reprogrammed 0000106 Louis J. Sheehan

Louis J. Sheehan

Stem cells’ powers of self-renewal, immortality and potential for medicine inspire those who study them. But progress toward understanding them has been slow it took 20 years just to figure out how to grow embryonic stem cells in the laboratory. More recently, though, molecular techniques have enabled swift movement on two fronts. Researchers are starting to see how stem cells can replenish their numbers while giving rise to specialized cells. Others are learning how to turn adult skin cells into cells more like their embryonic ancestors. These advances offer hope that scientists will soon harness the capabilities of stem cells, at last fulfilling the cells’ promise.

Illustrations by Bryan Christie

Back to the Womb

Reverting adult cells to an embryonic state without creating embryos is a tricky business

The diagnosis is not good; the patient will need surgery. So the doctor plucks a hair from the patient’s head and tells her to come back in a few weeks. When the patient returns, the surgeon patches up the faulty organ by implanting healthy cells generated in the lab from the patient’s hair follicle. After a few months, the new cells have integrated into the organ and the woman’s symptoms recede. A year later, she’s healthy and living a normal life.

This is the scenario that stem cell researchers hope will be commonplace 10 or 15 years from now. A patient’s own cells perhaps taken from hair follicles, blood or skin would be transformed into cells of the heart, brain or other organs. Doctors would then transplant these converted cells into the afflicted organ to treat the illness, whether it’s multiple sclerosis, Parkinson’s disease, heart failure or diabetes.

That dream came closer to reality last November. Two teams of scientists announced that they had wound back the clock on adult human skin cells, regressing those cells to an embryonic state. Just like embryonic stem cells, these reprogrammed cells seemed capable of becoming any of the 200-plus cell types in the human body, an ability called pluripotency (SN: 11/24/07, p. 323).

“The fact that you can do something to adult cells to reprogram them was absolutely novel,” says Jeanne Loring, director of the Center for Regenerative Medicine at the Scripps Research Institute in La Jolla, Calif. “It goes against everything that everyone had ever thought about the abilities of adult cells.”

Before, the only way to obtain pluripotent human cells had been to extract stem cells from 5-day-old embryos, which made the technique controversial. Reprogramming adult cells doesn’t involve making or destroying embryos, so the new cells called induced pluripotent stem cells, or iPS cells seemed to offer all the medical promise of embryonic stem cells without the political quagmire.

But nobody really knows how similar these new cells are to embryonic stem cells at the genetic level. Subtle differences in patterns of gene activity could undermine some of the new cells’ potential to treat diseases, or even cause the cells to behave abnormally if implanted. Before doctors can safely use reprogrammed cells in people, scientists need to know whether these new cells are truly genetic “twins” of real embryonic stem cells.

Cancer also poses a potential risk. To reprogram adult cells, scientists expose cells to four genetically engineered viruses. Each virus inserts a gene into the cells’ DNA at random locations. Such willy-nilly insertions sometimes disrupt the cells’ genes, including critical ones such as tumor suppressor genes. This viral disruption can cause cells to grow out of control and form a tumor another problem that must be solved before physicians can consider treating patients with reprogrammed cells.

Recent research is beginning to overcome these hurdles. Scientists are removing viruses one by one from their cell-conversion recipes. And new genetic studies suggest that these recipes do indeed fully reprogram adult cells fulfilling a possibility first suggested by the birth of Dolly.

Hitting the reset button

If a sheep could win a Nobel Prize, the prize for paving the way for reprogrammed stem cells would go to Dolly, the first cloned animal.

Lost in the media frenzy that followed Dolly’s birth in 1996 was a point more subtle than talk of clone armies and replacement pets. In Dolly, scientists saw proof that the DNA in a mature body cell could be “reset” to an embryonic state and then grow into every kind of cell in a newborn clone’s body, from heart muscle cells to nerve cells and bladder cells.


DNA METHYLATIONENLARGE | One way cells shut down unneeded genes is by attaching small molecules called methyl groups to the DNA, a process called methylation. Turning back the clock to return an adult cell to an embryonic state is largely a matter of removing this methylation.J. Korenblat

During cloning, inserting the nucleus of an adult cell and its DNA cargo into an emptied egg cell resets the adult DNA. Somehow, egg cells know how to perform this reprogramming feat.

By figuring out which proteins create and maintain this embryonic state, scientists thought they might be able to use those proteins to reset whole adult cells, not just the DNA.

