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Kowala A, Boot J, Meng J, Mein CA, Pourquié O, Connelly JT, Morgan JE, Lin YY. Engineered human myogenic cells in hydrogels generate innervated vascularized myofibers within dystrophic mouse muscle on long-term engraftment. Cell Rep Med 2025; 6:102019. [PMID: 40056909 PMCID: PMC11970389 DOI: 10.1016/j.xcrm.2025.102019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2023] [Revised: 07/29/2024] [Accepted: 02/17/2025] [Indexed: 03/21/2025]
Abstract
Transplantation of human myogenic progenitor cells (MPCs) is a promising therapeutic strategy for treating muscle-wasting diseases, e.g., Duchenne muscular dystrophy (DMD). To increase engraftment efficiency of donor stem cells, modulation of host muscles is required, significantly limiting their clinical translation. Here, we develop a clinically relevant transplantation strategy synergizing hydrogel-mediated delivery and engineered human MPCs generated from CRISPR-corrected DMD patient-derived pluripotent stem cells. We demonstrate that donor-derived human myofibers produce full-length dystrophin at 4 weeks and 5-6 months (long-term) after transplantation in the unmodulated muscles of the dystrophin-deficient mouse model of DMD. Remarkably, human myofibers are innervated by mouse motor neurons forming neuromuscular junctions and supported by vascularization after long-term engraftment in dystrophic mice. PAX7+ cells of human origin populate the satellite cell niche. There was no evidence of tumorigenesis in mice engrafted with hydrogel-encapsulated human MPCs. Our results provide a proof of concept in developing hydrogel-based cell therapy for muscle-wasting diseases.
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Affiliation(s)
- Anna Kowala
- Centre for Genomics and Child Health, Blizard Institute, Faculty of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London E1 2AT, UK; Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Faculty of Medicine and Dentistry, Queen Mary University of London, 2 Newark Street, London E1 2AT, UK; Centre for Predictive in vitro Models, Queen Mary University of London, Mile End Road, London E1 4NS, UK; UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
| | - James Boot
- Barts and the London Genome Centre, Faculty of Medicine and Dentistry, Blizard Institute, London, UK
| | - Jinhong Meng
- UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK; NIHR Biomedical Research Centre at Great Ormond Street Hospital, Great Ormond Street, London, UK
| | - Charles A Mein
- Barts and the London Genome Centre, Faculty of Medicine and Dentistry, Blizard Institute, London, UK
| | - Olivier Pourquié
- Department of Genetics, Harvard Medical School and Department of Pathology, Brigham and Women's Hospital, 60 Fenwood Road, Boston, MA, USA
| | - John T Connelly
- Centre for Predictive in vitro Models, Queen Mary University of London, Mile End Road, London E1 4NS, UK; Centre for Cell Biology and Cutaneous Research, Blizard Institute, Faculty of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London E1 2AT, UK
| | - Jennifer E Morgan
- UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK; NIHR Biomedical Research Centre at Great Ormond Street Hospital, Great Ormond Street, London, UK
| | - Yung-Yao Lin
- Centre for Genomics and Child Health, Blizard Institute, Faculty of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London E1 2AT, UK; Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Faculty of Medicine and Dentistry, Queen Mary University of London, 2 Newark Street, London E1 2AT, UK; Centre for Predictive in vitro Models, Queen Mary University of London, Mile End Road, London E1 4NS, UK.
