Potent Promise: Essential Stemness

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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

Essential Stemness

If you think the roadwork in your town is bad, that’s nothing compared with the traffic trouble inside a cell. DNA gets repaved with chemicals and proteins almost constantly, maintaining a DNA-protein-chemical infrastructure called chromatin. Chromatin construction helps determine whether a cell’s gene activation machinery can zip along like a car-pool van in the HOV lane or gets stuck in a bumper-to-bumper jam. And chromatin prevents cells from wandering off on the wrong developmental road, usually by turning off genes that misdirect cells.

But some cells do things differently. Embryonic stem cells are not stuck in one lane with only one route available. These cells are perpetually poised at a fork in the road, with all options open. They retain the ability to become any type of cell in the body, a property called pluripotency. At the same time, embryonic stem cells have the ability to copy themselves indefinitely. Understanding how these cells accomplish those two feats — dividing indefinitely and choosing multiple identities — is a long-standing mystery of biology.

When seeking the stem cells’ secrets, scientists have generally focused on finding the ingredients that confer pluripotency, with less concern for perpetual self-replication. But new research suggests that versatility and immortality are probably not separate traits. The key to making an embryonic stem cell, many researchers believe, lies in balancing the two.

New research shows, for example, that dividing forever may be the natural state of a stem cell’s affairs. The cells keep renewing themselves until a signal arrives to differentiate, or transform into another cell type. Another study finds that a family of chemicals involved in choosing identity is also important in guiding cell division. And a network of hundreds of genes involved in pluripotency may also be involved in self-renewal, further research suggests.

These and other studies are leading to deeper understandings of how embryonic stem cells accomplish their defining feats and are allowing scientists to better grasp the essence of stemness.

Stem cell turn signals

Researchers used to think that getting a stem cell to grow required hormones and growth factors, says Qi-Long Ying, a stem cell biologist at the University of Southern California’s Keck School of Medicine in Los Angeles. But maybe cells simply keep dividing unless instructed otherwise. In fact, embryonic stem cells will keep making more stem cells unless they get a signal to develop into another type of cell, Ying and colleagues reported in the May 22 Nature.

“Life is just self-renewal and differentiation,” Ying says. And stem cells are all about the interplay of those two processes.

For instance, by ultimately signaling a cell to turn on certain genes, proteins that take part in a series of reactions called the MAP kinase pathway exert an important influence on differentiation. And MAP kinase and its associates are also required for cell division. At low concentrations, the MAP kinase proteins tell the cell to divide; high levels prompt development of the stem cells into other cell types. So to stay a stem cell, the cell needs to turn down, but not off, activity of the MAP kinase pathway, Ying says. He suspects that several other factors may also walk such a tightrope to maintain stemness.

Other work, reported by an international group of researchers online August 24 in Nature, shows that a large network of many genes is responsible for the pluripotency of embryonic stem cells. Circumstantial evidence suggests that the same set of gene and protein interactions are involved in the cells’ self-renewal, says study coauthor Franz-Josef Müller of the Center for Integrative Psychiatry in Kiel, Germany.

“This is an active group of genes that are doing something together,” Müller says. “I’m pretty sure they’re not just suppressing differentiation signals.” But what exactly all the genes do is not clear.

Many of the factors identified in the networking study circle around the DNA. Some, like the two “master” ingredients Oct3/4 and SOX2, are what scientists call transcription factors, proteins that direct gene activity. (Those two proteins, along with KLF4 and c-Myc, are the transcription factors used to reprogram skin cells into pluripotent stem cells.)

Certain combinations of the A’s, C’s, G’s and T’s that make up the DNA alphabet form what amounts to a reserved parking sign for transcription factors. When the factors find a sign with their name on it, they latch on to the DNA and help to switch nearby genes on or off.

At least that is what would happen if DNA were naked, the equivalent of an empty parking lot. But it’s not. The situation is far more complex, thanks to the chromatin infrastructure that guides the cells’ machinery for activating genes.

During development, two groups of dueling proteins help direct gene activity. The Polycomb group shuts genes down; the trithorax group turns genes on. Both groups accomplish their task by pinning a chemical called a methyl group to one of DNA’s close associates, a protein known as histone H3. The Polycomb group attaches a methyl group to the protein building block lysine at position 27 in the chain of amino acids making up the histone. Trithorax proteins methylate a lysine as well, but at position 4 instead of 27.

It seems a subtle difference, but to a cell it’s a distinction as clear as that between red and green traffic signals.

In embryonic stem cells, genes that encode proteins important in development and about 2,500 other genes carry both Polycomb and trithorax methylation marks, says Bradley Bernstein, a genome scientist and chromatin biologist at Massachusetts GeneralHospital and HarvardMedicalSchool in Boston. So, much as Schrödinger’s hypothetical cat is paradoxically alive and dead at the same time, genes in embryonic stem cells are, in a sense, simultaneously on and off.

Chromatin is packed loosely around these genes, allowing easy access for turning genes on. In embryonic stem cells, the chromatin proteins “breathe,” Bernstein says, latching onto DNA and letting go — like pulling into a parking space, backing out, then parking again. That doesn’t happen in mature, differentiated cells. Once cells begin to specialize, the proteins tend to stay parked, Bernstein told colleagues gathered in Philadelphia in June for a meeting of the International Society for Stem Cell Research.

Although the dual methylation marks allow embryonic stem cells to keep their options open, all the doubly marked genes are switched off. As the cells differentiate, the marks made by the Polycomb proteins are erased, giving cells the green light to develop into particular cell types.

