Stem Cell Therapy the Huard Way

by Dan Stimson

Twelve years ago, MDA-funded scientists began an ambitious clinical trial in boys with Duchenne muscular dystrophy (DMD). In a procedure called myoblast transfer, they removed immature muscle cells (myoblasts) from close male relatives who didn't have DMD, and transplanted them directly into the boys' muscles. The myoblasts were supposed to regenerate the boys' damaged muscles, but they had little, if any, effect.

For cell biologist Johnny Huard (pronounced HEW-ard), who contributed to the preclinical research on myoblast transfer and helped to conduct an arm of the trial at Laval University in Quebec, the trial's failure left a lasting impression.

"We had 11 patients coming to the hospital regularly and I became good friends with them," Huard remembers. "For some of them, we increased their strength temporarily, but the treatment didn't hold and we weren't able to save their lives. I became very touched by those people."

Huard remained committed to finding a cure for DMD, but for a few years, he shifted his research away from cell transplantation.

Then, research in the late 1990s set the stage for a new look at cell transplantation for DMD and a host of other diseases. Scientists made several breakthrough discoveries about embryonic stem cells — cells that aren't just immature, but are nearly blank slates, capable of mass-producing the diverse cell types that make up the human body (see "Embryonic Stem Cells"). They came up with ways to grow stem cells in the laboratory, and found a collection of stem cell "markers" — proteins that could be used to identify stem cells and separate them from other cells.

Using those markers, Huard and other scientists began to fish for stem cells in adult tissues — including bone marrow, brain and muscle. By combining the markers with a new twist to his old myoblast isolation procedure, Huard recently hit pay dirt — a type of stem cell derived from adult muscle that appears to have an unmatched capacity for generating muscle. Armed with these new cells, he's gearing up for another try at cell transplantation for DMD.

Stem Cells With Muscle

Johnny Huard demonstrates the procedure he developed for isolating muscle-derived stem cells, which he hopes to use as a treatment for muscular dystrophies.

Myoblasts seemed to be effective at regenerating muscle tissue in mice with DMD, so scientists were baffled when the cells fizzled in humans.

Some scientists returned to animal studies in hopes of improving myoblast transfer and retesting it in people. Others spent years poring over tissue samples from the dozens of boys who had undergone the procedure. They found that some myoblasts were destroyed by the recipient's immune system, some appeared to become dormant, and others failed to make dystrophin — the protein that's missing in DMD.

Around that time, Huard was studying gene therapy for DMD, but in the dismal results of myoblast transfer, he saw a glimmer of hope. Although the myoblasts were duds overall, at least a small fraction of them, or perhaps some unknown cells inadvertently mixed in with them, appeared to have myogenic potential — the power to survive and produce muscle.

In hopes of finding those cells, he began to toy with his procedure for isolating myoblasts from mice — which involves taking a sample of skeletal (voluntary) muscle, and distilling it into a mixture of fluid and individual cells suspended in a flask. After about a day, some of the cells begin to attach to the flask — those are myoblasts.

"For years, we used to assume that if the cells didn't stick to the flask, they were dead, so we threw them away," Huard says.

But when he waited a week for the floating cells to stick, he found what he'd been looking for — cells with improved myogenic potential. And "when we started testing them for stem cell markers, we found out they were stem cells," he says.

Stem Cell Jump-Start

In 1999, MDA grantees Louis Kunkel and Emanuela Gussoni of Children's Hospital in Boston isolated stem cells from mouse muscle by revamping a procedure that had been used to isolate stem cells from bone marrow. Then, they used a bone marrow transplant procedure to deliver the cells to mice with DMD. Mark Keating believes the regenerative powers of newts might hold clues to repairing muscles destroyed by muscular dystrophy. The transplanted cells formed bone marrow in the mice, and a small fraction migrated away from the bone marrow to form new muscle fibers — but not enough to have a therapeutic effect (see "Renewing Muscles and Nerves," Quest, vol. 7, no. 2). "The procedure just had very low efficiency, and it needs to be improved," Kunkel says. Damaged muscles probably send out a distress signal that beckons stem cells from the bone marrow, but the signal is weak, Kunkel believes. Finding a way to boost that signal could be the key to making bone marrow transplants an effective treatment for DMD and other types of muscle damage, he says. In fact, many scientists have speculated that if there were a way to mobilize the stem cells in bone marrow — or in other adult tissues — it might be possible to perform stem cell therapy without a stem cell transplant. Late last year, scientists reported they'd

used that strategy to improve survival in mice following heart attack. Piero Anversa of New York Medical College in Valhalla and his team injected the mice with two signaling proteins called cytokines, then gave the mice heart attacks by tying off blood vessels to their hearts. The cytokines coaxed stem cells in the bone marrow to migrate through the bloodstream and repair the damaged cardiac muscle. The cues that attract stem cells from bone marrow to skeletal muscle are poorly understood, but research by Mark Keating, also of Children's Hospital in Boston, suggests that one day it might be possible to mobilize stem cells within muscle to regenerate lost muscle fibers. Keating studies regeneration in amphibians (frogs, newts and the like), which are known for their remarkable ability to grow back an entire amputated limb — muscles, nerves and all. "We don't think there's any reason mammals can't regenerate those things. The difference is in the molecular cues, and we're trying to discover those," Keating says. Amphibians owe much of their regenerative powers to a process called dedifferentiation, in which a cell regresses from a specific (differentiated) cell type such as a muscle cell or nerve cell to a type more like a stem cell. A little over a year ago, Keating showed that a gene called msx1 can promote dedifferentiation in mouse muscle cells. The gene is normally turned off in mature muscle cells, but when Keating turned it on, the cells split apart and began to divide. And last November, he reported that a liquid extract derived from newt limb had a similar effect. "This line of work has great promise for people with muscular dystrophy," Keating says. But he adds, "[it's] all being done in animals, so it's still far away from anything that's going to help patients."