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2
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Meng J, Moore M, Counsell J, Muntoni F, Popplewell L, Morgan J. Optimized lentiviral vector to restore full-length dystrophin via a cell-mediated approach in a mouse model of Duchenne muscular dystrophy. Mol Ther Methods Clin Dev 2022; 25:491-507. [PMID: 35615709 PMCID: PMC9121076 DOI: 10.1016/j.omtm.2022.04.015] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2021] [Accepted: 04/28/2022] [Indexed: 11/16/2022]
Abstract
Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the DMD gene. Restoration of full-length dystrophin protein in skeletal muscle would have therapeutic benefit, but lentivirally mediated delivery of such a large gene in vivo has been hindered by lack of tissue specificity, limited transduction, and insufficient transgene expression. To address these problems, we developed a lentiviral vector, which contains a muscle-specific promoter and sequence-optimized full-length dystrophin, to constrain dystrophin expression to differentiated myotubes/myofibers and enhance the transgene expression. We further explored the efficiency of restoration of full-length dystrophin in vivo, by grafting DMD myoblasts that had been corrected by this optimized lentiviral vector intramuscularly into an immunodeficient DMD mouse model. We show that these lentivirally corrected DMD myoblasts effectively reconstituted full-length dystrophin expression in 93.58% ± 2.17% of the myotubes in vitro. Moreover, dystrophin was restored in 64.4% ± 2.87% of the donor-derived regenerated muscle fibers in vivo, which were able to recruit members of the dystrophin-glycoprotein complex at the sarcolemma. This study represents a significant advance over existing cell-mediated gene therapy strategies for DMD that aim to restore full-length dystrophin expression in skeletal muscle.
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Affiliation(s)
- Jinhong Meng
- Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neuroscience Programme, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
- National Institute for Health Research, Great Ormond Street Institute of Child Health Biomedical Research Centre, University College London, London WC1N 1EH, UK
| | - Marc Moore
- Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neuroscience Programme, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
- Department of Biological Sciences, School of Life Sciences and the Environment, Royal Holloway University of London, Egham Hill, Egham TW20 0EX, UK
- National Institute for Health Research, Great Ormond Street Institute of Child Health Biomedical Research Centre, University College London, London WC1N 1EH, UK
| | - John Counsell
- Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neuroscience Programme, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
- UCL Division of Surgery and Interventional Science, Charles Bell House, 43-45 Foley Street, London W1W 7TY, UK
- National Institute for Health Research, Great Ormond Street Institute of Child Health Biomedical Research Centre, University College London, London WC1N 1EH, UK
| | - Francesco Muntoni
- Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neuroscience Programme, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
- National Institute for Health Research, Great Ormond Street Institute of Child Health Biomedical Research Centre, University College London, London WC1N 1EH, UK
| | - Linda Popplewell
- Department of Biological Sciences, School of Life Sciences and the Environment, Royal Holloway University of London, Egham Hill, Egham TW20 0EX, UK
| | - Jennifer Morgan
- Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neuroscience Programme, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
- National Institute for Health Research, Great Ormond Street Institute of Child Health Biomedical Research Centre, University College London, London WC1N 1EH, UK
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Al Tanoury Z, Rao J, Tassy O, Gobert B, Gapon S, Garnier JM, Wagner E, Hick A, Hall A, Gussoni E, Pourquié O. Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro. Development 2020; 147:dev187344. [PMID: 32541004 PMCID: PMC7328153 DOI: 10.1242/dev.187344] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2019] [Accepted: 05/29/2020] [Indexed: 12/12/2022]
Abstract
Satellite cells (SC) are muscle stem cells that can regenerate adult muscles upon injury. Most SC originate from PAX7+ myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7+ myogenic precursors. We show that the PAX7+ cells generated in culture can produce myofibers and self-renew in vitro and in vivo Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.
