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Liu Y, Zhou Z, Lu G, Zhang X, Shi D, Tong L, Chen D, Tuan RS, Li ZA. Musculoskeletal organoids: An emerging toolkit for establishing personalized models of musculoskeletal disorders and developing regenerative therapies. Acta Biomater 2025:S1742-7061(25)00362-9. [PMID: 40381929 DOI: 10.1016/j.actbio.2025.05.037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2025] [Revised: 05/09/2025] [Accepted: 05/14/2025] [Indexed: 05/20/2025]
Abstract
Musculoskeletal (MSK) conditions are the primary cause of physical disability globally. These disorders are physically and mentally debilitating and severely impact the patients' quality of life. As the median age of the world's population increases, there has been an intensifying urgency of developing efficacious therapies for various orthopaedic conditions. Furthermore, the highly heterogeneous nature of MSK conditions calls for a personalized approach to studying disease mechanisms and developing regenerative treatments. Organoids have emerged as an advanced approach to generating functional tissue/organ mimics in vitro, which hold promise in MSK regeneration, disease modeling, and therapeutic development. Herein, we review the preparation, characterization, and application of various MSK organoids. We highlight the potential of patient-specific organoids in the development of personalized medicine and discuss the challenges and opportunities in the future development of MSK organoids. STATEMENT OF SIGNIFICANCE: Despite decades of research, translation of MSK research into clinical applications remains limited, partially attributed to our inadequate understanding of disease mechanisms. To advance therapeutic development, there are critical needs for MSK disease models with higher clinical relevance and predictive power. Additionally, engineered constructs that closely mimic the structural and functional features of native MSK tissues are highly desirable. MSK organoids have emerged as a promising approach to meet the above requirements. To unleash the full potential of MSK organoids necessitates a comprehensive understanding of their categories, construction, development, functions, applications, and challenges. This review aims to fulfill this crucial need, aiming to accelerate the clinical translation of MSK organoid platforms to benefit millions of patients afflicted with MSK conditions.
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Affiliation(s)
- Yuwei Liu
- Department of Orthopedics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, PR China; Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong Special Administrative Region of China; Department of Sports Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518035, Guangdong, PR China
| | - Zhilong Zhou
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong Special Administrative Region of China
| | - Gang Lu
- Institute for Tissue Engineering and Regenerative Medicine, School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong Special Administrative Region of China; Center for Neuromusculoskeletal Restorative Medicine, Hong Kong Science Park, Hong Kong Special Administrative Region of China
| | - Xin Zhang
- Institute of Sports Medicine, Beijing Key Laboratory of Sports Injuries, Peking University Third Hospital, Beijing, 100191 PR China
| | - Dongquan Shi
- Division of Sports Medicine and Adult Reconstructive Surgery, Department of Orthopedic Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, 210008, PR China
| | - Liping Tong
- Research Center for Computer-aided Drug Discovery, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, PR China
| | - Di Chen
- Research Center for Computer-aided Drug Discovery, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, PR China; Department of Pharmacology, Faculty of Pharmaceutical Sciences, Shenzhen University of Advanced Technology, Shenzhen 518000, PR China.
| | - Rocky S Tuan
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong Special Administrative Region of China; Institute for Tissue Engineering and Regenerative Medicine, School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong Special Administrative Region of China; Center for Neuromusculoskeletal Restorative Medicine, Hong Kong Science Park, Hong Kong Special Administrative Region of China.
| | - Zhong Alan Li
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong Special Administrative Region of China; Institute for Tissue Engineering and Regenerative Medicine, School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong Special Administrative Region of China; Center for Neuromusculoskeletal Restorative Medicine, Hong Kong Science Park, Hong Kong Special Administrative Region of China; Peter Hung Pain Research Institute, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region of China.