Stem Cell Transplants

Huard, who's now at Children's Hospital of Pittsburgh, isn't the first scientist to isolate muscle-derived stem cells from mice (see "Stem Cell Jump-Start"). But, in his effort to reinvent myoblast transfer, he's the first to transplant muscle-derived stem cells directly into mouse muscle. With support from MDA, he hopes eventually to bring the procedure to clinical trials for DMD and other muscle diseases.

Muscle-derived stem cells, Huard believes, will succeed where myoblasts failed because stem cells occur a step or two before myoblasts in the chain of command for making new muscle.

Myoblasts form muscle; but the stem cells in muscle — perhaps reserved there from embryonic life — form myoblasts. To produce new muscle tissue, a myoblast fuses to an existing muscle fiber and thus ends its own short life. To produce a myoblast, a stem cell splits in two, making a new cell and preserving itself.

This means that stem cells can make new muscle and keep making it long after transplantation. It also means that muscle-derived stem cells lack most of the characteristics of mature muscle, including cell surface proteins that can trigger the immune system, leading to rejection of the transplanted cells. And finally, there's evidence that the stem cells in muscle don't just produce muscle fibers, but other cell types that might improve muscle regeneration.

One kind of stem cell transplant Huard is investigating — called an allogeneic transplant — is to use muscle-derived stem cells from healthy donors, the same way myoblast transfer was done. "In the best-case scenario," he says, "we could isolate cells from a single donor, grow millions of them in the lab, and then inject 50 different patients."

But there's a drawback to that approach: Even though stem cells are believed to possess few immune system triggers, many scientists fear that allogeneic stem cell transplants might provoke the same type of immune rejection observed in myoblast transfer.

So, Huard is looking into another possibility called an autologous transplant, in which the goal is to isolate stem cells from someone with the disease and transplant them into the same person. For someone with DMD or any other genetic disease, the stem cells could be fixed with a corrective gene before they're transplanted.

Promising Results

In December, at the annual meeting of the American Society for Cell Biology in Washington, Huard reported that he'd tested both kinds of transplant in mice. The results of those MDA-funded studies haven't yet been published, but are being reviewed by the Journal of Cell Biology.

Huard and his colleagues discuss the differences between muscle-derived stem cells and myoblasts — which could mean the difference between successful therapy and failure.

To test the autologous transplant approach, Huard and his colleagues isolated stem cells from the muscles of mice with DMD, grew the cells in large numbers, fixed them with a corrective dystrophin gene and reintroduced them into the diseased mice. The stem cells made dystrophin-positive muscle fibers, though not enough to restore normal muscle function.

The allogeneic transplants — injecting healthy donor-derived stem cells into the muscles of mice with DMD — worked better, in some cases restoring dystrophin to more than 25 percent of the cells in the injected muscle. That's probably enough to improve the muscle's function, Huard says, noting that many boys with Becker muscular dystrophy, a less severe version of DMD, have a similar fraction of dystrophin-positive fibers. "We're doing the strength analysis [on the mice] right now," he says.

Another interesting result of the allogeneic transplant is that "[the injected] stem cells didn't just make muscle fibers. They made blood vessels, and they made peripheral nerve," Huard said. For boys with DMD, this could turn out to be beneficial, especially in older boys who might have secondary damage to the vasculature and nerves connected to their muscles.

How Stem Cells Might Be Used to Treat Muscular Dystrophy
ALLOGENEIC TRANSPLANT (1) A sample of muscle is removed from a healthy donor (2) Stem cells are isolated and grown in the lab (3) Stem cells are injected into muscles of the person with the disease	AUTOLOGOUS TRANSPLANT (1) A sample of muscle is removed from the person with the disease (2) Stem cells are isolated and grown in the lab (3) The stem cells are treated with a virus that carries the corrective gene (4) The treated stem cells are injected back into the person's muscles

Next, Huard plans to test stem cell transplants in animals larger than mice, determine whether the cells carry any risk of forming tumors and, perhaps most importantly, find cells from human muscle tissue that are equivalent to the mouse stem cells he's been studying.

Without those crucial experiments, "stem cells aren't yet ready for clinical trials," he says. But he has consulted with the Food and Drug Administration in preparation for a trial in youngsters with DMD.

Eventually, Huard hopes to use a combination of muscle stem cell transplants to strengthen voluntary muscle and intravenous systemic delivery of stem cells to restore dystrophin in the heart and diaphragm.

Editor's note: A few private companies currently offer variations of myoblast transfer or stem cell therapy for DMD. These procedures, in which one "shot" of cells can carry a price tag of $100,000 or more, aren't approved by the FDA (hence, they're only available overseas). It's MDA's position that the treatments haven't been adequately tested for safety or effectiveness.