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Affiliation(s)
- Ziad Al Tanoury
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Department of Development and Stem Cells, CNRS (UMR 7104), Inserm U964, Université de Strasbourg, 67404, Illkirch Graffenstaden, France
- Department of Pathology, Brigham and Women's Hospital, 60 Fenwood Road, Boston, MA 02115, USA
- Department of Genetics, Harvard Medical School, 60 Fenwood Road, Boston, MA 02115, USA
| | - Jyoti Rao
- Department of Pathology, Brigham and Women's Hospital, 60 Fenwood Road, Boston, MA 02115, USA
- Department of Genetics, Harvard Medical School, 60 Fenwood Road, Boston, MA 02115, USA
| | - Olivier Tassy
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Department of Development and Stem Cells, CNRS (UMR 7104), Inserm U964, Université de Strasbourg, 67404, Illkirch Graffenstaden, France
| | - Bénédicte Gobert
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Department of Development and Stem Cells, CNRS (UMR 7104), Inserm U964, Université de Strasbourg, 67404, Illkirch Graffenstaden, France
- Anagenesis Biotechnologies, Parc d'innovation - BioParc 3, 850 Boulevard Sébastien Brandt, 67400 Illkirch Graffenstaden, France
| | - Svetlana Gapon
- Department of Pathology, Brigham and Women's Hospital, 60 Fenwood Road, Boston, MA 02115, USA
| | - Jean-Marie Garnier
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Department of Development and Stem Cells, CNRS (UMR 7104), Inserm U964, Université de Strasbourg, 67404, Illkirch Graffenstaden, France
| | - Erica Wagner
- Department of Pathology, Brigham and Women's Hospital, 60 Fenwood Road, Boston, MA 02115, USA
| | - Aurore Hick
- Anagenesis Biotechnologies, Parc d'innovation - BioParc 3, 850 Boulevard Sébastien Brandt, 67400 Illkirch Graffenstaden, France
| | - Arielle Hall
- Division of Genetics and Genomics, Boston Children's Hospital, 3 Blackfan Circle, CLS, Boston, MA 15021, USA
| | - Emanuela Gussoni
- Division of Genetics and Genomics, Boston Children's Hospital, 3 Blackfan Circle, CLS, Boston, MA 15021, USA
| | - Olivier Pourquié
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Department of Development and Stem Cells, CNRS (UMR 7104), Inserm U964, Université de Strasbourg, 67404, Illkirch Graffenstaden, France
- Department of Pathology, Brigham and Women's Hospital, 60 Fenwood Road, Boston, MA 02115, USA
- Department of Genetics, Harvard Medical School, 60 Fenwood Road, Boston, MA 02115, USA
- Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
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Barruet E, Garcia SM, Striedinger K, Wu J, Lee S, Byrnes L, Wong A, Xuefeng S, Tamaki S, Brack AS, Pomerantz JH. Functionally heterogeneous human satellite cells identified by single cell RNA sequencing. eLife 2020; 9:51576. [PMID: 32234209 PMCID: PMC7164960 DOI: 10.7554/elife.51576] [Citation(s) in RCA: 78] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2019] [Accepted: 03/27/2020] [Indexed: 12/19/2022] Open
Abstract
Although heterogeneity is recognized within the murine satellite cell pool, a comprehensive understanding of distinct subpopulations and their functional relevance in human satellite cells is lacking. We used a combination of single cell RNA sequencing and flow cytometry to identify, distinguish, and physically separate novel subpopulations of human PAX7+ satellite cells (Hu-MuSCs) from normal muscles. We found that, although relatively homogeneous compared to activated satellite cells and committed progenitors, the Hu-MuSC pool contains clusters of transcriptionally distinct cells with consistency across human individuals. New surface marker combinations were enriched in transcriptional subclusters, including a subpopulation of Hu-MuSCs marked by CXCR4/CD29/CD56/CAV1 (CAV1+). In vitro, CAV1+ Hu-MuSCs are morphologically distinct, and characterized by resistance to activation compared to CAV1- Hu-MuSCs. In vivo, CAV1+ Hu-MuSCs demonstrated increased engraftment after transplantation. Our findings provide a comprehensive transcriptional view of normal Hu-MuSCs and describe new heterogeneity, enabling separation of functionally distinct human satellite cell subpopulations.
