Mice made from induced stem cells

Technical feat shows that the different route to stem cells can indeed make a full mammal body.

David Cyranoski

Two teams of Chinese researchers have created live mice from induced pluripotent stem (iPS) cells, answering a lingering question about the developmental potential of the cells.

Since Shinya Yamanaka of Kyoto University in Japan created the first iPS cells¹ in 2006, researchers have wondered whether they could generate an entire mammalian body from iPS cells, as they have from true embryonic stem cells. Experiments reported online this week in Nature ² and in Cell Stem Cell ³ suggest that, at least for mice, the answer is yes.

For the first study, animal cloners Qi Zhou of the Institute of Zoology in Beijing and Fanyi Zeng of Shanghai Jiao Tong University started by creating iPS cells the same way as Yamanaka, by using viral vectors to introduce four genes into mouse fibroblast cells. The researchers hoped that the introduced factors would 'reprogram' the cells so that they could differentiate into any type of cell in the body.

To check whether the reprogramming had worked, Zhou and Zeng first carried out a standard set of tests, including analysing whether their iPS cells had the same surface markers as embryonic stem cells. Going a step further, they then created a 'tetraploid' embryo by fusing two cells of an early-stage fertilized embryo. A tetraploid embryo develops a placenta and other cells necessary for development, but not the embryonic cells that would become the body. It is, in essence, a car without a driver.

When implanted into these embryos, the iPS cells began to steer development. The developing embryo was transferred to a surrogate mother, and 20 days later a mouse was born. It was black, like the mice used to create the iPS cells and unlike the white mice used to create the tetraploid embryo. DNA tests confirmed the mouse, named Xiao Xiao or 'Tiny', had arisen from the iPS cells.

Rudolf Jaenisch, a cloning expert at the Massachusetts Institute of Technology in Cambridge, had tried to do the same experiment in 2007, but didn't succeed in getting beyond late-stage embryos⁴. "There were two possible explanations" for his team's failure, he says. "Either iPS cells aren't pluripotent so it was impossible, or we just hadn't tried hard enough. The first would have been more interesting, but I assumed it was the second explanation."

The Chinese team tried harder, tweaking the culture medium and analysing 250 developing embryos before getting their first mouse.

In the paper, the team reports 27 live births. With their best cell line and optimal recipe, they were able to get 22 live births from 624 injected embryos, a success rate of 3.5%.

Zeng says, however, that the mice seem to have a high death rate, with some dying after just two days, and others displaying physical abnormalities, details of which the team would not reveal. But some of their mice passed one of the most fundamental tests of health: all 12 mice that were mated produced offspring, and the offspring showed no abnormalities. The team says it now has hundreds of second-generation, and more than 100 third-generation, mice. The team found no tumours in the mice, although they have not systematically looked for them.

The leader of the second team, Shaorong Gao of the National Institute of Biological Sciences in Beijing, also credits persistence for success. His group, which used the same basic technique as Zeng and Zhou, transferred iPS cells to 187 tetraploid complementation embryos to get just two live births (a 1.1% efficiency rate), although one died in infancy. "The chance for generating such a cell line is rare but we tried very hard," he says. Gao's team is now trying to mate its surviving mouse.

Both groups are now trying to understand what differences between iPS cells and embryonic stem cells might explain the abnormalities, high death rates, low efficiency rates and the fact that most iPS cell lines don't seem to work in making mice. Zeng and Zhou found, for one thing, that timing was important: cells that formed iPS cell colonies quickly — after 14 days — were successful, whereas those that formed colonies after 20 or 36 days did not work. Gao suggests that "aberrant reprogramming" might be to blame, at least for the low efficiency rates.

Such mouse studies should help researchers to understand fundamental differences between human embryonic stem cells and iPS cells as well. Earlier this month, researchers at the University of California, Los Angeles, reported that human iPS cells that passed conventional pluripotency tests differed in gene expression from human embryonic stem cells⁵. "iPS cells might do things better or worse than embryonic stem cells," says team member Kathrin Plath. "I don't think we know the answer at this point." Because the tetraploid work cannot be done with human embryos, the Chinese studies can't say much about clinical applications of pluripotent cell lines, adds her colleague William Lowry.

Zhou and Zeng are pursuing several new avenues, including comparing the iPS mice with mice cloned with conventional techniques, and working to prove that the same experiment can be done with adult mice. (The fibroblasts used to create iPS cells in both studies came from late-stage embryos.)

This would essentially be a new way to clone adult mammals — reprogramming DNA from an adult and generating a genetically identical individual. As a potentially easier method that produces fewer abnormalities than conventional cloning, it might evoke interest among mavericks as a tool for human cloning. China recently strengthened its law prohibiting such cloning⁶.

Zhou says he hopes that researchers will take advantage of the technology as "an important model for understanding reprogramming". He adds: "It is not intended to be a first step towards using iPS cells to create a human being."

