SUMMARY Myogenic differentiation of human pluripotent stem cells (hPSCs) has been done by gene overexpression or directed differentiation. However, viral integration, long-term culture, and the presence of unwanted cells are the main obstacles. By using CRISPR/Cas9n, a double-reporter human embryonic stem cell (hESC) line was generated for PAX7/ MYF5, allowing prospective readout. This strategy allowed pathway screen to define efficient myogenic induction in hPSCs. Next, surface marker screen allowed identification of CD10 and CD24 for purification of myogenic progenitors and exclusion of non-myogenic cells. CD10 expression was also identified on human satellite cells and skeletal muscle progenitors. In vitro and in vivo studies using transgene and/or reporter-free hPSCs further validated myogenic potential of the cells by formation of new fibers expressing human dystrophin as well as donor-derived satellite cells in NSG-mdx4Cv mice. This study provides biological insights for myogenic differentiation of hPSCs using a double-reporter cell resource and defines an improved myogenic differentiation and purification strategy.

SummaryRecent reports have documented the differentiation of human pluripotent stem cells toward the skeletal myogenic lineage using transgene- and cell purification-free approaches. Although these protocols generate myocytes, they have not demonstrated scalability, safety, and in vivo engraftment, which are key aspects for their future clinical application. Here we recapitulate one prominent protocol, and show that it gives rise to a heterogeneous cell population containing myocytes and other cell types. Upon transplantation, the majority of human donor cells could not contribute to myofiber formation. As a proof-of-principle, we incorporated the inducible PAX7 lentiviral system into this protocol, which then enabled scalable expansion of a homogeneous population of skeletal myogenic progenitors capable of forming myofibers in vivo. Our findings demonstrate the methods for scalable expansion of PAX7+ myogenic progenitors and their purification are critical for practical application to cell replacement treatment of muscle degenerative diseases.

Volumetric muscle defect, caused by trauma or combat injuries, is a major health concern leading to severe morbidity. It is characterized by partial or full thickness loss of muscle and its bio-scaffold, resulting in extensive fibrosis and scar formation. Therefore, the ideal therapeutic option is to use stem cells combined with bio-scaffolds to restore muscle. For this purpose, muscle-derived stem cells (MDSCs) are a great candidate due to their unique multi-lineage differentiation potential. In this study, we evaluated the regeneration potential of MDSCs for muscle loss repair using a novel in situ fibrin gel casting. Muscle defect was created by a partial thickness wedge resection in the tibialis anterior (TA)muscles of NSG mice which created an average of 25% mass loss. If untreated, this defect leads to severe muscle fibrosis. Next, MDSCs were delivered using a novel in situ fibrin gel casting method. Our results demonstrated MDSCs are able to engraft and form new myofibers in the defect when casted along with fibrin gel. LacZ labeled MDSCs were able to differentiate efficiently into new myofibers and significantly increase muscle mass. This was also accompanied by significant reduction of fibrotic tissue in the engrafted muscles. Furthermore, transplanted cells also contributed to new vessel formation and satellite cell seeding. These results confirmed the therapeutic potential of MDSCs and feasibility of direct in situ casting of fibrin/MDSC mixture to repair muscle mass defects.

Dystrophin proteomic regulation in Muscular Dystrophies (MD) remains unclear. We report that a long noncoding RNA (lncRNA), H19 , associates with dystrophin and inhibits E3 ligase-dependent poly-ubiquitination at Lys3584 (referred to as Ub-DMD) and its subsequent protein degradation. In-frame deletions in BMD and a DMD non-silent mutation (C3340Y) result in defects in the protein’s ability to interact with H19 , causing elevated Ub-DMD levels and dystrophin degradation. Dmd C3333Y mice exhibited progressive muscular dystrophy, elevated serum CK, heart dilation, blood vessel irregularity, and respiratory failure with concurrently reduced dystrophin and increased Ub-DMD status. H19 RNA oligonucleotides conjugated with Agrin (AGR- H19 ) and Nifenazone competed-with/inhibited TRIM63. Dmd C3333Y animals, iPSC-derived skeletal muscle cells from BMD patients, or mdx mice subjected to exon-skipping exhibited inhibited dystrophin degradation, preserved skeletal/cardiac muscle histology, and improved strength/heart function following AGR- H19 or Nifenazone treatment. Our study paves the way to meaningful targeted therapeutics for BMD and certain DMD patients.