Muscular dystrophy describes a family of diseases that causes muscle loss. In the worst cases, the muscle loss is so great that the patients cannot walk or even breathe on their own. Stem cell research in animals has shown that cell therapies could regenerate the lost muscle. A new study by CiRA researchers shows how optimizing the time period of differentiating iPS cells maximizes the expression of the Myf5 gene to produce cells with excellent muscle regeneration potential in mice.

Most muscular dystrophies are the result of gene mutations. The mutation in Duchenne muscular dystrophy, one of the most severe childhood types, leads to an inability for muscle to produce the protein dystrophin, resulting in extremely fragile muscle. The body will continuously regenerate the lost muscle, but this is a Sisyphean task, because even the new muscle lacks dystrophin.

"In cell therapies for Duchenne muscular dystrophy, we are injecting muscle stem cells with the intact dystrophin gene. The regeneration of the transplanted cells leads to normal muscle. This approach may not fully recover all the muscle, but we expect it will significantly improve the patient's condition," explains CiRA Associate Professor Hidetoshi Sakurai, who led the study and has dedicated his laboratory to research on cell therapies for muscular dystrophy.

In principle, muscle stem cells can be acquired from donors, but it is difficult to multiply the cells to an amount needed for cell therapies. iPS cells, on the other hand, are much easier to multiply. Sakurai and his researchers therefore are investigating ways to generate muscle stem cell from iPS cells.

Ultimately, the quality of the muscle stem cells used for the therapy is defined by the functional recovery of the muscle. However, prior to injecting the cells into the patient, researchers must depend on other indicators, namely the expression of specific genes.

Pax7 is one such gene, and the gold standard in mouse experiments is the iPax7 cell, a type of muscle stem cell in which the Pax7 transgene is forcibly expressed. However, transgene technology is not suitable for human therapies because it risks causing cancer. Sakurai's team has been investigating other indicators that predict a high expression of Pax7.

"We found that tracing the Myf5 expression in iPS cells is a good marker of regenerative potential. By sustaining the Myf5 expression for a longer culture, we identified cells that had high regenerative potential," says Dr. Mingming Zhao, who conducted the bulk of the experiments for the study.

He found that sustaining Myf5 expression longer from iPS cells correlated with more Pax7 expression.

To test if these cells behaved like muscle stem cells, Zhao and colleagues implanted them into mice suffering from Duchenne muscular dystrophy. Muscles did indeed regenerate and did so with an intact dystrophin protein, suggesting the new muscles were inoculated from the fragility caused by the disease. The muscles also resulted in better muscular function in the mice.

Although a direct comparison with iPax cells was not made, Zhao says the number of generated muscle fibers in his experiments was comparable with other research using iPax cells.

"Transplanting our Myf5 cells resulted in 100 muscle fibers per three hundred thousand cells transplantation, which is a very good number," he says.

Sakurai adds that the differentiation protocol to manufacture the Myf5 cells from iPS cells follows myogenic developmental steps, including the dermomyotome.

"The differentiation to dermomyotome is important for preparing muscle stem cells. Further optimization of our system could lead to effective cell therapies for muscular diseases," he says.

• doi: 10.1016/j.stemcr.2020.06.004