ARTICLE IN BRIEF
Investigators were able to produce and engraft human muscle stem cell progenitors into a mouse model of muscular dystrophy, producing enough dystrophin to enable the muscle fibers to contract normally.
For the first time, human stem cells capable of generating normal muscle fibers have been produced in sufficient quantities to treat muscular dystrophy (MD) in mice. The grafted cells enabled muscle fibers in the mice to contract normally, thereby raising hopes for the development of the first effective treatment of the disease in humans.
“This is a major advance,” said Rita Perlingeiro, PhD, principal investigator and lead author of the paper published in the May 4 issue of Cell Stem Cell. “With this technique it is possible to produce enough human muscle stem progenitor cells to achieve a therapeutically effective response. And because these cells are derived from reprogrammed skin cells, there are none of the ethical concerns associated with using embryonic stem cells.”
The researchers produced rapidly dividing populations of skeletal myogenic progenitor cells by using the paired box protein 7 (Pax7) gene to modify two human induced pluripotent stem (iPS) cell lines derived from skin cells, and an existing human embryonic stem cell line. The gene produces the Pax7 protein, essential for skeletal muscle regeneration. [Dr. Perlingeiro's work was supported by a two-year NIH Challenge Grant.]
People with MD have a mutation in the gene for dystrophin, a protein that connects muscle fibers to the surrounding tissue. The dystrophin gene is the longest human gene, and mutations reduce or eliminate dystrophin production, leaving muscle fibers weak and easily fatigued. In more severe forms of MD, such as Duchenne MD, muscle fibers die, causing progressive paralysis and premature death.
Dr. Perlingeiro and her colleagues used adult fibroblasts from normal human donors and converted them into iPS cells that could be expanded in vitro, producing a large number of cells for implantation.
When injected into dystrophic mice, the Pax7-induced iPS-derived myogenic cells engraft into muscle and produce dystrophin, enabling the muscle fibers to contract normally.
Because iPS cells are derived from a patient's own skin cells, returning them to the body won't require immunosuppressive drugs to prevent rejection, according to Dr. Perlingeiro, associate professor and Lillehei Endowed Scholar at the Lillehei Heart Institute and department of medicine at the University of Minnesota in Minneapolis. However, the sudden appearance of dystrophin protein in a body that has never produced any could cause an immune reaction.
“If we choose to replace the dystrophin gene that's missing or not producing properly, you'd probably have a reaction because these patients have never experienced dystrophin,” she said. “The immune system won't be reactive to the cells, because they're their own cells, but they might be reactive to the protein.”
ARE THERE SUFFICIENT MUSCLE FIBERS?
Commenting on the study, Jennifer E. Morgan, PhD, of the Dubowitz Neuromuscular Centre at the University College London Institute of Child Health, said the technique devised by Dr. Perlingeiro and her colleagues is “certainly a proof of principle, but it is still intramuscular injection.” And, she added, they did not demonstrate the ability to produce sufficient quantities of skeletal muscle fibers.
“They say that the myogenic precursor cells are able to engraft efficiently, producing abundant human-derived, dystrophin-positive myofibers that exhibit superior strength,” said Dr. Morgan, who was not involved with the study and is studying another technique for treating MD. “They get about 100 muscle fibers at the most, and that is not abundant in my mind. I don't think that would give rise to functional improvement. That worries me a little bit.”
Dr. Morgan co-authored several papers in Lancet Neurology, Lancet, and Molecular Therapy that describe a technique of delivering antisense oligonucleotides to the binding site of pre-messenger RNA so that exon 51 of the dystrophin gene is masked and ignored during gene expression, giving rise to a shorter but still functional dystrophin protein. The technique has been shown to be effective in a mouse model of MD, and in human Duchenne MD muscle cells in vivo.
An effective MD therapy almost certainly would have to be administered systemically, Dr. Morgan said, since it would be impossible to inject stem cells into all the muscles in the body. Injections into specific muscle groups — the muscles of the hand, for example — “would immeasurably improve quality of life for some patients,” she acknowledged, but a comprehensive treatment would have to involve all muscle fibers.
Giulio Cossu, MD, who has been investigating ways to deliver genetically altered stem cells systemically in children with Duchenne MD, agrees that intramuscular injection would be limited to local therapeutic applications since stem cells do not migrate more than a millimeter or two from the injection site. This kind of injection can be used in specific cases, said Dr. Cossu, of the department of cell and developmental biology at University College London, and the division of regenerative medicine at San Raffaele Scientific Institute in Milan, Italy.
For example, researchers in France are conducting a clinical trial using myoblasts from non-affected muscles to treat the dysphagia caused by oculopharyngeal MD by injecting cultured cells from the patients into pharyngeal muscles. [For more on the study, see http://1.usa.gov/Kg6m6d.] But comprehensive treatment will require a way to correct mutations in all muscles — the goal of exon skipping. Nevertheless, he was impressed by the work done by Dr. Perlingeiro and her colleagues.
“This result is not unexpected but it is important because it moves the field forward toward clinical application,” he said. “Also, they have not found tumors developing after selecting myogenic progenitors, and while that is by no means enough to guarantee safety in clinical trials, it is a good indication you can select the cell population you want and be relatively safe.”
Maura Parker, PhD, associate in clinical research at the Fred Hutchinson Cancer Research Center in Seattle, has been investigating ways to use stem cells to repair dystrophic muscle and co-authored a recent paper in Skeletal Muscle on the successful transplantation of myogenic stem cells delivered intramuscularly in a canine model of Duchenne MD. She is enthusiastic about Dr. Perlingeiro's work.
“I'm not sure mouse-to-mouse transplantation is going to be the best way to go into a clinical trial, but seeing how human cells work in mice was a great middle step,” she said.
The use of iPS cells to treat muscular dystrophy has the potential to bypass the problems encountered by early myoblast transfer therapy, proposed in the 1970s as a way to restore dystrophin expression through intramuscular injections of myoblasts — immature muscle cells — from healthy donors. Clinical trials in the early 1990s failed to produce benefit due to poor dispersion of injected cells, immune reactions, and the death of injected cells.
Stem cells derived from bone marrow, blood, muscle and other tissues capable of generating muscle showed promise, but proved to be limited in their ability to regenerate when implanted in the host. Jacques P. Tremblay, PhD, of Laval University in Quebec, developed a protocol of immunosuppression to cope with the danger of rejection, and explored other techniques for promoting the engraftment and survival of transplanted myoblasts, but recent research efforts have turned more toward the use of iPS skin cells, despite the challenges of programming them to produce normal muscle.
“When taken from the patient, skin cells need to be genetically modified to become iPS cells, then modified to become myogenic cells, and then modified again to correct the mutation causing their particular form of muscular dystrophy,” said Dr. Parker. “That's a lot of modification!”