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Franken M, van der Wal E, Zheng D, den Hamer B, van der Vliet PJ, Lemmers RJLF, van den Heuvel A, Dorn AL, Duivenvoorden CGA, in ’t Groen SLM, Freund C, Eussen B, Tawil R, van Engelen BGM, Pijnappel WWMP, van der Maarel SM, de Greef JC. Three-dimensional tissue engineered skeletal muscle modelling facioscapulohumeral muscular dystrophy. Brain 2025; 148:1723-1739. [PMID: 39556762 PMCID: PMC12074006 DOI: 10.1093/brain/awae379] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2024] [Revised: 10/16/2024] [Accepted: 10/20/2024] [Indexed: 11/20/2024] Open
Abstract
Facioscapulohumeral muscular dystrophy (FSHD) is caused by sporadic misexpression of the transcription factor double homeobox 4 (DUX4) in skeletal muscles. So far, monolayer cultures and animal models have been used to study the disease mechanism of FSHD and for development of FSHD therapy, but these models do not fully recapitulate the disease and there is a lack of knowledge on how DUX4 misexpression leads to skeletal muscle dysfunction. To overcome these barriers, we have developed a 3D tissue engineered skeletal muscle (3D-TESM) model by generating genetically matched myogenic progenitors from human induced pluripotent stem cells of three mosaic FSHD patients. 3D-TESMs derived from genetically affected myogenic progenitors recapitulated pathological features including DUX4 and DUX4 target gene expression, smaller myofibre diameters and reduced absolute forces upon electrical stimulation. RNA-sequencing data illustrated increased expression of DUX4 target genes in 3D-TESMs compared with 2D myotubes, and cellular differentiation was improved by 3D culture conditions. Treatment of 3D-TESMs with three different small molecules identified in drug development screens in 2D muscle cultures showed no improvements, and sometimes even declines, in contractile force and sarcomere organization. These results suggest that these compounds either have a detrimental effect on the formation of 3D-TESMs, an effect that might have been overlooked or was challenging to detect in 2D cultures and in vivo models, and/or that further development of the 3D-TESM model is needed. In conclusion, we have developed a 3D skeletal muscle model for FSHD that can be used for preclinical research focusing on DUX4 expression and downstream pathways of FSHD in relationship to contractile properties. In the future, we expect that this model can also be used for preclinical drug screening.
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Affiliation(s)
- Marnix Franken
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Erik van der Wal
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Dongxu Zheng
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Bianca den Hamer
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Patrick J van der Vliet
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Richard J L F Lemmers
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Anita van den Heuvel
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Alexandra L Dorn
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Cas G A Duivenvoorden
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Stijn L M in ’t Groen
- Department of Clinical Genetics, Erasmus University Medical Center, 3015 GD, Rotterdam, The Netherlands
- Department of Pediatrics, Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands
- Center for Lysosomal and Metabolic Diseases, Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands
| | - Christian Freund
- Leiden hiPSC Centre, Department of Anatomy and Embryology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Bert Eussen
- Department of Clinical Genetics, Erasmus University Medical Center, 3015 GD, Rotterdam, The Netherlands
| | - Rabi Tawil
- Department of Neurology, University of Rochester Medical Center, Rochester, NY 14642, USA
| | - Baziel G M van Engelen
- Department of Neurology, Donders Centre of Neuroscience, Radboud University Medical Centre, 6525 GA, Nijmegen, The Netherlands
| | - W W M Pim Pijnappel
- Department of Clinical Genetics, Erasmus University Medical Center, 3015 GD, Rotterdam, The Netherlands
- Department of Pediatrics, Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands
- Center for Lysosomal and Metabolic Diseases, Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands
| | - Silvère M van der Maarel
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Jessica C de Greef
- Department of Human Genetics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
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Hamer MS, Rossi FMV. Multitasking muscle: engineering iPSC-derived myogenic progenitors to do more. Front Cell Dev Biol 2025; 12:1526635. [PMID: 39911186 PMCID: PMC11794491 DOI: 10.3389/fcell.2024.1526635] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2024] [Accepted: 12/23/2024] [Indexed: 02/07/2025] Open
Abstract
The generation of myogenic progenitors from iPSCs (iMPs) with therapeutic potential for in vivo tissue regeneration has long been a goal in the skeletal muscle community. Today, protocols enable the production of potent, albeit immature, iMPs that resemble Pax7+ adult muscle stem cells. While muscular dystrophies are often the primary therapeutic target for these cells, an underexplored application is their use in treating traumatic muscle injuries. Notably absent from recent reviews on iMPs is the concept of engineering these cells to perform functions post-transplantation that non-transgenic cells cannot. Here, we highlight protocols to enhance the generation, purification, and maturation of iMPs, and introduce the idea of engineering these cells to perform functions beyond their normal capacities, envisioning novel therapeutic applications.