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Affiliation(s)
- Emilie Barruet
- Departments of Surgery and Orofacial Sciences, Division of Plastic and Reconstructive Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, United States
| | - Steven M Garcia
- Departments of Surgery and Orofacial Sciences, Division of Plastic and Reconstructive Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, United States
| | - Katharine Striedinger
- Departments of Surgery and Orofacial Sciences, Division of Plastic and Reconstructive Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, United States
| | - Jake Wu
- Departments of Surgery and Orofacial Sciences, Division of Plastic and Reconstructive Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, United States
| | - Solomon Lee
- Departments of Surgery and Orofacial Sciences, Division of Plastic and Reconstructive Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, United States
| | - Lauren Byrnes
- University of California San Francisco, San Francisco, United States
| | - Alvin Wong
- Departments of Surgery and Orofacial Sciences, Division of Plastic and Reconstructive Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, United States
| | - Sun Xuefeng
- Department of Orthopedic Surgery, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, United States
| | - Stanley Tamaki
- Departments of Surgery and Orofacial Sciences, Division of Plastic and Reconstructive Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, United States
| | - Andrew S Brack
- Department of Orthopedic Surgery, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, United States
| | - Jason H Pomerantz
- Departments of Surgery and Orofacial Sciences, Division of Plastic and Reconstructive Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, United States
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5
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Meng J, Sweeney NP, Doreste B, Muntoni F, McClure M, Morgan J. Restoration of Functional Full-Length Dystrophin After Intramuscular Transplantation of Foamy Virus-Transduced Myoblasts. Hum Gene Ther 2020; 31:241-252. [PMID: 31801386 PMCID: PMC7047098 DOI: 10.1089/hum.2019.224] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2019] [Accepted: 11/24/2019] [Indexed: 12/12/2022] Open
Abstract
Stem cell therapy is a promising strategy to treat muscle diseases such as Duchenne muscular dystrophy (DMD). To avoid immune rejection of donor cells or donor-derived muscle, autologous cells, which have been genetically modified to express dystrophin, are preferable to cells derived from healthy donors. Restoration of full-length dystrophin (FL-dys) using viral vectors is extremely challenging, due to the limited packaging capacity of the vectors, but we have recently shown that either a foamy viral or lentiviral vector is able to package FL-dys open-reading frame and transduce myoblasts derived from a DMD patient. Differentiated myotubes derived from these transduced cells produced FL-dys. Here, we transplanted the foamy viral dystrophin-corrected DMD myoblasts intramuscularly into mdx nude mice, and showed that the transduced cells contributed to muscle regeneration, expressing FL-dys in nearly all the muscle fibers of donor origin. Furthermore, we showed that the restored FL-dys recruited members of the dystrophin-associated protein complex and neuronal nitric oxide synthase within donor-derived muscle fibers, evidence that the restored dystrophin protein is functional. Dystrophin-expressing donor-derived muscle fibers expressed lower levels of utrophin than host muscle fibers, providing additional evidence of functional improvement of donor-derived myofibers. This is the first in vivo evidence that foamy virus vector-transduced DMD myoblasts can contribute to muscle regeneration and mediate functional dystrophin restoration following their intramuscular transplantation, representing a promising therapeutic strategy for individual small muscles in DMD.
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Affiliation(s)
- Jinhong Meng
- Developmental Neuroscience Programme, Molecular Neurosciences Section, Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
- National Institute for Health Research, Great Ormond Street Institute of Child Health Biomedical Research Centre, University College London, London, United Kingdom
| | - Nathan Paul Sweeney
- Jefferiss Research Trust Laboratories, Imperial College London, London, United Kingdom
| | - Bruno Doreste
- Developmental Neuroscience Programme, Molecular Neurosciences Section, Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
- National Institute for Health Research, Great Ormond Street Institute of Child Health Biomedical Research Centre, University College London, London, United Kingdom
| | - Francesco Muntoni
- Developmental Neuroscience Programme, Molecular Neurosciences Section, Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
- National Institute for Health Research, Great Ormond Street Institute of Child Health Biomedical Research Centre, University College London, London, United Kingdom
| | - Myra McClure
- Jefferiss Research Trust Laboratories, Imperial College London, London, United Kingdom
| | - Jennifer Morgan
- Developmental Neuroscience Programme, Molecular Neurosciences Section, Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
- National Institute for Health Research, Great Ormond Street Institute of Child Health Biomedical Research Centre, University College London, London, United Kingdom
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Mueller AL, Bloch RJ. Skeletal muscle cell transplantation: models and methods. J Muscle Res Cell Motil 2019; 41:297-311. [PMID: 31392564 DOI: 10.1007/s10974-019-09550-w] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2019] [Accepted: 08/01/2019] [Indexed: 02/07/2023]
Abstract
Xenografts of skeletal muscle are used to study muscle repair and regeneration, mechanisms of muscular dystrophies, and potential cell therapies for musculoskeletal disorders. Typically, xenografting involves using an immunodeficient host that is pre-injured to create a niche for human cell engraftment. Cell type and method of delivery to muscle depend on the specific application, but can include myoblasts, satellite cells, induced pluripotent stem cells, mesangioblasts, immortalized muscle precursor cells, and other multipotent cell lines delivered locally or systemically. Some studies follow cell engraftment with interventions to enhance cell proliferation, migration, and differentiation into mature muscle fibers. Recently, several advances in xenografting human-derived muscle cells have been applied to study and treat Duchenne muscular dystrophy and Facioscapulohumeral muscular dystrophy. Here, we review the vast array of techniques available to aid researchers in designing future experiments aimed at creating robust muscle xenografts in rodent hosts.
