Scientists at the University of Minnesota have succeeded in almost completely regenerating skeletal muscle in a mouse model of muscular dystrophy, using genetically unmodified mouse pluripotent stem cells (PSCs) collected from an unexpected source. Sunny Sun-Kin Chan, Ph.D., Michael Kyba, Ph.D., and colleagues, isolated skeletal muscle stem cells from PSC-derived teratomas—a benign tumor that can produce cells of any type—which they transplanted into damaged tibialis anterior (TA) muscle tissue in the experimental mice.
They found that even small numbers of cells readily engrafted, regenerated up to 80% of the skeletal muscle volume, and significantly improved muscle strength and resistance to fatigue. In fact, the transplanted teratoma-derived muscle stem cells were found to exhibit a far greater regenerative potential than muscle stem cells generated in the lab from genetically modified pluripotent stem cells. The transplants also generated a source of new muscle stem cells to maintain the reconstructed muscle tissue and help it to repair after subsequent injury.
“The goal of this research was to seek in unexplored places a source of cells that, when transplanted, would rebuild skeletal muscle and demonstrate significant improvements in muscle strength and resilience,” says Michael Kyba, Ph.D., a professor in the University of Minnesota Medical School's department of pediatrics. Dr. Kyba is lead author of the team’s published paper in Cell Stem Cell. “The fact that teratomas harbor cells of such greater potency than those that spontaneously differentiate when we culture them in a dish is remarkable,” says Dr. Chan, who is an assistant professor at the Medical School’ department of pediatrics. “Indeed, beauty can be found in the most unexpected of places.”
The team’s published paper is entitled “Skeletal Muscle Stem Cells from PSC-Derived Teratomas Have Functional Regenerative Capacity.”
Pluripotent stem cells don’t differentiate spontaneously into skeletal muscle cells in vitro, and to date, generating functional, force-producing muscle tissue through cell transplantation has required genetic modification of the pluripotent cells prior to transplantation, the authors explain. “With conventional tissue culture, it has proven difficult to efficiently derive myogenic progenitors from pluripotent stem cells, and, to date, the only functional force-generating repopulating cells reported have been derived through genetic modification.”
Given that PSC-derived teratomas implanted in vivo can generate highly complex tissues, including glands and hair follicles, the team investigated teratomas to see if they produced skeletal muscle progenitor cells. They created teratomas using undifferentiated pluripotent stem cells injected into immunodeficient mice and then sorted the different cell types in the benign tumors to identify myogenic progenitors by gene expression. When the team then isolated the teratoma-derived muscle stem cells and grew them in culture, they found that the cells retained their ability to differentiate into muscle cells, even through multiple rounds of replication. “This is notable because myoblasts from adults are reported to lose myogenic potential with extended passage,” they write.
To see how the teratoma-derived skeletal muscle cells would behave in vivo, the team transplanted relatively small numbers of the stem cells—about 40,000—directly into the damaged TA muscle tissue of the muscular dystrophy mouse model. They found that within a month, the cell transplants had produced “muscle regeneration on a scale that significantly surpassed previous reports.” In fact, within about three months, the transplant had rebuilt about 80% of the muscle volume, without any sign of new teratomas developing in more than 100 transplantations, even a year later.
Encouragingly, the regenerated muscle fibers matured and connected with nerves. The new tissue significantly improved muscle force generation. Assessments confirmed that in comparison with muscle in control animals, the regenerated tissue in the transplant recipients produced threefold greater measurements of tetanic force and significantly increased specific force. And, in addition to generating new, mature muscle tissue, the transplants also produced a subpopulation of functional muscle stem cells that could maintain the muscle over the long term, and even regenerate the same muscle tissue in response to subsequent injuries, “as would be expected for normal quiescent skeletal muscle stem cells,” the researchers write. “On a per cell basis, these cells have remarkable in vivo regenerative potential, developing into muscle fibers with similar efficiency as freshly isolated satellite cells and capable of regenerating 80% or more of the TA muscle.” Effectively, the teratoma-derived cells are “embryonic in nature but, after transplantation into adult muscle, undergo an in vivo maturation into quiescent muscle stem cells.”
The authors conclude that their approach represents, “a simple and efficient method for generating skeletal, myogenic progenitors from pluripotent stem cells.” Differentiation via teratoma also demonstrates numerous advantages, they suggest. “It is technically simple, inexpensive, and capable of producing large quantities of skeletal muscle stem cells. However its most striking feature is the scale of contribution to tissue regeneration after transplantation.”
Previous reported attempts to regenerate muscle using in vitro–differentiated pluripotent cells have managed to generate fewer than 200 fibers, and even then, only following the transplantation of more than 1 million cells. “In contrast,” the researchers comment, “myogenic progenitors derived from teratomas produce thousands of fibers…but with orders of magnitude fewer cells transplanted.”
They do acknowledge that although no secondary teratomas developed in their transplant recipient mice, “caution would demand a highly methodical evaluation of safety before considering the use of in vitro differentiation via teratoma to obtain human skeletal muscle stem cells for clinical use.” Much more research will also be needed to confirm whether human teratomas produce skeletal muscle progenitors that have the same regenerative potential as the mouse skeletal muscle stem cells, and to determine whether the human system can be developed into a robust, scalable platform. Even so, they conclude, “taken together, differentiation via teratoma represents an interesting and accessible means of generating cells for skeletal muscle regeneration.”