References:

1. Takahashi, K. & Yamanaka, S. Cell 126, 663-676 (2006). | Article | PubMed | ISI | ChemPort |
2. Zhao, X.-Y. et al. Nature advance online publication doi:10.1038/nature08267 (2009).
3. Kang, L. et al. Cell Stem Cell doi:10.1016/j.stem.2009.07.001 (2009).
4. Wernig, M. et al. Nature 448, 318-324 (2007). | Article | PubMed | ISI | ChemPort |
5. Chin, M. H. et al. Cell Stem Cell 5, 111-123 (2009). | Article | PubMed | ChemPort |
6. Cyranoski, D. Nature 459, 146-147 (2009). | Article | PubMed | ChemPort |

Brain Researchers Open Door to Editing Memory

By BENEDICT CAREY

Suppose scientists could erase certain memories by tinkering with a single substance in the brain. Could make you forget a chronic fear, a traumatic loss, even a bad habit.

Researchers in Brooklyn have recently accomplished comparable feats, with a single dose of an experimental drug delivered to areas of the brain critical for holding specific types of memory, like emotional associations, spatial knowledge or motor skills.

The drug blocks the activity of a substance that the brain apparently needs to retain much of its learned information. And if enhanced, the substance could help ward off dementias and other memory problems.

So far, the research has been done only on animals. But scientists say this memory system is likely to work almost identically in people.

The discovery of such an apparently critical memory molecule, and its many potential uses, are part of the buzz surrounding a field that, in just the past few years, has made the seemingly impossible suddenly probable: neuroscience, the study of the brain.

“If this molecule is as important as it appears to be, you can see the possible implications,” said Dr. Todd C. Sacktor, a 52-year-old neuroscientist who leads the team at the SUNY Downstate Medical Center, in Brooklyn, which demonstrated its effect on memory. “For trauma. For addiction, which is a learned behavior. Ultimately for improving memory and learning.”

Artists and writers have led the exploration of identity, consciousness and memory for centuries. Yet even as scientists sent men to the moon and spacecraft to Saturn and submarines to the ocean floor, the instrument responsible for such feats, the human mind, remained almost entirely dark, a vast and mostly uncharted universe as mysterious as the New World was to explorers of the past.

Now neuroscience, a field that barely existed a generation ago, is racing ahead, attracting billions of dollars in new financing and throngs of researchers. The National Institutes of Health last year spent $5.2 billion, nearly 20 percent of its total budget, on brain-related projects, according to the Society for Neuroscience.

Endowments like the Wellcome Trust and the Kavli Foundation have poured in hundreds of millions of dollars more, establishing institutes at universities around the world, including Columbia and Yale.

The influx of money, talent and technology means that scientists are at last finding real answers about the brain — and raising questions, both scientific and ethical, more quickly than anyone can answer them.

Millions of people might be tempted to erase a severely painful memory, for instance — but what if, in the process, they lost other, personally important memories that were somehow related? Would a treatment that “cleared” the learned habits of addiction only tempt people to experiment more widely?

And perhaps even more important, when scientists find a drug to strengthen memory, will everyone feel compelled to use it?

The stakes, and the wide-open opportunities possible in brain science, will only accelerate the pace of discovery.

“In this field we are merely at the foothills of an enormous mountain range,” said Dr. Eric R. Kandel, a neuroscientist at Columbia, “and unlike in other areas of science, it is still possible for an individual or small group to make important contributions, without any great expenditure or some enormous lab.”

Dr. Sacktor is one of hundreds of researchers trying to answer a question that has dumbfounded thinkers since the beginning of modern inquiry: How on earth can a clump of tissue possibly capture and store everything — poems, emotional reactions, locations of favorite bars, distant childhood scenes? The idea that experience leaves some trace in the brain goes back at least to Plato’s Theaetetus metaphor of a stamp on wax, and in 1904 the German scholar Richard Semon gave that ghostly trace a name: the engram.

What could that engram actually be?

The answer, previous research suggests, is that brain cells activated by an experience keep one another on biological speed-dial, like a group of people joined in common witness of some striking event. Call on one and word quickly goes out to the larger network of cells, each apparently adding some detail, sight, sound, smell. The brain appears to retain a memory by growing thicker, or more efficient, communication lines between these cells.

The billion-dollar question is how?

In the decades since this process was described in the 1960s and 1970s, scientists have found scores of molecules that play some role in the process. But for years the field struggled to pinpoint the purpose each one serves. The problem was not that such substances were so hard to find — on the contrary.

In a 1999 paper in the journal Nature Neuroscience, two of the most prominent researchers in brain science, Dr. Jeff W. Lichtman and Joshua R. Sanes of Harvard, listed 117 molecules that were somehow involved when one cell creates a lasting speed-dial connection with a neighbor, a process known as “long-term potentiation.”

