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Tiny Cells, Huge Possibilities

After eight years of ethical and political disagreement, a change in the White House may touch off an explosion of stem cell research at Johns Hopkins and elsewhere. But already, new and less-controversial advances in stem cell creation are making the search for cures even more promising.

By Michael Anft

As election returns rolled in on the night of November 4, researchers at labs on the Johns Hopkins medical campus raised glasses and celebrated the victory of Barack Obama. These weren't the street revelers who overtook downtowns across the country — no one would peg this reserved bunch as Obama's base. In fact, many were noncitizens who couldn't even vote. Some of the celebrants might have applauded his economic plans, his directional shift on Iraq, or something not having to do with their work. But most were single-issue enthusiasts. Obama's promise that, under his administration, the rigors of science would be undertaken without interference — to "restore science to its rightful place, and wield technology's wonders to raise health care's quality," as he put it a couple of months later at his inauguration — is what lit up the white-coat crowd. That, and the minuscule speck of biology lurking in the nucleus of Obama's lofty language: the stem cell.

In the months following that night, researchers who study the stem cell have remained buoyant. "It almost feels like a liberation for people who do science," says Chi V. Dang, Med '82, a witness to the jubilation as well as the vice dean of research at the School of Medicine. He also serves as director of the Johns Hopkins Institute for Cell Engineering, where scientists grow stem cells every day to seek new answers to old biological questions. Dang conducts research on cancer in his own lab using stem cells — the building blocks of life that construct blood vessels, the nervous system, organs, and everything else that makes us human.

Since first being grown in a lab in 1998, human stem cells have been touted as the key to understanding the mechanisms that underlie long-incurable diseases, an answer to cancer, a way to get paralyzed limbs moving, and a step toward using cells as medicine. One day, scientists envision, therapies wrought from various types of stem cells will be used to treat entire classes of diseases and injuries — perhaps turning chemical-based drugs and the side effects that come with them into relics. Scientists have coined a name for the field that taps stem cells' promise in rebuilding human tissue: regenerative medicine.

Stem cells have been regarded as the best — perhaps the last — hope for patients with certain diseases such as diabetes; if stem cells could be used inside the body to grow the pancreatic islet cells that produce insulin — cells that are ravaged by an immune response in diabetics — then, one day, injecting them into patients might reverse the disorder. The same might be true for Parkinson's disease, which afflicts 2 percent of people over age 65, and thousands of younger people. If stem cell therapies could be developed that would repair or replace damaged or dead dopamine neurons in the brain, sufferers would no longer lose control of their movements. Stem cell research affords scientists the chance to see if they can develop personalized "drugs" that would work throughout the body, or to uncover the secrets behind the development of human tissues and the origins of disease. No other therapy or group of therapies comes close to its potential impact.

Like other scientific powerhouses unobservable to the naked eye — think of the atom or the bacterium — the stem cell has become magnified not only by the microscope but by public debate. In 2001, then president George W. Bush, siding with social and religious conservatives who believe life begins at conception, ordered restrictions on federal funding of stem cell research derived from human embryos. In the process of extracting stem cells, scientists destroy days-old embryos that have been fertilized in test tubes, originally to artificially impregnate women. Typically, those embryos left over from in vitro fertilization are frozen or thrown out as medical waste.

In the intervening years, dozens of scientists moved abroad to do embryonic stem cell research in countries such as Great Britain, Singapore, and Sweden, where laws and funding favored them. Some stayed behind to work with more than 60 government-approved embryonic stem cell lines created before Bush's order took effect, even though most of those lines proved to be tainted by mouse viruses, or developed genetic changes that occur over time. Other bioscientists decided to do research that had nothing to do with stem cells. They avoided hassles with paperwork and keeping their federally supported labs separate from labs where researchers performed nonsanctioned stem cell experiments fueled by a smattering of private grants and outlays from several states, including California (which pledged to invest $3 billion) and Maryland.

Because of the strictures, many bench workers and professors believe that the pace of embryonic stem cell work has moved too slowly.

How handcuffed have researchers been? The National Institutes of Health — the biggest repository of federal research dollars, with an annual grant budget of nearly $30 billion — approved only about $2 billion for all types of stem cell research last year. That's less than 7 percent of NIH's total expenditures, for the area of research that scientists are calling "the gateway to 21st-century medicine." Obama vowed to overturn restrictions and free up more federal dollars for stem cell research. His opponent, John McCain, vacillated on the issue. Hence, the euphoria among bioresearchers that night in November. In March, Obama validated their exuberance, allowing researchers to apply for federal grant dollars to work with embryonic stem cell lines created with the help of private or state money.

