which is a perfect copy of the original."

Vacanti says that the cloned mouse fetus stopped developing normally halfway through the pregnancy. "There was some sort of glitch – which is probably a good thing due to the ethical issues that would occur if we were able to create a live clone," he says. Obokata says the aim of the research is not to create clones, but to take control of these earliest of stem cells and encourage them to grow into whatever tissue is needed. The team is now investigating just how to do that and has started a project on human cells. "We are placing cells in different environments to see how finely we can regulate what they develop into. We don't want to grow a tooth in the middle of your liver," Vacanti says.

Preliminary work suggests that STAP cells are strongly influenced by the environment in which they are placed. "We might need to artificially mimic these environments, but perhaps we don't even need to do that," says Vacanti. "We might just be able to place a STAP cell into a muscle and it will turn into muscle."

The ultimate goal is not to replace whole organs but to seed damaged organs with healthy cells. With many organs, such as the liver and kidneys, you can survive with around 20 per cent of the normal amount of tissue, so why go to the trouble of creating a whole complex organ when regular infusions of healthy cells might work just as well.

Theoretically, says Vacanti, if you have cancer of the liver, you could take a healthy liver cell, transform it into a STAP cell, and grow it into replacements. "I believe people may be barking up the wrong tree by trying to replace entire organs," he says. "We can use the technology that we have demonstrated today and come up with ways to perfuse tissues with healthy cells. That way we can boost function and transform it from a failing organ to an organ which will survive."

"This is particularly good work because they used an incredibly simple approach," says Mason. "Making a personalised stem cell line would be cheap and easy. It would take days rather than the weeks or months it takes to make iPS cells, making it much more cost-effective – a big plus."
"The reprogramming step seems to be quite simple, it could be very inexpensive technology for reproductive medicine," agrees Lanza. "But it has more potential for abuse than iPS cells. It'll be interesting how this all plays out, but if it's possible to do this in humans, it changes everything."

Progress getting faster

Now there is a potentially quick and easy way to turn adult cells into any cell in the body (see main
story), when could we see the benefits?

Though it will be a while, newly discovered STAP cells could get going faster than earlier types of stem cells. The first to be developed, in 1998, were human embryonic stem cells. And although a more versatile kind known as human induced pluripotent stem (iPS) cells came along in 2006, they have not yet replaced ESCs.

"Every breakthrough has to catch up with the years of accumulated scientific and clinical knowledge that the earlier discovery has generated," says Chris Mason, a regenerative medicine specialist at University College London. But this knowledge pool is accelerating.

For example, it took 12 years to get to the point where ESCs could be trialled in people. In contrast, iPS cells will be injected into people for the first time later this year, eight years after inception, in a trial to see if they can treat a type of blindness.

"It is likely that this will shorten the development pathway for STAP cells," says Mason. "However, it will still be many years before the technology could potentially be in everyday clinical practice."

World reaction

"These observations are very exciting and quite extraordinary. They open up other potential avenues for reprogramming of cells"

Ian Wilmut, leader of the team that created Dolly the cloned sheep, University of Edinburgh, UK
"The findings are important to understand nuclear reprogramming. Practically, I see this a new approach to generate iPS-like cells"

Shinya Yamanaka, co-recipient of a 2012 Nobel prize for developing induced pluripotent stem cells, Kyoto University, Japan

"The authors have done a good deal of work to validate their findings. The big question will be whether, if reproduced, this biological 'trick-of-nature' can be harnessed therapeutically"
Evan Snyder, director of the Center for Stem Cell and Regenerative Biology, Sanford-Burnham Medical Research Institute, La Jolla, California

"If this can be reproduced in humans, it will be a paradigm changer"

Robert Lanza, chief scientific officer at Advanced Cell Technology in Marlborough, Massachusetts
"The papers are written carefully. The data let me feel that creating [an] embryo from the cells can be true. We'll need to see – someone needs to repeat it"

Keisuke Kaji, head of the biological reprogramming group, University of Edinburgh, UK
"Its speed, its simplicity, it's really unexpected"

Chris Mason, professor of regenerative medicine, University College London


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