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Mutations in the DMD gene cause fatal Duchenne Muscular Dystrophy (DMD). An attractive therapeutic approach is autologous cell transplantation utilizing myogenic progenitors derived from induced pluripotent stem cells (iPSCs). Given that a significant number of DMD mutations occur between exons 45 and 55, we developed a gene knock-in approach to correct any mutations downstream of exon 44. We applied this approach to two DMD patient-specific iPSC lines carrying mutations in exons 45 and 51 and confirmed mini-DYSTROPHIN (mini-DYS) protein expression in corrected myotubes by western blot and immunofluorescence staining. Transplantation of gene-edited DMD iPSC-derived myogenic progenitors into NSG/mdx4Cv mice produced donor-derived myofibers, as shown by the dual expression of human DYSTROPHIN and LAMIN A/C. These findings further provide proof-of-concept for the use of programmable nucleases for the development of autologous iPSC-based therapy for muscular dystrophies. [ABSTRACT FROM AUTHOR]

Pluripotent stem cell (PSC)-based cell therapy is an attractive option for the treatment of multiple human disorders, including muscular dystrophies. While in vitro differentiating PSCs can generate large numbers of human lineage-specific tissue, multiple studies evidenced that these cell populations mostly display embryonic/fetal features. We previously demonstrated that transplantation of PSC-derived myogenic progenitors provides long-term engraftment and functional improvement in several dystrophic mouse models, but it remained unknown whether donor-derived myofibers mature to match adult tissue. Here, we transplanted iPAX7 myogenic progenitors into muscles of non-dystrophic and dystrophic mice and compared the transcriptional landscape of human grafts with respective in vitro-differentiated iPAX7 myotubes as well as human skeletal muscle biospecimens. Pairing bulk RNA sequencing with computational deconvolution of human reads, we were able to pinpoint key myogenic changes that occur during the in vitro–to–in vivo transition, confirm developmental maturity, and consequently evaluate their applicability for cell-based therapies. [ABSTRACT FROM AUTHOR]

The diaphragm muscle is essential for breathing, and its dysfunctions can be fatal. Many disorders affect the diaphragm, including muscular dystrophies. Despite the clinical relevance of targeting the diaphragm, there have been few studies evaluating diaphragm function following a given experimental treatment, with most of these involving anti-inflammatory drugs or gene therapy. Cell-based therapeutic approaches have shown success promoting muscle regeneration in several mouse models of muscular dystrophy, but these have focused mainly on limb muscles. Here we show that transplantation of as few as 5000 satellite cells directly into the diaphragm results in consistent and robust myofiber engraftment in dystrophin- and fukutin-related protein-mutant dystrophic mice. Transplanted cells also seed the stem cell reservoir, as shown by the presence of donor-derived satellite cells. Force measurements showed enhanced diaphragm strength in engrafted muscles. These findings demonstrate the feasibility of cell transplantation to target the diseased diaphragm and improve its contractility. [ABSTRACT FROM AUTHOR]

Pluripotent stem (PS) cells enable the scalable production of tissue-specific derivatives with therapeutic potential for various clinical applications, including muscular dystrophies. Given the similarity to human counterparts, the non-human primate (NHP) is an ideal preclinical model to evaluate several questions, including delivery, biodistribution, and immune response. While the generation of human-induced PS (iPS)-cell-derived myogenic progenitors is well established, there have been no data for NHP counterparts, probably due to the lack of an efficient system to differentiate NHP iPS cells towards the skeletal muscle lineage. Here, we report the generation of three independent Macaca fascicularis iPS cell lines and their myogenic differentiation using PAX7 conditional expression. The whole-transcriptome analysis confirmed the successful sequential induction of mesoderm, paraxial mesoderm, and myogenic lineages. NHP myogenic progenitors efficiently gave rise to myotubes under appropriate in vitro differentiation conditions and engrafted in vivo into the TA muscles of NSG and FKRP-NSG mice. Lastly, we explored the preclinical potential of these NHP myogenic progenitors in a single wild-type NHP recipient, demonstrating engraftment and characterizing the interaction with the host immune response. These studies establish an NHP model system through which iPS-cell-derived myogenic progenitors can be studied. [ABSTRACT FROM AUTHOR]