"A Brief History of Cell Engineering"
"When you're told you can do ethical research in an unfettered way, you have the opportunity to play out any scenario, to fulfill the potential that comes from the art of doing research," says Dang.

But the liberation Dang feels isn't limited to new opportunities to plumb the depths of stem cells taken from destroyed embryos. Two years ago, stem cell scientists envisioned an arc connecting only embryonic stem cells to cures. But, as often happens in science, the line from the past to the future is jagged and frequently interrupted, and can take a sharp turn or two. Researchers in Japan and the United States have devised a way to unleash the power of the stem cell without destroying embryos — the concern that led Bush to limit stem cell research in the first place. Theologians and bioethicists who are squeamish about embryos have no problem with this newer type of stem cell. Researchers are intrigued by it as well. The converted adult cells, they say, offer advantages over cells taken from embryos because they could be altered, then injected into their donor. By using a person's own stem cells as medicine, doctors could eliminate worries about a patient's immune system rejecting the cells, as they might if they were derived from embryos.

If nothing else, so-called induced stem cells call into question what the future of research might be. They give scientists a lot to ponder: Will they still need embryonic stem cells for certain types of experiments that lead to treatments, or to learn the biomechanics of disease? Or will they be able to skirt ethical misgivings by exclusively using cells that come from a patient's own body?

Taking cells from a patient is simple enough. A surgeon simply removes a plug of skin with a scalpel and caps it in a test tube. But to reprogram those cells requires scientists at the bench to do nothing less than turn back time, if only to get a glimpse of the future.

Cassandra Obie has grown cultures for Johns Hopkins researchers for decades. But last May, while manning her well-lit workspace at the Johns Hopkins Institute for Genetic Medicine, she began receiving a different kind of request. Physicians treating people with amyotrophic lateral sclerosis (ALS, more commonly known as Lou Gehrig's disease), Huntington's disease, Parkinson's, and schizophrenia sent her biopsies — "a three-millimeter standard punch of skin" for each patient, she says — and asked her to grow primary tissue cells called fibroblasts from them. The cells would be used for research in mouse models; the patients agreed to undergo the biopsies in hopes that the work would provide clues, and perhaps cures, to their diseases.

Obie, a senior research specialist, takes a small piece of the biopsied tissue apart with a scalpel, then digs the scalpel into a culture dish, forcing the skin cells to adhere to the cuts she makes. The process of taking a cell back to its infancy — of turning back the clock — is rooted in the technical, not the miraculous. "You have to embed it. If you see the piece of skin floating around," she says, "you won't grow fibroblasts."

Within three days to a week, she'll see fibroblasts, thin fibers that stretch out in all directions like microscopic tree branches from pieces of biopsied skin. Once a week, she gives them a bath of fresh medium — made up of the fetal serum of a cow, as well as a combination of antibiotics and non-essential amino acids — before returning them to an incubator. After cells have grown to cover more than half the dish, she scoops them out and places them into tissue culture flasks. When the flasks become full, she sends the five- to six-week-old cells upstairs to another lab in the Broadway Research Building, where Institute for Cell Engineering investigators wait for them to be made young again.

That's Jason Chiang's job. A graduate student in neuroscience in the lab of associate professor Hongjun Song (and a physician in his native Taiwan), Chiang transforms fibroblasts into induced pluripotent stem cells (iPS cells) — a mouthful that means adult tissue cells that have been reprogrammed into cells that, like embryonic stem cells, can make a variety of cell types. Chiang watches as the fibroblasts develop dots — concentrated bits of energy that will help them grow — under the microscope.

It takes a month for fibroblasts to morph into iPS cells, which very closely resemble embryonic stem cells, or ESCs. Chiang varies the medium they grow in daily, moving them to new dishes as they multiply. By changing the medium over the next three to four weeks, he guides them to "differentiate" into neural stem cells — the ones Chiang and Song need to investigate the workings of the brain and diseases that affect it. Otherwise, they could become stem cells that form any other part of the body. Chiang makes sure to use only the first batches of cells from a dish, ones that haven't lived long enough to develop traits that could lead to bad science. "We try to avoid genetic mutations" that could wreck results, Chiang says.

In all, Chiang has made about 1 million neural stem cells in the last year or more so that he can inject a small amount of them into the embryos of mice. After the mice are born, the researchers will observe them for signs that the stem cells have been integrated into the brain. If they have, Song says, "it can give us a preclinical model for human diseases."

