Researchers have grown the first functioning human muscle tissue from skin cells reprogrammed into stem cells. [Duke University]
Researchers have grown the first functioning human muscle tissue from skin cells reprogrammed into stem cells. [Duke University]

Duke University researchers have grown the first functional human skeletal muscle tissue entirely from induced human pluripotent stem cells (hPSCs). The achievement could feasibly allow scientists to generate skeletal muscle tissue for disease modeling and drug studies, and potentially for developing hPSC-based therapies for muscle-wasting diseases.

“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,” comments Lingjun Rao, Ph.D., first author of the team’s published report in Nature Communications. “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.” The team’s paper, released today, is entitled “Engineering Human Pluripotent Stem Cells into a Functional Skeletal Muscle Tissue.”

Previous research has shown that skeletal muscle cells can be derived from hPSCs using either small-molecule differentiation or direct reprogramming techniques, explains lead Duke researcher Nenad Bursac, Ph.D., professor of biomedical engineering at Duke University, and colleagues. Small-molecule differentiation has proven to be slow and inefficient, but direct reprogramming protocols that involve overexpression of the myogenic transcription factor Pax7 have been far more efficient and have generated yields of 90% pure myogenic cells. “Specifically, Pax7 overexpression generates a population of myogenic progenitors that can expand in vitro and populate the stem cell niche when implanted into native muscle, suggesting their resemblance with primary satellite cells.” 

Duke engineers have grown the first functioning human muscle from nonmuscle cells—skin cells reverted to their primordial stem cell state. The advance holds promise for cellular therapies, drug discovery, and studying rare diseases. [Duke University]

Several studies have shown that hPSC-derived myogenic cells can fuse with host myofibers and improve muscle function following in vivo transplantation, but it's not yet known if these these cells alone can generate 3D functional skeletal muscle, according to the authors. Studies also suggest that hPSC-derived myotube structures are relatively immature. 

Prof. Bursac’s laboratory had previously created functioning human muscle tissue, or myobundles, from primary myoblasts obtained from human muscle biopsies. Culturing the myoblasts in a 3D matrix allowed the cells to align and form functional human muscle fibers. The technique did have some drawbacks, however, which means it wasn't suitable for generating large quantities of muscle for in vitro studies or in vivo regenerative therapies.

As an alternative approach, the team cultured induced hPSCs derived from adult nonmuscle tissues in the presence of Pax7. They found that the cultured cells proliferated and developed into expandable myogenic progenitors, or induced myogenic progenitor cells (iMPSCs), which resemble native adult muscle stem cells. Grown in 2D culture, the hPSC-derived iMPSC differentiated into functional myotubes and cells that expressed Pax7, which are “the two main constituents of native skeletal muscle.”

The iMPC-derived myotubes didn’t survive for long in 2D culture, however, so the team next embedded the cells in a fibrin-based hydrogel to provide support and allow the tissue to develop into 3D skeletal muscle (iSKM) bundles. Within just a few weeks, these bundles differentiated into functional muscle tissue that exhibited the characteristic sarcomeric structure of natural muscle. These iSKM bundles were also able to contract and react to electrical stimuli and biochemical signals. “When cultured in a 3D hydrogel environment, iMPCs structurally reorganize to generate aligned functional skeletal muscle tissues (iSKM bundles) that can generate twitch and tetanic contractions and Ca2+ transients in response to electrical and neurotransmitter stimulation,” the authors comment.

Encouragingly, cultured muscle tissue transplanted into immunocompromised mice survived and continued to function. The transplanted tissue became vascularized, and integrated into the animals’ own muscle. “Overall, these results showed that human iSKM bundles can successfully engraft and retain functionality following implantation into skin and muscle environments in mice,” the team writes.

Although hPSC-derived muscle isn’t as robust as native muscle tissue, it does still hold potential for multiple applications, the researchers suggest. Unlike muscle tissue derived using previous approaches, the hPSC-derived muscle fibers develop satellite-like cells that are needed for normal adult muscles to repair damage. Greater numbers of muscle cells can also be grown from a smaller starting culture of hPSCs than from biopsied cells. These features could make hPCS-grown muscle suitable for developing regenerative therapies and for creating disease models. “Together, 3D culture environment of iSKM bundles appears superior to standard 2D culture for in vitro myogenic differentiation of hPSCs,” the authors state. ”… however, further optimization will be required for iSKM bundles to match the maturation state and functionality of primary human engineered muscle.”

Bursac says his team is particularly excited by the prospect of studying rare diseases. “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 nonmuscle 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.”

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