Facioscapulohumeral muscular dystrophy (FSHD) is a genetically dominant progressive myopathy caused by improper silencing of the DUX4 gene, leading to fibrosis, muscle atrophy, and fatty replacement. Approaches focused on muscle regeneration through the delivery of stem cells represent an attractive therapeutic option for muscular dystrophies. To investigate the potential for cell transplantation in FSHD, we have used the doxycycline-regulated iDUX4pA-HSA mouse model in which low-level DUX4 can be induced in skeletal muscle. We find that mouse pluripotent stem cell (PSC)-derived myogenic progenitors engraft in muscle actively undergoing DUX4-mediated degeneration. Donor-derived muscle tissue displayed reduced fibrosis and importantly, engrafted muscles showed improved contractile specific force compared to non-transplanted controls. These data demonstrate the feasibility of replacement of diseased muscle with PSC-derived myogenic progenitors in a mouse model for FSHD, and highlight the potential for the clinical benefit of such a cell therapy approach. [ABSTRACT FROM AUTHOR]

Background: Satellite cells (SCs) are indispensable for muscle regeneration and repair; however, due to low frequency in primary muscle and loss of engraftment potential after ex vivo expansion, their use in cell therapy is currently unfeasible. To date, an alternative to this limitation has been the transplantation of SC-derived myogenic progenitor cells (MPCs), although these do not hold the same attractive properties of stem cells, such as selfrenewal and long-term regenerative potential. Methods: We develop a method to expand wild-type and dystrophic fresh isolated satellite cells using transient expression of Pax3. This approach can be combined with genetic correction of dystrophic satellite cells and utilized to promote muscle regeneration when transplanted into dystrophic mice. Results: Here, we show that SCs from wild-type and dystrophic mice can be expanded in culture through transient expression of Pax3, and these expanded activated SCs can regenerate the muscle. We test this approach in a gene therapy model by correcting dystrophic SCs from a mouse lacking dystrophin using a Sleeping Beauty transposon carrying the human µDYSTROPHIN gene. Transplantation of these expanded corrected cells into immune-deficient, dystrophin-deficient mice generated large numbers of dystrophin-expressing myofibers and improved contractile strength. Importantly, in vitro expanded SCs engrafted the SC compartment and could regenerate muscle after secondary injury. Conclusion: These results demonstrate that Pax3 is able to promote the ex vivo expansion of SCs while maintaining their stem cell regenerative properties. [ABSTRACT FROM AUTHOR]

Muscle regeneration occurs through activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to make new myofibers. We used a transgenic Pax7- ZsGreen reporter mouse to prospectively isolate stem cells of skeletal muscle by flow cytometry. We show that Pax7- expressing cells (satellite cells) in the limb, head, and diaphragm muscles are homogeneous in size and granularity and uniformly labeled by certain cell surface markers, including CD34 and CD29. The frequency of the satellite cells varies between muscle types and with age. Clonal analysis demonstrated that all colonies arising from single cells within the Pax7-sorted fraction have myogenic potential. In response to injury, Pax7+ cells reduce CD34, CD29, and CXCR4 expression, increase in size, and acquire Sca-1. When directly isolated and cultured in vitro, Pax7+ cells display the hallmarks of activation and proliferate, initially as suspension aggregates and later distributed between suspension and adherence. During in vitro expansion, Pax7 (ZsGreen) and CD34 expression decline, whereas expression of PSA-NCAM is acquired. The nonmyogenic, Pax7neg cells expand as Sca1+ PDGRα+ PSA-NCAMneg cells. Satellite cells expanded exclusively in suspension can engraft and produce dystrophin+ fibers in mdx-/- mice. These results establish a novel animal model for the study of muscle stem cell physiology and a culture system for expansion of engraftable muscle progenitors. [ABSTRACT FROM AUTHOR]

Mutations in the fukutin-related protein (FKRP) cause Walker-Warburg syndrome (WWS), a severe form of congenital muscular dystrophy. Here, we established a WWS human induced pluripotent stem cell-derived myogenic model that recapitulates hallmarks of WWS pathology. We used this model to investigate the therapeutic effect of metabolites of the pentose phosphate pathway in human WWS. We show that functional recovery of WWS myotubes is promoted not only by ribitol but also by its precursor ribose. Moreover, we found that the combination of each of these metabolites with NAD+ results in a synergistic effect, as demonstrated by rescue of α-dystroglycan glycosylation and laminin binding capacity. Mechanistically, we found that FKRP residual enzymatic capacity, characteristic of many recessive FKRP mutations, is required for rescue as supported by functional and structural mutational analyses. These findings provide the rationale for testing ribose/ribitol in combination with NAD+ to treat WWS and other diseases associated with FKRP mutations. [ABSTRACT FROM AUTHOR]