Song says that iPS cells offer researchers several advantages over ESCs. Since the stem cells are derived from a patient's own cells, it is less likely the patient's immune system will reject them. They also are much less likely to form tumors than ESCs. After creating pancreatic islet cells during the iPS-making process, Song explains, "we could replace a [diabetes] patient's bad cells with ones that could make insulin. They would become part of the patient's pancreatic tissue. So, you would only need to inject them once" — which would limit possible complications and help people who have been financially battered by having to pay thousands of dollars each year renewing their prescriptions.

The newer type of stem cells also makes patient-specific genetic manipulations possible, meaning that some diseases might one day be reversed, says Ted Dawson, co-director of the Institute for Cell Engineering. "We may be able to use these cells to deal with a translocated chromosome in an individual with leukemia," he says. "Eventually, we might be able to take a skin biopsy from a patient, correct the abnormality in the lab, give the patient chemotherapy to kill off the blood marrow cells, and then inject the patient with the corrected cells."

Because iPS cells are derived from a patient, they could be used in a dish to test drugs to see if they will stamp out a disease. "If you can get the dopamine neurons of a patient with Parkinson's, you can see what works on them and what doesn't," says Song.

For science to garner as many answers as possible, it needs all the tools it can wrap its gloved hands around, say researchers. Song and his wife, Guoli Ming, also an associate professor in neurology, investigate how to activate adult stem cells that weren't made in a lab but naturally live on in the bodies of people — even the elderly — long after the brain and nervous system have been formed. Researchers hope that tweaking this third type of stem cell can help them cure disease.

Adult stem cells can regenerate themselves even after they are done forming nerves and organs — a power that distinguishes them from embryonic stem cells and iPS cells. Unlike the other two types, adult cells have been used for years in bone marrow transplants to treat people with blood disorders. Experimentally, researchers at the Johns Hopkins-affiliated Kennedy Krieger Institute are searching for ways to activate adult stem cells in the spinal cord to help some of the 250,000 paralyzed people nationally regain some mobility.

By experimenting with all types of stem cells, Song says his lab has begun to learn how plastic existing stem cells in the brain are and how anti-depression drugs activate them. The lab's goal now is to learn how to use cells' flexibility to develop treatments for conditions such as autism and schizophrenia.

"That shows you how broad an impact one stem cell inquiry can have," he says. "Can you imagine the impact we can have when science has thousands going at the same time?"

The 2007 announcement of Japanese researcher Shinya Yamanaka's discovery that regular human adult cells could be returned to their stem cell state — and the success in replicating his experiment since then — would seem, in one light, to make the controversial embryonic stem cell pass´┐Ż. But it's not that simple. For science to garner as many answers as possible to the riddles of disease, it needs all the tools it can wrap its gloved hands around, say Johns Hopkins researchers who use stem cells to unravel them. That includes cells made from embryos.

"If there's such a thing as a gold standard in this type of research, the embryonic stem cell is it," says Jeffrey Rothstein, a professor of neurology at the School of Medicine and a longtime researcher of ALS. An adult cell has been affected by its environment and gone through changes as it has become damaged or developed resistance to damage. It has been exposed to viruses. Will all that affect stem cells derived from it? Given that stem cells may one day be used to grow replacement organs and to cure ailments of the circulatory and nervous systems, any unintended consequences are likely to be harmful. For now, it is best for scientists to play out their differences in the labs, so humans aren't endangered by whatever may lurk deep inside the cell. At this point, iPS cells are intriguing but essentially limited.

"It's a bit like a brand new tire versus a retread," Rothstein explains. "They look the same, but one is new and the other is made of old, used stuff that may not last as long. Maybe the glue will come loose one day and the tread will fall off. We just don't know." What's important, say Rothstein and others, is that scientists have enough funding to do a wide variety of research, including comparing the two types of stem cells and seeing what the differences are.

"A Cluster of Ethical Questions"
Some scientists worry that iPS cells made from older tissue may exhibit signs of advanced aging. Dolly, the lamb cloned in Scotland from the cells of a six-year-old sheep, lived only half a typical ovine lifespan. Was it because she was cloned using the cells of an older sheep? The issue of iPS cells' "pluripotence" — the ability to become any type of cell in the body — is debatable as well. While embryonic cells unquestionably demonstrate pluripotence, it isn't fully known whether reprogrammed adult cells are equally versatile, or whether their genetic material is faithfully replicated. What's more, iPS cells don't "live" nearly as long as those made from embryos. And doing what Obie and Chiang do takes a lot of money. "The technology is still new," says Song. "It is very expensive to reprogram cells."

