Long before stem cell became a household term, people had observed that plants and animals can, within limits, repair damage they sustain.

Wounds heal, broken bones knit, and lost blood is replenished. Mowed grass soon regrows, and barren trees sprout new leaves in spring. Cut off a limb of a salamander or some other amphibians, and it regrows. But the limbs of other animals, once gone, can’t grow back.

Where do the new cells come from, and why are some organisms so much better at regenerating tissue than others? How can this regenerative power be used to repair muscle tissue that’s wasted by neuromuscular diseases?

The answers lie in the biology of a species’ stem cells, the cells from which all other cells are derived — from which all other cells “stem.”

Of course, a human or animal can grow from a single fertilized egg, which comes from the fusion of only two cells. Somehow, the programming for every tissue, organ and system is contained in that microscopic structure, the embryo.

Embryos give rise to all kinds of cells, so they’re an obvious source of stem cells for tissue regeneration, but there are problems. For one thing, it’s their very flexibility that makes it possible for cells to appear in the wrong place at the wrong time — say, teeth growing in a muscle. Worse yet, their unimpeded growth potential can lead to tumors.

And, harvesting cells from embryos that have the potential to become human raises philosophical and moral questions for some.

Fortunately, embryos are far from the only source of cells that can generate a variety of tissues throughout an organism’s life.

Bone marrow contains cells long known to replenish blood. And more recently, progenitor cells that can mature and adopt specialized roles — “differentiate” — have been found in the umbilical cords of newborn babies, as well as in less expected places, such as adult brains. Cells that can become muscle have been found in muscle tissue itself, as well as in the bone marrow and along blood vessel walls.

In 1961, the electron microscope began revealing structures previously unknown to biologists. In New York, at the Rockefeller Institute, Alexander Mauro was taking a closer look at skeletal muscle fibers (cells). Dotting the surface of the long, striplike fibers that make up voluntary muscles, he saw, were small, rounded cells, lying apparently dormant most of the time but occasionally proliferating.

Because of their location around the periphery of the fibers, Mauro called these satellite cells and suspected they might be myoblasts (from Greek roots meaning muscle and bud).

Later work would show that these satellite cells could differentiate and become more like muscle, and that they moved in to perform needed repairs on damaged fibers (see illustration below). Their numbers didn’t seem to diminish much over time, leading scientists to suspect that they were a type of stem cell — self-renewing in their flexible, undifferentiated state, but also capable of differentiating when necessary.

The advances in molecular biology of the 1980s brought unprecedented understanding of the genetic muscle disease Duchenne muscular dystrophy. By 1987, MDA-backed researchers had identified the lack of the muscle protein known as dystrophin as the source of the problem in DMD, and they knew that dystrophin was normally located just inside the fiber-surrounding membrane. There, it helps provide stability to the cell.

Then, in 1989, two research teams showed that, when normal myoblasts were injected into dystrophin-deficient mice, their muscles were rescued from a Duchenne-like dystrophy. The prospect of transferring satellite cells, with functional dystrophin genes, from healthy donors into boys with DMD, began to be taken seriously.

In the early 1990s, the Association funded five research teams to conduct small clinical trials of a procedure that came to be known as myoblast transfer.

The myoblast transfer trials differed with respect to the number and timing of the cell injections (ranging from one set of injections to injections every month for six months); the sites of injection (leg versus biceps muscles); and the immunosuppressant medications given (none, cyclosporine or cyclophosphamide).

But the results were the same: The procedure was safe, with no ill effects sustained by any of the participants. But one to six months after the injections, very little donor-derived dystrophin was seen, and no one got stronger.

“The tissue and the disease may be very different,” says Emanuela Gussoni of mouse and human DMD. Gussoni, an investigator on the San Francisco myoblast transfer trial, says the tissue matching to prevent immunologic rejection of the donated cells was far easier to control in the mice than it is in people.

MDA grantee Michael Rudnicki says the problem may have been the cells themselves. The lab-grown myoblasts, while they may have originally been satellite cells, were too far along in their development to merge with existing fibers in the patients, Rudnicki says.

Still, a few of the transplanted cells survived beyond the first two days after injection, even in the early 1990s trials. The question is, Which ones?

The goal is to find “cells that are at the right stage and have the right capabilities,” says Sharon Hesterlee, MDA’s director of research development. “It doesn’t matter where we find them.”

Then there’s the environment the new cells enter. A muscle that’s full of scar tissue or one that’s completely intact probably isn’t receptive to new cells. The ideal environment for fiber repair may be cells that have begun to break down, because it’s thought that these “distress signals” orchestrate the repair process.

The impressive regenerative process seen among salamanders may have much to tell us about repair and regeneration, but the messages aren't yet decoded.

The holy grail of stem cell transplantation can be phrased as “right cells, right time, right environment.” Some two dozen MDA research grantees have joined the quest.

In 1990, 28-year-old Emanuela Gussoni arrived at Stanford University in California, having completed a doctoral degree in neuroscience from the University of Milan in Italy and postgraduate studies in neuromuscular disease at Milan’s Carlo Besta Neurological Institute.

