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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/mdx 4Cv 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.

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.

During cardiac development and morphogenesis, cardiac progenitor cells differentiate into cardiomyocytes that expand in number and size to generate the fully formed heart. Much is known about the factors that regulate initial differentiation of cardiomyocytes, and there is ongoing research to identify how these fetal and immature cardiomyocytes develop into fully functioning, mature cells. Accumulating evidence indicates that maturation limits proliferation and conversely proliferation occurs rarely in cardiomyocytes of the adult myocardium. We term this oppositional interplay the proliferation-maturation dichotomy. Here we review the factors that are involved in this interplay and discuss how a better understanding of the proliferation-maturation dichotomy could advance the utility of human induced pluripotent stem cell-derived cardiomyocytes for modeling in 3-dimensional engineered cardiac tissues to obtain truly adult-level function.

This commentary provides a brief overview of the steps necessary for the generation of an induced pluripotent stem (iPS) cell-derived clinical grade product. This process requires extensive, proper documentation as well as a thoughtful and systematic optimization of the manufacturing methods to ensure maintenance of the key biological features of the product, compliance with current good manufacturing practices (cGMP), and most importantly patient safety. The scale-up and optimization also ideally include the identification of efficient and cost-effective purification/isolation and expansion of the target cell population., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Pluripotent stem cells have a multitude of potential applications in the areas of disease modeling, drug screening, and cell-based therapies for genetic diseases, including muscular dystrophies. The advent of induced pluripotent stem cell technology allows for the facile derivation of disease-specific pluripotent stem cells for any given patient. Targeted in vitro differentiation of pluripotent stem cells into the muscle lineage is a key step to enable all these applications. Transgene-based differentiation using conditional expression of the transcription factor PAX7 leads to the efficient derivation of an expandable and homogeneous population of myogenic progenitors suitable for both in vitro and in vivo applications. Here, we describe an optimized protocol for the derivation and expansion of myogenic progenitors from pluripotent stem cells using conditional expression of PAX7. Importantly, we further describe an optimized procedure for the terminal differentiation of myogenic progenitors into more mature myotubes, which are better suited for in vitro disease modeling and drug screening studies., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Muscular dystrophy encompasses a large number of heterogeneous genetic disorders characterized by progressive and devastating muscle wasting. Cell-based replacement strategies aimed at promoting skeletal muscle regeneration represent a candidate therapeutic approach to treat muscular dystrophies. Due to the difficulties of obtaining large numbers of stem cells from a muscle biopsy as well as expanding these in vitro, pluripotent stem cells (PSCs) represent an attractive cell source for the generation of myogenic progenitors, given that PSCs can repeatedly produce large amounts of lineage-specific tissue, representing an unlimited source of cells for therapy. In this review, we focus on the progress to date on different methods for the generation of human PSC-derived myogenic progenitor cells, their regenerative capabilities upon transplantation, their potential for allogeneic and autologous transplantation, as well as the specific challenges to be considered for future therapeutic applications., (© 2022. The Author(s).)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Limb girdle muscular dystrophy type 2A (LGMD2A), caused by mutations in the Calpain 3 (CAPN3) gene, is an incurable autosomal recessive disorder that results in muscle wasting and loss of ambulation. To test the feasibility of an autologous induced pluripotent stem cell (iPSC)-based therapy for LGMD2A, here we applied CRISPR-Cas9-mediated genome editing to iPSCs from three LGMD2A patients to enable correction of mutations in the CAPN3 gene. Using a gene knockin approach, we genome edited iPSCs carrying three different CAPN3 mutations, and we demonstrated the rescue of CAPN3 protein in myotube derivatives in vitro. Transplantation of gene-corrected LGMD2A myogenic progenitors in a novel mouse model combining immunodeficiency and a lack of CAPN3 resulted in muscle engraftment and rescue of the CAPN3 mRNA. Thus, we provide here proof of concept for the integration of genome editing and iPSC technologies to develop a novel autologous cell therapy for LGMD2A., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Myotonic dystrophy 1 (DM1) is a multisystem disorder primarily affecting the central nervous system, heart and skeletal muscle. It is caused by an expansion of the CTG trinucleotide repeats in the 3' untranslated region of the DMPK gene. Although patient myoblasts have been used for studying the disease in vitro , the invasiveness as well as the low accessibility to muscle biopsies motivate the development of alternative reliable myogenic models. Here, we established two DM1 induced pluripotent stem (iPS) cell lines from patient-derived fibroblasts and, using the PAX7 conditional expression system, differentiated these into myogenic progenitors and, subsequently, terminally differentiated myotubes. Both DM1 myogenic progenitors and myotubes were found to express the intranuclear RNA foci exhibiting sequestration of MBNL1. Moreover, we found the DM1-related mis-splicing, namely BIN1 exon 11 in DM1 myotubes. We used this model to test a specific therapy, antisense oligonucleotide treatment, and found that this efficiently abolished RNA foci and rescued BIN1 mis-splicing in DM1 iPS cell-derived myotubes. Together, our results demonstrate that myotubes derived from DM1 iPS cells recapitulate the critical molecular features of DM1 and are sensitive to antisense oligonucleotide treatment, confirming that these cells can be used for in vitro disease modeling and candidate drug testing or screening.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Derivation of functional skeletal muscle stem cells from pluripotent cells without genetic modification has proven elusive. Here we show that teratomas formed in adult skeletal muscle differentiate in vivo to produce large numbers of α7-Integrin+ VCAM-1+ myogenic progenitors. When FACS-purified and transplanted into diseased muscles, mouse teratoma-derived myogenic progenitors demonstrate very high engraftment potential. As few as 40,000 cells can reconstitute ∼80% of the tibialis anterior muscle volume. Newly generated fibers are innervated, express adult myosins, and ameliorate dystrophy-related force deficit and fatigability. Teratoma-derived myogenic progenitors also contribute quiescent PAX7+ muscle stem cells, enabling long-term maintenance of regenerated muscle and allowing muscle regeneration in response to subsequent injuries. Transcriptional profiling reveals that teratoma-derived myogenic progenitors undergo embryonic-to-adult maturation when they contribute to the stem cell compartment of regenerated muscle. Thus, teratomas are a rich and accessible source of potent transplantable skeletal muscle stem cells. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Pluripotent stem (PS)-cell-derived cell types hold promise for treating degenerative diseases. However, PS cell differentiation is intrinsically heterogeneous; therefore, clinical translation requires the development of practical methods for isolating progenitors from unwanted and potentially teratogenic cells. Muscle-regenerating progenitors can be derived through transient PAX7 expression. To better understand the biology, and to discover potential markers for these cells, here we investigate PAX7 genomic targets and transcriptional changes in human cells undergoing PAX7-mediated myogenic commitment. We identify CD54, integrin α9β1, and Syndecan2 (SDC2) as surface markers on PAX7-induced myogenic progenitors. We show that these markers allow for the isolation of myogenic progenitors using both fluorescent- and CGMP-compatible magnetic-based sorting technologies and that CD54+α9β1+SDC2+ cells contribute to long-term muscle regeneration in vivo. These findings represent a critical step toward enabling the translation of PS-cell-based therapies for muscle diseases., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cell-based therapies are considered as one of the most promising approaches for the treatment of degenerating pathologies including muscle disorders and dystrophies. Advances in the approach of reprogramming somatic cells into induced pluripotent stem (iPS) cells allow for the possibility of using the patient's own pluripotent cells to generate specific tissues for autologous transplantation. In addition, patient-specific tissue derivatives have been shown to represent valuable material for disease modeling and drug discovery. Nevertheless, directed differentiation of pluripotent stem cells into a specific lineage is not a trivial task especially in the case of skeletal myogenesis, which is generally poorly recapitulated during the in vitro differentiation of pluripotent stem cells.Here, we describe a practical and efficient method for the derivation of skeletal myogenic precursors from differentiating human pluripotent stem cells using controlled expression of PAX7. Flow cytometry (FACS) purified myogenic precursors can be expanded exponentially and differentiated in vitro into myotubes, enabling researchers to use these cells for disease modeling as well as therapeutic purposes.