Polycomb and trithorax methylation marks may act as homing beacons to transcription factors searching for their parking spaces. Researchers at HarvardUniversity investigated how nine different transcription factors behave and interact in embryonic stem cells. In the March 21 Cell, the team reported that Oct3/4, SOX2, NANOG, KLF4, and three other transcription factors tend to carpool, selecting genes marked by both Polycomb and trithorax. Those genes are generally active in embryonic stem cells but get turned off as cells differentiate.

On the other hand, genes that have only one type of methylation mark tend to attract single transcription factors, or smaller groups of transcription factors. Those genes are usually shut off in stem cells but get turned on as cells differentiate. A small number of genes have no methyl groups on their histones. Those genes are largely ignored by transcription factors, the Harvard group reports.

Still other genes seem to be turned on at low levels in stem cells all the time, Bernstein says. Those genes may help direct the cell down a developmental path.

This type of promiscuous gene activity is also found in oocytes — immature egg cells, says John Gurdon, a developmental biologist at the University of Cambridge in England. Oocytes will often turn on genes normally found in muscle cells or other adult cells. And the oocytes do it without the help of transcription factors, Gurdon says. That means the earliest cells open up the entire genome, stripping the DNA and histones bare. Development then becomes an exercise in shutting down things that aren’t wanted.

Red light, stop

One method of shutting things down is to stick methyl groups on DNA. The groups gum up the works, closing the on-ramps to the gene-activation fast lane. In embryonic stem cells, some regulatory regions called CpG islands get away scot-free, while other areas of the genome are heavily methylated, Bernstein says.

Louise Laurent, a stem cell biologist at the University of California, San Diego, and her colleagues examined DNA methylation patterns in several different embryonic stem cell lines. The researchers compared the stem cells with many types of differentiated cells to see if stem cells contain other hidden methylation patterns that distinguish them from adult cells.

The group did the same sort of comparison for another type of master regulator in the cells. Those regulators are tiny snippets of RNA only 20 letters, or bases, long. Their diminutive size has earned them the name microRNAs, but the molecules do a big job, controlling much of the protein production in the cell. Usually microRNAs act a lot like building inspectors, shutting down protein-building until certain conditions are met. Each microRNA may help regulate production of hundreds to thousands of proteins. 

On human chromosome 14, the team found a cluster of microRNAs located “bang, one right after the other,” Laurent says. This cluster is turned off in embryonic stem cells. The team soon discovered why. Located nearby is a gene, called maternally expressed gene 3, that makes RNA, but no protein. Only the copy of the gene inherited from the mother is turned on because a special chemical alteration keeps the copy from the father switched off. Scientists call this imprinting.

Imprinting is akin to a genetic custody fight. People inherit two copies of almost all genes, one from mom and one from dad. Most of the time, both copies get to make RNA and proteins, but in a few cases, it’s important that only one copy be active. In those cases, cells decide which parent’s gene will get the honor, by serving the other parent’s gene with a methylation mark. In the case of maternally expressed gene 3, the father’s gene is shut off by methylation while mom’s gene makes RNA. The cluster of microRNAs is imprinted in the same way so that only the mother’s copy is active. The situation may be reversed for other genes.

Many important genes are imprinted, and disrupting this balance leads to diseases and disorders, such as Angelman syndrome and Prader-Willi syndrome.

Curiously, in every embryonic stem cell line the team examined, both the mother’s and father’s chromosome carried a methylation mark. That is not supposed to happen. It’s as if a judge decides that neither parent should get custody and the child ends up in an orphanage instead. The consequence is that the microRNA cluster is silenced in embryonic stem cells.

The result was unexpected, and Laurent is still trying to sort out how the methylation later gets erased from the mother’s chromosome, allowing the microRNAs to be made. The scientists also don’t know why embryonic stem cells handle imprinting so differently from other cells.

“One possibility is that we don’t really understand imprinting as well as we thought we did,” Laurent says. “The other possibility is that imprinting in embryonic stem cells is not stable.”

Driving forward

These types of dysfunctional family battles could help explain why some cloned animals have health problems. Imprinting defects might also limit the use of stem cells as therapies for people.

Just as embryonic stem cells do things differently from mature cells, embryonic stem cells from other species also have particular characteristics, Ying says. Even though human embryonic stem cells and mouse embryonic stem cells both come from embryos at what appears to be the same stage of development, the cells differ in their abilities. Human embryonic stem cells can produce placenta, while mouse embryonic cells can’t. That seems to indicate that human cells are at a slightly earlier stage of development with more possibilities open to them, but Ying says most data suggest human cells are slightly more advanced than mouse cells in a developmental sense.

Ying and his colleagues have succeeded in isolating embryonic stem cells from rat embryos, a feat scientists have been trying to accomplish for more than 30 years. Rat cells are different from either human or mouse cells and must be grown under special conditions, Ying says. He has been able to make the rat cells do almost everything human and mouse embryonic stem cells can do, including producing about 95 percent of the cell types in the animal. But the rat cells haven’t yet formed cells that will produce sperm and eggs, crucial for classification as true embryonic stem cells.

But Ying thinks focusing on differences will teach only a limited amount about how stem cells work. He wants to compare human, rat and mouse embryonic stem cells to see what traits are alike. Stem cells are just too important for evolution to have taken a different tack in every species, he says.

“The real mechanism must be shared between species, so we’re trying to look at what’s common,” Ying says.

Even with rats and mice as guides, it may still be years before scientists know all the secret ingredients and tricks embryonic stem cells use to achieve stemness.