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Affiliation(s)
- Mark Stephen Hamer
- School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada
| | - Fabio M. V. Rossi
- Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada
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Wada E, Susumu N, Okuzaki Y, Hotta A, Sakurai H, Hayashi YK. Optimized simple culture protocol for inducing mature myotubes from MYOD1-overexpressed human iPS cells. Sci Rep 2024; 14:28783. [PMID: 39567611 PMCID: PMC11579357 DOI: 10.1038/s41598-024-79745-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2024] [Accepted: 11/12/2024] [Indexed: 11/22/2024] Open
Abstract
The forced expression system of MYOD1, a master gene for myogenic differentiation, can efficiently and rapidly reproduce muscle differentiation of human induced pluripotent stem cells (hiPSCs). Despite these advantages of the MYOD1 overexpression system, developed myotubes are relatively immature and do not recapitulate several aspects of striated muscle fibers. Here, we developed a simple optimized protocol using an alternative culture medium for maximizing the advantages of the MYOD1 overexpression system, and successfully improved the formation of multinucleated mature myotubes within 10 days. In this study, we generated hiPSCs derived from healthy donors and an individual with congenial muscular dystrophy caused by LMNA mutation (laminopathy), and compared disease-associated phenotypes in differentiated myotubes generated by the conventional method and by our new optimized culture method. Using our optimized method, abnormal myonuclear shape was pronounced in the patient-derived iPSCs. In addition, abnormal accumulation of the nuclear membrane protein emerin was observed in LMNA-mutant hiPSCs. Our new culture method is expected to be widely applicable as a MYOD1 overexpression model of hiPSC-derived skeletal muscle cells for the analysis of a variety of muscle diseases.
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Affiliation(s)
- Eiji Wada
- Department of Pathophysiology, Tokyo Medical University, Tokyo, Japan
| | - Nao Susumu
- Department of Pathophysiology, Tokyo Medical University, Tokyo, Japan
| | - Yuya Okuzaki
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Graduate School of Bioagricultural Sciences, Nagoya University, Aichi, Japan
| | - Akitsu Hotta
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Hidetoshi Sakurai
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Yukiko K Hayashi
- Department of Pathophysiology, Tokyo Medical University, Tokyo, Japan.
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Mehmood H, Kasher PR, Barrett-Jolley R, Walmsley GL. Aligning with the 3Rs: alternative models for research into muscle development and inherited myopathies. BMC Vet Res 2024; 20:477. [PMID: 39425123 PMCID: PMC11488271 DOI: 10.1186/s12917-024-04309-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2024] [Accepted: 09/30/2024] [Indexed: 10/21/2024] Open
Abstract
Inherited and acquired muscle diseases are an important cause of morbidity and mortality in human medical and veterinary patients. Researchers use models to study skeletal muscle development and pathology, improve our understanding of disease pathogenesis and explore new treatment options. Experiments on laboratory animals, including murine and canine models, have led to huge advances in congenital myopathy and muscular dystrophy research that have translated into clinical treatment trials in human patients with these debilitating and often fatal conditions. Whilst animal experimentation has enabled many significant and impactful discoveries that otherwise may not have been possible, we have an ethical and moral, and in many countries also a legal, obligation to consider alternatives. This review discusses the models available as alternatives to mammals for muscle development, biology and disease research with a focus on inherited myopathies. Cell culture models can be used to replace animals for some applications: traditional monolayer cultures (for example, using the immortalised C2C12 cell line) are accessible, tractable and inexpensive but developmentally limited to immature myotube stages; more recently, developments in tissue engineering have led to three-dimensional cultures with improved differentiation capabilities. Advances in computer modelling and an improved understanding of pathogenetic mechanisms are likely to herald new models and opportunities for replacement. Where this is not possible, a 3Rs approach advocates partial replacement with the use of less sentient animals (including invertebrates (such as worms Caenorhabditis elegans and fruit flies Drosophila melanogaster) and embryonic stages of small vertebrates such as the zebrafish Danio rerio) alongside refinement of experimental design and improved research practices to reduce the numbers of animals used and the severity of their experience. An understanding of the advantages and disadvantages of potential models is essential for researchers to determine which can best facilitate answering a specific scientific question. Applying 3Rs principles to research not only improves animal welfare but generates high-quality, reproducible and reliable data with translational relevance to human and animal patients.
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Affiliation(s)
- Hashir Mehmood
- Department of Musculoskeletal and Ageing Science, Institute of Life Course and Medical Sciences, Faculty of Health and Lifesciences, University of Liverpool, William Henry Duncan Building, 6 West Derby Street, Liverpool, L7 8TX, UK
- Division of Neuroscience, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK
| | - Paul R Kasher
- Division of Neuroscience, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK
- Geoffrey Jefferson Brain Research Centre, Manchester Academic Health Science Centre, Northern Care Allianceand the, University of Manchester , Manchester, M6 8HD, UK
| | - Richard Barrett-Jolley
- Department of Musculoskeletal and Ageing Science, Institute of Life Course and Medical Sciences, Faculty of Health and Lifesciences, University of Liverpool, William Henry Duncan Building, 6 West Derby Street, Liverpool, L7 8TX, UK
| | - Gemma L Walmsley
- Department of Musculoskeletal and Ageing Science, Institute of Life Course and Medical Sciences, Faculty of Health and Lifesciences, University of Liverpool, William Henry Duncan Building, 6 West Derby Street, Liverpool, L7 8TX, UK.