The gene for dystrophin, the protein missing in Duchenne MD, had been identified only four years earlier, by MDA grantee Louis Kunkel and colleagues at Harvard-associated Children’s Hospital in Boston, and doctors were gearing up to make use of the new discovery.

In California, as elsewhere, the plan was to undertake myoblast transfer, transplanting cells from close relatives into boys with DMD, with the goal of replacing missing dystrophin and rescuing muscle tissue from destruction.

Gussoni joined a team that included MDA grantees Larry Steinman and Helen Blau at Stanford, and Robert Miller at California Pacific Medical Center in San Francisco.

“My role was to detect any immune response between donor and patient,” she says, “and to design an assay to detect donor transcripts [dystrophin from donors].”

She detected both. In three out of eight boys in the San Francisco trial, she found evidence of dystrophin production from donor cells, although the amount was miniscule compared to the number of injected myoblasts. About 1 percent of the muscle fibers in the injected area showed some normal dystrophin one month after the transfer.

Then, in 1992, just before Gussoni was scheduled to return to Italy, she received an MDA grant that allowed her to stay at Stanford for two more years. After that she moved to Harvard to work with Kunkel, who also had conducted myoblast transfer experiments.

Surviving Myoblasts

Gussoni and Kunkel asked themselves questions about the myoblast transfer trials, none of which had harmed anyone, but none of which had shown much dystrophin production or even much cell survival.

In 1997, with Kunkel and Blau, she published an updated analysis of the fate of the transplanted myoblasts in the San Francisco trial of the early ‘90s.

By then molecular detection techniques had improved. “We re-examined the muscle biopsies using a new method,” Gussoni says. “We had originally injected 80 million to 100 million myoblasts and gotten few dystrophin-positive fibers, so the question was, Where did the cells go?”

Using the new techniques, they found a lot of the cells still there, in fact, more than they had originally thought. “Many were not expressing dystrophin, but they were there,” she says.

Gussoni and others theorized that only the slowly dividing cells survived. “That provided a hint that maybe we should use a different kind of progenitor cells, not the classic ‘myoblasts’ we used.”

For Gussoni and many others, muscle “side population” cells are a major focus. These cells reside in the muscles themselves and probably give rise to satellite cells, as well as integrating directly into damaged fibers at times. (The term side population cells comes from methods used to isolate them.)

Muscle Preparation

In addition to the status of the donor cells, the status of the recipient’s muscle also has to be taken into account, Gussoni notes.

“We have to pay attention to what cells we put in, but also how to condition the muscle to accept the transplant.” Radiation has been shown to help the process in mice, but that has obvious drawbacks and probably can’t be used in people.

“Immune parameters are important,” Gussoni says. “The children in our clinical trial were immunosuppressed, and now we know that some immunosuppressant medications interfere with differentiation [maturation] of muscle cells. Cyclosporine [the drug they used] does that.”

But just matching fathers or siblings to patients isn’t enough to prevent an immune response.

“These days we would not have gone to human myoblast trials from the mouse data that we had,” she says.

Cell Integration

For Gussoni, a crucial question is, What forces determine cell integration? “The cell has to have something on it that can be recognized by a fiber,” she says. “I don’t think the process is random. When you look at muscle fibers after cell delivery, there are groups of cells that have engrafted. It’s not a random uptake.”

The role of intercellular signals that say “repairs needed” and “repairs offered,” once understood, Gussoni believes, will clarify where we go from here.

"I think that when the results of those first experiments from the 1990s came in — that myoblast transplants don’t work — alarm bells rang,” says Michael Rudnicki, an MDA research grantee at the Ottawa Health Research Institute, where he heads the Program in Molecular Medicine.

In 1988, after earning a doctoral degree in biology at the University of Ottawa, Rudnicki went to the Massachusetts Institute of Technology’s Whitehead Institute in Cambridge for postdoctoral studies.

There he trained with famed molecular geneticist Rudolf Jaenisch, who, in the 1970s, had been among the first scientists to insert DNA into mouse embryos and breed mice with conditions mimicking human disorders.

Rudnicki joined this effort, producing and analyzing mice with genetic mutations. He concentrated on figuring out the function of the myoD proteins, which push undifferentiated cells toward becoming muscle.

In Rudnicki’s view, a principal reason for the failure of most of the myoblasts to survive and join the recipients’ muscle fibers is the nature of the cells that were chosen.

“The state of the art for satellite cell biology was really descriptive histology [study of tissues],” he says. “There were a few exceptions, cell biologists working in the area. But they were primarily not involved with genetics or molecular mechanisms.

“It was thought that myoblasts were the same as satellite cells,” Rudnicki says of the early 1990s, “and that is absolutely incorrect.”

A muscle satellite cell, Rudnicki says, “is defined by its anatomical location. It’s beneath the basal lamina [a tough sheath surrounding each fiber], closely nested in a cleft against a muscle fiber. Within that population, some portion of the cells may have more robust self-renewal and expansion capacity than others, but satellite cells clearly are upstream [earlier in their development] from myoblasts.”