Looking at the genes active in embryonic stem cells, Shinya Yamanaka and Kazutoshi Takahashi from Kyoto University in Japan found a set of four genes that, when inserted into the cells via viruses, did the trick in mouse cells in 2006 and, later, human cells: Oct3/4, SOX2, c-Myc and KLF4.

Almost immediately, stem cell researchers around the world flocked to the newly discovered recipe and began experimenting with reprogrammed cells. But work on the more controversial embryonic stem cells wasn’t abandoned. Unaltered, true embryonic cells still serve as a gold standard of “stemness” for sizing up the new cells.

“You never could have made a [reprogrammed] cell without an embryonic stem cell to compare to, to tell you what a pluripotent cell was,” says cardiologist Robb MacLellan of the UCLA David Geffen School of Medicine. Reprogrammed cells are “still very preliminary, and they will need a lot of work before you can say that they would be better or equivalent to embryonic stem cells.”

Scientists also assess the reprogrammed cells’ stemness by using them to treat disease in animals. If iPS cells are truly the same as embryonic stem cells, they should behave the same once converted to heart or nerve cells and implanted in the body.

For sickle cell anemia, at least, some evidence suggests that reprogrammed cells can do the job. Rudolf Jaenisch and his colleagues at the Massachusetts Institute of Technology corrected the faulty gene that causes sickle cell anemia in reprogrammed mouse skin cells. After coaxing the cells to develop into mature bone marrow cells, the scientists injected the cells back into the mice. Once the new cells took residence in the mice’s bone marrow, those cells began producing normal blood cells and the mice’s conditions improved, Jaenisch’s team reported last December in Science.

Reprogrammed cells can also become nerve cells and treat mice with a condition analogous to Parkinson’s disease, Jaenisch’s team showed in research published in the April 15 Proceedings of the National Academy of Sciences. In the experiments, reprogrammed cells once again appeared to behave just as embryonic stem cells would.

Another encouraging sign came from comparing which genes are switched off in reprogrammed cells and in embryonic stem cells. As an embryo develops in the womb, cells descended from embryonic stem cells gradually become more and more specialized into, say, liver or bone cells. Specialization primarily consists of shutting down unneeded genes turning off liver genes in bone cells and vice versa.

One way cells shut down unneeded genes is by attaching small molecules called methyl groups to the DNA, a process called methylation. Turning back the clock to return an adult cell to an embryonic state is largely a matter of removing this methylation. In most studies, methylation patterns of reprogrammed cells have closely mirrored those of embryonic stem, or ES, cells, leading some scientists to view the two as functionally identical.

“They’re essentially the same cells,” says Michael West, a stem cell researcher and chief executive of BioTime, a biotechnology company based in Alameda, Calif. “They’re called iPS cells, but they are ES cells as far as I’m concerned.”

Not all scientists agree. “I would say they’re not entirely the same, but they’re pretty close,” says Sheng Ding, a stem cell researcher at the Scripps Research Institute. “There’s still sort of imperfect gene activation.”

A study appearing online August 24 in Nature could finally settle the matter. Loring of Scripps and her colleagues profiled the activity of thousands of genes and proteins in reprogrammed cells, embryonic stem cells, neural stem cells and other stem cells. Comparing these activity profiles revealed a set of 19 proteins that clearly delineate between cells that are pluripotent and others, such as neural stem cells, which are not.

“We can always tell you if a cell falls into that category or not. It’s very clear,” Loring says. When her team screened reprogrammed cells based on these key proteins, those cells fell cleanly into the same group as embryonic stem cells.

“People who have looked at [reprogrammed] cells before have gotten the impression that they are different, but this shows that they’re the same,” she says. “If they hadn’t been labeled, I wouldn’t have been able to tell them apart from [embryonic stem] cells.”

The results suggest that, on a genetic level, converted adult cells are so much like true stem cells that they’re interchangeable. Except, that is, for those pesky viruses.

Breaking a few eggs

Progress on exorcising viruses from reprogramming recipes has been swift.

In January, research groups led by Yamanaka and Jaenisch separately succeeded in converting cells without the virus carrying c-Myc, the groups reported in Nature Biotechnology and Cell Stem Cell. Because c-Myc is known to increase the risk of cancer on its own, it was the first gene scientists aimed to eliminate.

The conversion of mouse skin cells took longer without c-Myc 21 days instead of six but the cells otherwise appeared to be reprogrammed. During previous experiments, mice grown from embryos containing reprogrammed cells developed tumors because of c-Myc, but in these two experiments, the hybrid mice were tumor free. Yamanaka’s group also showed that the c-Myc–free technique can reset human skin cells.

