Summary One major challenge in realizing cell-based therapy for treating muscle-wasting disorders is the difficulty in obtaining therapeutically meaningful amounts of engraftable cells. We have previously described a method to generate skeletal myogenic progenitors with exceptional engraftability from pluripotent stem cells via teratoma formation. Here, we show that these cells are functionally expandable in vitro while retaining their in vivo regenerative potential. Within 37 days in culture, teratoma-derived skeletal myogenic progenitors were expandable to a billion-fold. Similar to their freshly sorted counterparts, the expanded cells expressed PAX7 and were capable of forming multinucleated myotubes in vitro . Importantly, these cells remained highly regenerative in vivo . Upon transplantation, the expanded cells formed new DYSTROPHIN + fibers that reconstituted up to 40% of tibialis anterior muscle volume and repopulated the muscle stem cell pool. Our study thereby demonstrates the possibility of producing large quantities of engraftable skeletal myogenic cells for transplantation.

Differentiation of pluripotent stem cells (PSCs) is a promising approach to obtaining large quantities of skeletal myogenic progenitors for disease modeling and cell-based therapy. However, generating skeletal myogenic cells with high regenerative potential is still challenging. We recently reported that skeletal myogenic progenitors generated from mouse PSC-derived teratomas possess robust regenerative potency. We have also found that teratomas derived from human PSCs contain a skeletal myogenic population. Here, we showed that these human PSC-derived skeletal myogenic progenitors had exceptional engraftability. A combination of cell surface markers, CD82, ERBB3, and NGFR enabled efficient purification of skeletal myogenic progenitors. These cells expressed PAX7 and were able to differentiate into MHC+ multinucleated myotubes. We further discovered that these cells are expandable in vitro. Upon transplantation, the expanded cells formed new dystrophin+ fibers that reconstituted almost ¾ of the total muscle volume, and repopulated the muscle stem cell pool. Our study, therefore, demonstrates the possibility of producing large quantities of engraftable skeletal myogenic cells from human PSCs.