Because iPS cells are reprogrammed using genes associated with cancer, researchers worry that therapies might pose a danger to patients in the longer term — something that would limit their clinical applications until scientists can figure out how to use different genes. Already, possibly with the aid of political change, ESCs are ahead on the clinical front. Days after Obama's inauguration, the Food and Drug Administration announced its approval of the first clinical human trials using embryonic stem cells. The California firm that won approval had been asking the agency since 2005 for permission to run trials on people with spinal cord injuries. During the trials, they will be injected with stem cells capable of reversing paralysis in experimental rodents.

Physicians who deal with particularly difficult diseases, like ALS, say that keeping all types of stem cells on the lab bench makes the most sense. Some type of stem cell therapy may be the last hope for sufferers of a disease that strips bodies of their ability to feel or move but leaves minds intact to assess the damage. The FDA hasn't approved an ALS drug since 1995. Unlike Parkinson's disease, which centers upon one cell — the dopamine neuron — ALS attacks a complex system of cells, making it incredibly difficult to understand. While Ted Dawson and others can envision the development of treatments for Parkinson's, ALS researchers worry about how to identify how ALS comes into being, how to stop its progression, how to repair existing damage, and how to eliminate it from a patient's system. The use of iPS cells as the lone research tool likely won't do much to speed up the pace.

"We still need to work through some of the technical issues," says Rothstein. "It would take three years with the technology we have now to connect one nerve in the spinal cord to one in the foot. One of the problems [with iPS cells] is that they grow so slowly."

Other roadblocks exist, and they point up how far researchers have to go. If physicians had stem cell therapies at their disposal, they might be able to inject people with cells that could help them rebuild nerves and tissue, but they'd still lack the targeting methods necessary to make sure they do the right job in the right place — another aspect of stem cell research that needs to be figured out. "It's like having your computer screen go black," says Rothstein. "You could go to Radio Shack and buy these little processors, and then open up the back of your computer and toss them in there. But would they work?"

To come up with better models for ALS and some genetic neurological disorders, Douglas Kerr, an associate professor of neurology, says that ongoing research with human ESCs is indispensable. Stem cells from ESCs feature all the guides for the growth of new axons and neurons, he says: "It's only been in the last five years that we've figured out how to generate the right kinds of stem cells. We're really in time zero. We've learned how to generate millions of specific cells, but research is just in its infancy."

Elsewhere on campus, the same recognition that the stem cell field is too new to limit experimental designs is replicated many times over. In Song's lab, both embryonic and induced stem cells are used "because we don't know which ones are going to be most effective in research," says Jason Chiang. "We're still not sure whether iPS cells will behave the same as ESCs in a mouse." Until scientists from all disciplines know and understand the differences, they will need to experiment, comparatively and otherwise, with all types of stem cells, he says.

Song adds that keeping all options open also could grow the research field and guarantee there will be enough scientists around to follow the most promising leads. Somehow settling the bio-ethical concerns — Will science devise a safe way to extract stem cells without killing embryos? Will new methods to derive stem cells from umbilical cord blood solve the problem? — could lead to more federal research money, which will push the field farther faster, he says.

"How do we train the next generation of researchers without it? There have been so many people scared off. This field is so broad that there are many avenues of research," Song says. "We need as many people on it as possible."

The march of science may now be ready to break into a sprint, putting it in position to catch up with the wishes of the public, which, polls show, overwhelmingly favors increasing federal spending on stem cell research. Chi Dang says that Johns Hopkins is ready to accommodate more stem cell researchers. There's 40,000 square feet of lab space set aside for the purpose, plus what he estimates to be tens of millions of dollars in additional funding that could finance a dozen or more investigators. Many of them will work with embryonic stem cells. "What we're starting to see now is a real variety of grant applications," says Dang. "The projects we will work on now will try new things — things we couldn't have imagined doing a year or so ago."

The ascendancy of the Obama administration may indeed answer the wishes of researchers, who say that ethical questions should be broadened to consider the good that embryos slated for eventual destruction could do for humankind.

"If we accept as a society that in vitro fertilization is a good thing, then the real question here is whether we should throw away unused embryos, because we'll have plenty of them," Dang says. "That's where we'll find a lot of answers."

Michael Anft is senior writer for Johns Hopkins Magazine.

Go to "A Brief History of Cell Engineering"
Go to "A Cluster of Ethical Questions"
Return to April 2009 Table of Contents

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