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Biomedical engineers from Duke University have developed the first functioning human skeletal muscle from induced pluripotent stem cells.

The researchers built upon previous work where they grew the first functioning human muscle tissue from cells called myoblasts obtained from muscle biopsies that had already progressed beyond the stem cell stage, but hadn’t yet become mature muscle fibers.

They grew the cells by many folds and then put them into a supportive 3D scaffolding filled with a nourishing gel that allowed them to form aligned and functioning human muscle fibers.

However, until this study researchers had been unable to grow the intermediate cells into functioning skeletal muscle.

“It's taken years of trial and error, making educated guesses and taking baby steps to finally produce functioning human muscle from pluripotent stem cells,” Lingjun Rao, a postdoctoral researcher at Duke University and first author of the study, said in a statement. “What made the difference are our unique cell culture conditions and 3D matrix, which allowed cells to grow and develop much faster and longer than the 2D culture approaches that are more typically used.”

During the new study, the team began with human induced pluripotent stem cells that were taken from adult non-muscle tissues including skin or blood.

“Starting with pluripotent stem cells that are not muscle cells, but can become all existing cells in our body, allows us to grow an unlimited number of myogenic progenitor cells,” Nenad Bursac, professor of biomedical engineering at Duke University, said in a statement. “These progenitor cells resemble adult muscle stem cells called 'satellite cells' that can theoretically grow an entire muscle starting from a single cell.”

The cells were reprogrammed to revert to a primordial state and then grown while being flooded with a molecule called Pax7—which signals the cells to start becoming muscle.

As the cells proliferated, they become similar to but not as robust as adult muscle stem cells.

The team halted the Pax7 signaling molecule once the cells were on their way to becoming muscle and started to give the cells the support and nourishment necessary for them to mature.

After two to four weeks of 3D culture, the muscle cells formed muscle fibers that contract and react to external stimuli such as electrical pulses and biochemical signals mimicking neuronal inputs similar to native muscle tissue.

The researchers also implanted the newly grown muscle fibers into adult mice and found that they survive and function for at least three weeks while progressively integrating into the native tissue through vascularization.

This resulted in muscle that is not as strong as native muscle tissue and falls short of the muscle grown in the previous study that started from muscle biopsies. However, the researchers said the muscle still holds potential that the older, stronger relative does not.

The pluripotent stem cell-derived muscle fibers develop reservoirs of "satellite-like cells" that are necessary for normal adult muscles to repair damage, while the muscle from the previous study had much fewer of these cells. The new method is also capable of growing many more cells from a smaller starting batch than the biopsy method.   

The ability to start from cellular scratch using non-muscle tissue will enable scientists to grow more muscle cells, provide an easier path to genome editing and cellular therapies and develop individually tailored models of rare muscle diseases for drug discovery and basic biology studies.

“The prospect of studying rare diseases is especially exciting for us,” Bursac said. “When a child's muscles are already withering away from something like Duchenne muscular dystrophy, it would not be ethical to take muscle samples from them and do further damage.

“But with this technique, we can just take a small sample of non-muscle tissue, like skin or blood, revert the obtained cells to a pluripotent state, and eventually grow an endless amount of functioning muscle fibers to test,” he added.

The next step for the team will be to refine the technique to grow muscles that are more robust and begin work to develop new models of rare muscle diseases.

The study was published in Nature Communications.    

 

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