Duchenne muscular dystrophy is a progressive and incurable neuromuscular disease caused by genetic and biochemical defects of the dystrophin-glycoprotein complex. Here we show the regenerative potential of myogenic progenitors derived from corrected dystrophic induced pluripotent stem cells generated from fibroblasts of mice lacking both dystrophin and utrophin. We correct the phenotype of dystrophic induced pluripotent stem cells using a Sleeping Beauty transposon system carrying the micro-utrophin gene, differentiate these cells into skeletal muscle progenitors and transplant them back into dystrophic mice. Engrafted muscles displayed large numbers of micro-utrophin-positive myofibers, with biochemically restored dystrophin-glycoprotein complex and improved contractile strength. The transplanted cells seed the satellite cell compartment, responded properly to injury and exhibit neuromuscular synapses. We also detect muscle engraftment after systemic delivery of these corrected progenitors. These results represent an important advance towards the future treatment of muscular dystrophies using genetically corrected autologous induced pluripotent stem cells.

A major obstacle in the application of cell-based therapies for the treatment of neuromuscular disorders is obtaining the appropriate number of stem/progenitor cells to produce effective engraftment. The use of embryonic stem (ES) or induced pluripotent stem (iPS) cells could overcome this hurdle. However, to date, derivation of engraftable skeletal muscle precursors that can restore muscle function from human pluripotent cells has not been achieved. Here we applied conditional expression of PAX7 in human ES/iPS cells to successfully derive large quantities of myogenic precursors, which, upon transplantation into dystrophic muscle, are able to engraft efficiently, producing abundant human-derived DYSTROPHIN-positive myofibers that exhibit superior strength. Importantly, transplanted cells also seed the muscle satellite cell compartment, and engraftment is present over 11 months posttransplant. This study provides the proof of principle for the derivation of functional skeletal myogenic progenitors from human ES/iPS cells and highlights their potential for future therapeutic application in muscular dystrophies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Direct reprogramming of adult fibroblasts to a pluripotent state has opened new possibilities for the generation of patient- and disease-specific stem cells. However the ability of induced pluripotent stem (iPS) cells to generate tissue that mediates functional repair has been demonstrated in very few animal models of disease to date. Here we present the proof of principle that iPS cells may be used effectively for the treatment of muscle disorders. We combine the generation of iPS cells with conditional expression of Pax7, a robust approach to derive myogenic progenitors. Transplantation of Pax7-induced iPS-derived myogenic progenitors into dystrophic mice results in extensive engraftment, which is accompanied by improved contractility of treated muscles. These findings demonstrate the myogenic regenerative potential of iPS cells and provide rationale for their future therapeutic application for muscular dystrophies.