In later experiments by another group, the c-Myc–free technique enhanced with valproic acid converted more than 100 times as many cells after a week as the technique alone did. This improvement offset much of the efficiency lost by removing c-Myc, a team led by Douglas Melton of the Harvard Stem Cell Institute in Cambridge, Mass., reported in the July Nature Biotechnology.

Using simple chemicals like valproic acid to replace the virus-carried genes could be much safer than dealing with viruses, since the pharmaceutical industry has decades of experience developing and testing small-molecule drugs. These drugs can activate the cell’s same native molecular machinery as inserted genes would, thus reprogramming the cells in similar ways.

Replacing the three other virus-gene packages in the reprogramming recipe with chemicals like valproic acid (a compound commonly used in antiseizure medications) is one approach scientists are pursuing to make the conversion process more palatable to the FDA, which would have to approve any medical use of reprogrammed cells. “The FDA brings a whole extra level of scrutiny when there’s this genetic modification of cells” by viral insertion of genes, West says.

In unpublished research, Ding’s group recently replaced two more of the virus-delivered genes with simple chemicals. “Before it was four genes, now we’re down to one, and it’s only been about two years” since reprogrammed mouse cells were first created, Ding says. “We were surprised that it wasn’t really difficult at all.”

Scientists are exploring other ways to solve the virus issue as well, Loring notes. One approach could be to use a different kind of virus. Some viruses deliver genes into a cell without integrating those genes into the cell’s DNA. The genes remain free-floating in the cell body, where they can be translated into proteins. Eventually, the cell’s enzymes degrade the genes, removing the potential danger created by random insertions of foreign genes into the cell’s DNA.

Scientists could also inject the reprogramming proteins directly into the cells, rather than adding the genes that encode those proteins. But the problem with direct protein injection as well as the nonintegrating viruses is timing.

“We don’t know really how long these inducing factors have to be around in order to reprogram the cells,” Loring explains. Without some intervention by scientists, injected proteins would be degraded by the cell in a matter of hours. “If you don’t do it for long enough, you might as well not have done it.”

Loring is exploring yet another idea: microRNAs, short RNA molecules that silence specific genes. Each cell in a person’s body contains a complete set of his or her genetic code, including the four genes in the original reprogramming recipe. So adult cells already have these genes they’re just switched off. Loring hopes to reactivate the cell’s own reprogramming genes by using microRNAs to silence the proteins that keep those genes turned off.

Her lab recently identified specific microRNAs that could perform this task. While the experiments are ongoing, Loring says that these microRNAs could be delivered into cells without viruses.

Techniques for each of these approaches are developing rapidly, and many scientists expect that, one way or another, the virus issue will soon be resolved. “It’s happening pretty fast,” Ding says. “One, two years this will be done.”

The hard part

Of course, that’s not to say that stem cell therapies will be readily available in only one or two years.

Even after scientists can make virus-free, embryonic-like reprogrammed cells, perfecting the techniques for using these cells in patients will take years.

“You cannot use [converted] cells on their own for anything,” Ding notes. “You still have to differentiate the cells into something for it to be useful.”

Because reprogrammed cells can become any type of cell in the body, such cells transplanted directly into mice (and presumably people) can develop into teratomas ghastly tumors consisting of jumbles of hair, teeth, heart and other tissue types, all growing unchecked.

Before transplantation, scientists must first grow the reprogrammed cells in lab dishes under carefully controlled conditions to steer the cells into becoming heart cells, pancreas cells or whatever is needed for the therapy.

These steering techniques involve complex mixtures of signaling molecules. Scientists already know how to direct human stem cells into becoming cells of the heart, brain, pancreas and others, but in experiments on animals, cells made with these techniques don’t cope well after transplantation. Typically, the newly minted cells don’t integrate well into the organs, and most of the transplanted cells die.

“The actual survival of the cells is so poor,” MacLellan says. “This is going to be a huge issue before it will be clinically applicable.” These techniques will have to be refined further before they’ll be ready for widespread clinical use, Ding says. And that research is slow going.

Researchers estimate that it might take five or 10 years before they’ll be ready to begin clinical trials on people using cells derived from reprogrammed cells.

Scientists also want to know the full spectrum of cells in a patient’s body that can be reprogrammed. If and when therapies based on converted cells become commonplace, having to extract deep skin cells from each patient wouldn’t be particularly convenient.

Ding suggests an easier approach: Perhaps scientists could convert cells from follicles of a patient’s hair.

Louis J. Sheehan



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