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Affiliation(s)
- Amber L Mueller
- Department of Physiology, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD, 21201, USA
| | - Robert J Bloch
- Department of Physiology, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD, 21201, USA.
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7
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Miyagoe-Suzuki Y, Takeda S. Skeletal muscle generated from induced pluripotent stem cells - induction and application. World J Stem Cells 2017; 9:89-97. [PMID: 28717411 PMCID: PMC5491631 DOI: 10.4252/wjsc.v9.i6.89] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/29/2017] [Revised: 04/20/2017] [Accepted: 05/19/2017] [Indexed: 02/06/2023] Open
Abstract
Human induced pluripotent stem cells (hiPS cells or hiPSCs) can be derived from cells of patients with severe muscle disease. If skeletal muscle induced from patient-iPSCs shows disease-specific phenotypes, it can be useful for studying the disease pathogenesis and for drug development. On the other hand, human iPSCs from healthy donors or hereditary muscle disease-iPSCs whose genomes are edited to express normal protein are expected to be a cell source for cell therapy. Several protocols for the derivation of skeletal muscle from human iPSCs have been reported to allow the development of efficient treatments for devastating muscle diseases. In 2017, the focus of research is shifting to another stage: (1) the establishment of mature myofibers that are suitable for study of the pathogenesis of muscle disease; (2) setting up a high-throughput drug screening system; and (3) the preparation of highly regenerative, non-oncogenic cells in large quantities for cell transplantation, etc.
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Alexander MS, Rozkalne A, Colletta A, Spinazzola JM, Johnson S, Rahimov F, Meng H, Lawlor MW, Estrella E, Kunkel LM, Gussoni E. CD82 Is a Marker for Prospective Isolation of Human Muscle Satellite Cells and Is Linked to Muscular Dystrophies. Cell Stem Cell 2016; 19:800-807. [PMID: 27641304 DOI: 10.1016/j.stem.2016.08.006] [Citation(s) in RCA: 88] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2015] [Revised: 05/17/2016] [Accepted: 08/05/2016] [Indexed: 12/23/2022]
Abstract
Cell-surface markers for prospective isolation of stem cells from human skeletal muscle have been difficult to identify. Such markers would be powerful tools for studying satellite cell function during homeostasis and in pathogenesis of diseases such as muscular dystrophies. In this study, we show that the tetraspanin KAI/CD82 is an excellent marker for prospectively isolating stem cells from human fetal and adult skeletal muscle. Human CD82+ muscle cells robustly engraft into a mouse model of muscular dystrophy. shRNA knockdown of CD82 in myogenic cells reduces myoblast proliferation, suggesting it is functionally involved in muscle homeostasis. CD82 physically interacts with alpha7beta1 integrin (α7β1-ITG) and with α-sarcoglycan, a member of the Dystrophin-Associated Glycoprotein Complex (DAPC), both of which have been linked to muscular dystrophies. Consistently, CD82 expression is decreased in Duchenne muscular dystrophy patients. Together, these findings suggest that CD82 function may be important for muscle stem cell function in muscular disorders.