When a satellite cell becomes activated (as it does when fiber repairs are called for), Rudnicki says, it starts making proteins called myf5 and myoD. At that point, it becomes a true muscle precursor cell. “Then, if we put that into a culture dish, we get what we would call a primary myoblast,” he says.

Once a cell has reached that stage, it can’t go back to being a satellite cell, and it can’t fix a damaged muscle fiber. “It’s a one-way street,” Rudnicki says. Not all the satellite cells taken from donors and grown in a lab dish have reached that point of no return, but most of them have, he says. And as time goes by in the lab, more and more of them reach it.

Pre-myoblast satellite cells would be needed for repair of damaged tissue.

Rudnicki believes that cell stage isn’t the only important factor in cell transplantation. The environment into which the cells are placed may be as important.

Dealing with the immune system’s response to the new cells and, in Duchenne MD, the dystrophin that the system may regard as “foreign” if the body hasn’t encountered it before, is crucial, he notes.

He hypothesizes that younger donor cell recipients may have more receptive muscles and a more tolerant immune system, although he isn’t certain. But Rudnicki is certain that scarring in the muscle must be minimized for cell transplantation to work.

“I think that’s one area that we need to pay more attention to,” he says. “We need to learn more about the mechanisms that cause scarring and learn how to stop that.”

And, Rudnicki notes, there’s also more than one reason to understand how cells choose a muscle career path.

“In my view,” he says, “cell transplant therapy is still a very technically challenging approach. If we could identify a drug that would, even in a modest way, stimulate the activity, expansion or self-renewal of [the patient’s own] muscle satellite cells, we could perhaps make Duchenne muscular dystrophy into a chronic disease rather than a lethal disease. I think that’s an approach we shouldn’t forget about.”

Molecular biologist Margaret Goodell, an MDA research grantee at Baylor College of Medicine in Houston, became interested in muscular dystrophy through her long-standing research on blood cell regeneration.

Since 2002, she’s been an associate professor at Baylor’s Center for Cell and Gene Therapy, where she received a Michael E. DeBakey Excellence in Research Award last year.

Goodell, born in Baltimore in 1965, completed a doctoral degree in biology in 1991 at the University of Cambridge in England. She then relocated to Cambridge, Mass., to begin postdoctoral research at the Whitehead Institute for Biomedical Research, studying cells that form blood.

Blood cells regenerate from stem cells found in the bone marrow, she explains. “We became interested in the possibility that you could use bone marrow stem cells for the regeneration of other tissues.”

Goodell’s lab studies a type of bone marrow cell called a hematopoietic stem cell (HSC). Normally, hematopoietic (blood-forming) cells generate the many different types of red and white blood cells.

But over the past five years, a number of laboratories, including Goodell’s, have reported that, in very rare instances, HSCs also appear to convert to mature muscle cells. “What we’re trying to do is find out why it’s at a low efficiency and see if we can boost that efficiency,” she says.

To detect the conversion, researchers typically place stem cells that have been tagged with a visible marker, such as a fluorescent protein, into a laboratory mouse or a tissue culture dish growing muscle cells.

“If you stimulate the muscle to regenerate, you will see that a small proportion of the muscle cells have incorporated the fluorescent tag,” indicating that the stem cells have become muscle cells, Goodell explains.

The research is still in its infancy, and researchers continue to debate the means by which the bone marrow cells turn into muscle. Some scientists think the stem cells’ genetic program becomes “rewired” so that, instead of maturing into blood cells, they become muscle cells. But Goodell suspects something else may be happening.

“My lab thinks that it’s fusion — that some type of blood-borne cell fuses to the muscle cells.” Whatever the mechanism, the bottom line, Goodell says, is that bone marrow cells or their descendants can wind up in the muscle. And this may offer a path to therapy for people with muscle diseases.

Goodell’s team has homed in on a particular type of bone-marrow-derived blood cell — the macrophage — which they think is the predominant blood cell type that fuses with muscles.

Macrophages are large, amoebalike cells that crawl through the tissues of the body, scavenging bacteria and other foreign particles. They often fuse with one another, and muscles normally grow by the fusion of muscle stem cells to mature muscle fibers. So, Goodell says, it’s reasonable that macrophages might also fuse with muscle cells, and experiments in her lab support that notion.

“We’ve been, first of all, trying to identify proteins that are involved in the fusion between the macrophage and the muscle cell, and we’re trying to see whether their expression can be modulated to enhance the process’s efficiency.”

There’s still much to be learned before Goodell’s research reaches patients. “If it ever does lead to a therapy, we’re probably talking about at least 10 years,” she cautions.

Nonetheless, she’s excited at the prospect of treating muscular dystrophy with blood cells. “It’s a way to get at virtually all of the muscles in the body, not just the major ones that we can see. Every muscle fiber is fed by the bloodstream in one way or another, so if you can really get something delivered through the bloodstream rather than in some localized way, it’s potentially a very powerful therapy.”