They did not see that these findings were necessarily clarifying the picture of how memories are formed. But an oddball substance right there on their own list, it turned out, had unusual properties.

A Helpful Nudge

“You know, my dad was the one who told me to look at this molecule — he was a scientist too, my dad, he’s dead now but he had these instincts — so anyway that’s how it all started,” Dr. Sacktor was saying. He was driving from his home in Yonkers to his laboratory in the East Flatbush neighborhood of Brooklyn, with three quiches and bag of bagels bouncing in the back seat. Lunch for the lab.

The father’s advice led the son, eventually, to a substance called PKMzeta. In a series of studies, Dr. Sacktor’s lab found that this molecule was present and activated in cells precisely when they were put on speed-dial by a neighboring neuron.

In fact, the PKMzeta molecules appeared to herd themselves, like Army Rangers occupying a small peninsula, into precisely the fingerlike connections among brain cells that were strengthened. And they stayed there, indefinitely, like biological sentries.

In short: PKMzeta, a wallflower in the great swimming party of chemicals that erupts when one cell stimulates another, looked as if it might be the one that kept the speed-dial function turned on.

“After that,” Dr. Sacktor said, “we began to focus solely on PKMzeta to see how critical it really was to behavior.”

Running a lab is something like fielding a weekend soccer team. Players come and go, from Europe, India, Asia, Grand Rapids. You move players around, depending on their skills. And you bring lunch, because doctoral students logging 12-hour days in a yellowing shotgun lab in East Flatbush need to eat.

“People think that state schools like ours are low-key, laid back, and they’re right, we are,” said Robert K. S. Wong, chairman of the physiology and pharmacology department at SUNY Downstate, who brought Dr. Sacktor with him from Columbia. “You have less pressure to apply for grants, and you can take more time, I think, to work out your ideas.”

To find out what, if anything, PKMzeta meant for living, breathing animals, Dr. Sacktor walked a flight downstairs to the lab of André A. Fenton, also of SUNY Downstate, who studies spatial memory in mice and rats.

Dr. Fenton had already devised a clever way to teach animals strong memories for where things are located. He teaches them to move around a small chamber to avoid a mild electric shock to their feet. Once the animals learn, they do not forget. Placed back in the chamber a day later, even a month later, they quickly remember how to avoid the shock and do so.

But when injected — directly into their brain — with a drug called ZIP that interferes with PKMzeta, they are back to square one, almost immediately. “When we first saw this happen, I had grad students throwing their hands up in the air, yelling,” Dr. Fenton said. “Well, we needed a lot more than that” one study.

They now have it. Dr. Fenton’s lab repeated the experiment, in various ways; so has a consortium of memory researchers, each using a different method. Researchers led by Yadin Dudai at the Weizmann Institute of Science in Israel found that one dose of ZIP even made rats forget a strong disgust they had developed for a taste that had made them sick — three months earlier.

A Conscience Blocker?

“This possibility of memory editing has enormous possibilities and raises huge ethical issues,” said Dr. Steven E. Hyman, a neurobiologist at Harvard. “On the one hand, you can imagine a scenario in which a person enters a setting which elicits traumatic memories, but now has a drug that weakens those memories as they come up. Or, in the case of addiction, a drug that weakens the associations that stir craving.”

Researchers have already tried to blunt painful memories and addictive urges using existing drugs; blocking PKMzeta could potentially be far more effective.

Yet any such drug, Dr. Hyman and others argue, could be misused to erase or block memories of bad behavior, even of crimes. If traumatic memories are like malicious stalkers, then troubling memories — and a healthy dread of them — form the foundation of a moral conscience.

For those studying the biology of memory, the properties of PKMzeta promise something grander still: the prospect of retooling the engram factory itself. By 2050 more than 100 million people worldwide will have Alzheimer’s disease or other dementias, scientists estimate, and far more will struggle with age-related memory decline.

“This is really the biggest target, and we have some ideas of how you might try to do it, for instance to get cells to make more PKMzeta,” Dr. Sacktor said. “But these are only ideas at this stage.”

A substance that improved memory would immediately raise larger social concerns, as well. “We know that people already use smart drugs and performance enhancers of all kinds, so a substance that actually improved memory could lead to an arms race,” Dr. Hyman said.

Many questions in the science remain. For instance, can PKMzeta really link a network of neurons for a lifetime? If so, how? Most molecules live for no more than weeks at a time.

And how does it work with the many other substances that appear to be important in creating a memory?

“There is not going to be one, single memory molecule, the system is just not that simple,” said Thomas J. Carew, a neuroscientist at the University of California, Irvine, and president of the Society for Neuroscience. “There are going to be many molecules involved, in different kinds of memories, all along the process of learning, storage and retrieval.”

Yet as scientists begin to climb out of the dark foothills and into the dim light, they are now poised to alter the understanding of human nature in ways artists and writers have not.

courtesy: Newyorktimes