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Affiliation(s)
- Matthew S Alexander
- Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115, USA; The Stem Cell Program at Boston Children's Hospital, Boston, MA 02115, USA
| | - Anete Rozkalne
- Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115, USA
| | - Alessandro Colletta
- Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115, USA; Boston University School of Medicine, Boston, MA 02215, USA
| | - Janelle M Spinazzola
- Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115, USA; The Stem Cell Program at Boston Children's Hospital, Boston, MA 02115, USA
| | - Samuel Johnson
- Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115, USA
| | - Fedik Rahimov
- Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115, USA; The Stem Cell Program at Boston Children's Hospital, Boston, MA 02115, USA
| | - Hui Meng
- Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Michael W Lawlor
- Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Elicia Estrella
- Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115, USA
| | - Louis M Kunkel
- Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115, USA; The Stem Cell Program at Boston Children's Hospital, Boston, MA 02115, USA; The Manton Center for Orphan Disease Research, Boston Children's Hospital, Boston, MA 02115, USA
| | - Emanuela Gussoni
- Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115, USA; The Stem Cell Program at Boston Children's Hospital, Boston, MA 02115, USA.
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9
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Xu X, Wilschut KJ, Kouklis G, Tian H, Hesse R, Garland C, Sbitany H, Hansen S, Seth R, Knott PD, Hoffman WY, Pomerantz JH. Human Satellite Cell Transplantation and Regeneration from Diverse Skeletal Muscles. Stem Cell Reports 2016; 5:419-34. [PMID: 26352798 PMCID: PMC4618654 DOI: 10.1016/j.stemcr.2015.07.016] [Citation(s) in RCA: 98] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2015] [Revised: 07/31/2015] [Accepted: 07/31/2015] [Indexed: 12/24/2022] Open
Abstract
Identification of human satellite cells that fulfill muscle stem cell criteria is an unmet need in regenerative medicine. This hurdle limits understanding how closely muscle stem cell properties are conserved among mice and humans and hampers translational efforts in muscle regeneration. Here, we report that PAX7 satellite cells exist at a consistent frequency of 2-4 cells/mm of fiber in muscles of the human trunk, limbs, and head. Xenotransplantation into mice of 50-70 fiber-associated, or 1,000-5,000 FACS-enriched CD56(+)/CD29(+) human satellite cells led to stable engraftment and formation of human-derived myofibers. Human cells with characteristic PAX7, CD56, and CD29 expression patterns populated the satellite cell niche beneath the basal lamina on the periphery of regenerated fibers. After additional injury, transplanted satellite cells robustly regenerated to form hundreds of human-derived fibers. Together, these findings conclusively delineate a source of bona-fide endogenous human muscle stem cells that will aid development of clinical applications.
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Affiliation(s)
- Xiaoti Xu
- Division of Plastic and Reconstructive Surgery, Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Karlijn J Wilschut
- Division of Plastic and Reconstructive Surgery, Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Gayle Kouklis
- Division of Plastic and Reconstructive Surgery, Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Hua Tian
- Division of Plastic and Reconstructive Surgery, Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Robert Hesse
- Division of Plastic and Reconstructive Surgery, Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Catharine Garland
- Division of Plastic and Reconstructive Surgery, Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Hani Sbitany
- Division of Plastic and Reconstructive Surgery, Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Scott Hansen
- Division of Plastic and Reconstructive Surgery, Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Rahul Seth
- Department of Otolaryngology-Head and Neck Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - P Daniel Knott
- Department of Otolaryngology-Head and Neck Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - William Y Hoffman
- Division of Plastic and Reconstructive Surgery, Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Jason H Pomerantz
- Division of Plastic and Reconstructive Surgery, Departments of Surgery and Orofacial Sciences, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
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Hattori Y, Kim H, Tsuboi N, Yamamoto A, Akiyama S, Shi Y, Katsuno T, Kosugi T, Ueda M, Matsuo S, Maruyama S. Therapeutic Potential of Stem Cells from Human Exfoliated Deciduous Teeth in Models of Acute Kidney Injury. PLoS One 2015; 10:e0140121. [PMID: 26509261 PMCID: PMC4625005 DOI: 10.1371/journal.pone.0140121] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2015] [Accepted: 09/21/2015] [Indexed: 12/15/2022] Open
Abstract
Background Acute kidney injury (AKI) is a critical condition associated with high mortality. However, the available treatments for AKI are limited. Stem cells from human exfoliated deciduous teeth (SHED) have recently gained attention as a novel source of stem cells. The purpose of this study was to clarify whether SHED have a therapeutic effect on AKI induced by ischemia-reperfusion injury. Methods The left renal artery and vein of the mice were clamped for 20 min to induce ischemia. SHED, bone marrow derived mesenchymal stem cells (BMMSC) or phosphate-buffered saline (control) were administered into the subrenal capsule. To confirm the potency of SHED in vitro, H2O2 stimulation assays and scratch assays were performed. Results The serum creatinine and blood urea nitrogen levels of the SHED group were significantly lower than those of the control group, while BMMSC showed no therapeutic effect. Infiltration of macrophages and neutrophils in the kidney was significantly attenuated in mice treated with SHED. Cytokine levels (MIP-2, IL-1β, and MCP-1) in mice kidneys were significantly reduced in the SHED group. In in vitro experiments, SHED significantly decreased MCP-1 secretion in tubular epithelial cells (TEC) stimulated with H2O2. In addition, SHED promoted wound healing in the scratch assays, which was blunted by anti-HGF antibodies. Discussion SHED attenuated the levels of inflammatory cytokines and improved kidney function in AKI induced by IRI. SHED secreted factors reduced MCP-1 and increased HGF expression, which promoted wound healing. These results suggest that SHED might provide a novel stem cell resource, which can be applied for the treatment of ischemic kidney injury.
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Affiliation(s)
- Yuka Hattori
- Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, Japan
| | - Hangsoo Kim
- Department of Nephrology, Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, Japan
| | - Naotake Tsuboi
- Department of Nephrology, Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, Japan
| | - Akihito Yamamoto
- Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, Japan
| | - Shinichi Akiyama
- Department of Nephrology, Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, Japan
| | - Yiqin Shi
- Department of Nephrology, Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, Japan
| | - Takayuki Katsuno
- Department of Nephrology, Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, Japan
| | - Tomoki Kosugi
- Department of Nephrology, Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, Japan
| | - Minoru Ueda
- Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, Japan
| | - Seiichi Matsuo
- Department of Nephrology, Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, Japan
| | - Shoichi Maruyama
- Department of Nephrology, Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, Japan
- * E-mail:
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11
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Meng J, Bencze M, Asfahani R, Muntoni F, Morgan JE. The effect of the muscle environment on the regenerative capacity of human skeletal muscle stem cells. Skelet Muscle 2015; 5:11. [PMID: 25949786 PMCID: PMC4422426 DOI: 10.1186/s13395-015-0036-8] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2015] [Accepted: 03/12/2015] [Indexed: 12/31/2022] Open
Abstract
Background Muscle stem cell transplantation is a possible treatment for muscular dystrophy. In addition to the intrinsic properties of the stem cells, the local and systemic environment plays an important role in determining the fate of the grafted cells. We therefore investigated the effect of modulating the host muscle environment in different ways (irradiation or cryoinjury or a combination of irradiation and cryoinjury) in two immunodeficient mouse strains (mdx nude and recombinase-activating gene (Rag)2-/γ chain-/C5-) on the regenerative capacity of two types of human skeletal muscle-derived stem cell (pericytes and CD133+ cells). Methods Human skeletal muscle-derived pericytes or CD133+ cells were transplanted into muscles of either mdx nude or recombinase-activating gene (Rag)2-/γ chain-/C5- host mice. Host muscles were modulated prior to donor cell transplantation by either irradiation, or cryoinjury, or a combination of irradiation and cryoinjury. Muscles were analysed four weeks after transplantation, by staining transverse cryostat sections of grafted muscles with antibodies to human lamin A/C, human spectrin, laminin and Pax 7. The number of nuclei and muscle fibres of donor origin and the number of satellite cells of both host and donor origin were quantified. Results Within both host strains transplanted intra-muscularly with both donor cell types, there were significantly more nuclei and muscle fibres of donor origin in host muscles that had been modulated by cryoinjury, or irradiation+cryoinjury, than by irradiation alone. Irradiation has no additive effects in further enhancing the transplantation efficiency than cryodamage. Donor pericytes did not give rise to satellite cells. However, using CD133+ cells as donor cells, there were significantly more nuclei, muscle fibres, as well as satellite cells of donor origin in Rag2-/γ chain-/C5- mice than mdx nude mice, when the muscles were injured by either cryodamage or irradiation+cryodamage. Conclusions Rag2-/γ chain-/C5- mice are a better recipient mouse strain than mdx nude mice for human muscle stem cell transplantation. Cryodamage of host muscle is the most effective method to enhance the transplantation efficiency of human skeletal muscle stem cells. This study highlights the importance of modulating the muscle environment in preclinical studies to optimise the efficacy of transplanted stem cells. Electronic supplementary material The online version of this article (doi:10.1186/s13395-015-0036-8) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Jinhong Meng
- The Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neurosciences Programme, UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UK
| | - Maximilien Bencze
- The Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neurosciences Programme, UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UK
| | - Rowan Asfahani
- The Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neurosciences Programme, UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UK
| | - Francesco Muntoni
- The Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neurosciences Programme, UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UK
| | - Jennifer E Morgan
- The Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neurosciences Programme, UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UK
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12
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Duan D. Duchenne muscular dystrophy gene therapy in the canine model. HUM GENE THER CL DEV 2015; 26:57-69. [PMID: 25710459 PMCID: PMC4442571 DOI: 10.1089/humc.2015.006] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2015] [Accepted: 01/13/2015] [Indexed: 12/12/2022] Open
Abstract
Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disease caused by dystrophin deficiency. Gene therapy has significantly improved the outcome of dystrophin-deficient mice. Yet, clinical translation has not resulted in the expected benefits in human patients. This translational gap is largely because of the insufficient modeling of DMD in mice. Specifically, mice lacking dystrophin show minimum dystrophic symptoms, and they do not respond to the gene therapy vector in the same way as human patients do. Further, the size of a mouse is hundredfolds smaller than a boy, making it impossible to scale-up gene therapy in a mouse model. None of these limitations exist in the canine DMD (cDMD) model. For this reason, cDMD dogs have been considered a highly valuable platform to test experimental DMD gene therapy. Over the last three decades, a variety of gene therapy approaches have been evaluated in cDMD dogs using a number of nonviral and viral vectors. These studies have provided critical insight for the development of an effective gene therapy protocol in human patients. This review discusses the history, current status, and future directions of the DMD gene therapy in the canine model.
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Affiliation(s)
- Dongsheng Duan
- Department of Molecular Microbiology and Immunology, Department of Neurology School of Medicine, University of Missouri , Columbia, MO 65212
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13
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Meng J, Chun S, Asfahani R, Lochmüller H, Muntoni F, Morgan J. Human skeletal muscle-derived CD133(+) cells form functional satellite cells after intramuscular transplantation in immunodeficient host mice. Mol Ther 2014; 22:1008-17. [PMID: 24569833 DOI: 10.1038/mt.2014.26] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2013] [Accepted: 02/16/2014] [Indexed: 12/28/2022] Open
Abstract
Stem cell therapy is a promising strategy for treatment of muscular dystrophies. In addition to muscle fiber formation, reconstitution of functional stem cell pool by donor cells is vital for long-term treatment. We show here that some CD133(+) cells within human muscle are located underneath the basal lamina of muscle fibers, in the position of the muscle satellite cell. Cultured hCD133(+) cells are heterogeneous and multipotent, capable of forming myotubes and reserve satellite cells in vitro. They contribute to extensive muscle regeneration and satellite cell formation following intramuscular transplantation into irradiated and cryodamaged tibialis anterior muscles of immunodeficient Rag2-/γ chain-/C5-mice. Some donor-derived satellite cells expressed the myogenic regulatory factor MyoD, indicating that they were activated. In addition, when transplanted host muscles were reinjured, there was significantly more newly-regenerated muscle fibers of donor origin in treated than in control, nonreinjured muscles, indicating that hCD133(+) cells had given rise to functional muscle stem cells, which were able to activate in response to injury and contribute to a further round of muscle regeneration. Our findings provide new evidence for the location and characterization of hCD133(+) cells, and highlight that these cells are highly suitable for future clinical application.
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Affiliation(s)
- Jinhong Meng
- The Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, UK
| | - Soyon Chun
- The Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, UK
| | - Rowan Asfahani
- The Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, UK
| | - Hanns Lochmüller
- Institute of Genetic Medicine, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne, UK
| | - Francesco Muntoni
- The Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, UK
| | - Jennifer Morgan
- The Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, UK
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