- Department of Small Animal Clinical Sciences, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Leahurst Campus, South Wirral, Neston, CH64 7TE, UK.
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Zhao M, Taniguchi Y, Shimono C, Jonouchi T, Cheng Y, Shimizu Y, Nalbandian M, Yamamoto T, Nakagawa M, Sekiguchi K, Sakurai H. Heparan Sulfate Chain-Conjugated Laminin-E8 Fragments Advance Paraxial Mesodermal Differentiation Followed by High Myogenic Induction from hiPSCs. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2308306. [PMID: 38685581 PMCID: PMC11234437 DOI: 10.1002/advs.202308306] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/01/2023] [Revised: 03/26/2024] [Indexed: 05/02/2024]
Abstract
Human-induced pluripotent stem cells (hiPSCs) have great therapeutic potential. The cell source differentiated from hiPSCs requires xeno-free and robust methods for lineage-specific differentiation. Here, a system is described for differentiating hiPSCs on new generation laminin fragments (NGLFs), a recombinant form of a laminin E8 fragment conjugated to the heparan sulfate chains (HS) attachment domain of perlecan. Using NGLFs, hiPSCs are highly promoted to direct differentiation into a paraxial mesoderm state with high-efficiency muscle lineage generation. HS conjugation to the C-terminus of Laminin E8 fragments brings fibroblast growth factors (FGFs) bound to the HS close to the cell surface of hiPSCs, thereby facilitating stronger FGF signaling pathways stimulation and initiating HOX gene expression, which triggers the paraxial mesoderm differentiation of hiPSCs. This highly efficient differentiation system can provide a roadmap for paraxial mesoderm development and an infinite source of myocytes and muscle stem cells for disease modeling and regenerative medicine.
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Affiliation(s)
- Mingming Zhao
- Department of Clinical ApplicationCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
- Center for Medical EpigeneticsSchool of Basic Medical SciencesChongqing Medical University1 Yixueyuan Road, Yuzhong DistrictChongqing400016China
| | - Yukimasa Taniguchi
- Division of Matrixome Research and ApplicationInstitute for Protein ResearchOsaka University3‐2 Yamadaoka, SuitaOsaka565‐0871Japan
| | - Chisei Shimono
- Division of Matrixome Research and ApplicationInstitute for Protein ResearchOsaka University3‐2 Yamadaoka, SuitaOsaka565‐0871Japan
| | - Tatsuya Jonouchi
- Department of Clinical ApplicationCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
| | - Yushen Cheng
- Department of Life Science FrontiersCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
| | - Yasuhiro Shimizu
- Division of Matrixome Research and ApplicationInstitute for Protein ResearchOsaka University3‐2 Yamadaoka, SuitaOsaka565‐0871Japan
| | - Minas Nalbandian
- Department of Clinical ApplicationCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
| | - Takuya Yamamoto
- Department of Life Science FrontiersCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
| | - Masato Nakagawa
- Department of Life Science FrontiersCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
| | - Kiyotoshi Sekiguchi
- Division of Matrixome Research and ApplicationInstitute for Protein ResearchOsaka University3‐2 Yamadaoka, SuitaOsaka565‐0871Japan
| | - Hidetoshi Sakurai
- Department of Clinical ApplicationCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
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Arai Y, Ito H, Shimizu T, Shimoda Y, Song D, Matsuo-Takasaki M, Hayata T, Hayashi Y. Patient-derived and gene-edited pluripotent stem cells lacking NPHP1 recapitulate juvenile nephronophthisis in abnormalities of primary cilia and renal cyst formation. Front Cell Dev Biol 2024; 12:1370723. [PMID: 38989059 PMCID: PMC11233770 DOI: 10.3389/fcell.2024.1370723] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Accepted: 05/21/2024] [Indexed: 07/12/2024] Open
Abstract
Juvenile nephronophthisis is an inherited renal ciliopathy with cystic kidney disease, renal fibrosis, and end-stage renal failure in children and young adults. Mutations in the NPHP1 gene encoding nephrocystin-1 protein have been identified as the most frequently responsible gene and cause the formation of cysts in the renal medulla. The molecular pathogenesis of juvenile nephronophthisis remains elusive, and no effective medicines to prevent end-stage renal failure exist even today. No human cellular models have been available yet. Here, we report a first disease model of juvenile nephronophthisis using patient-derived and gene-edited human induced pluripotent stem cells (hiPSCs) and kidney organoids derived from these hiPSCs. We established NPHP1-overexpressing hiPSCs from patient-derived hiPSCs and NPHP1-deficient hiPSCs from healthy donor hiPSCs. Comparing these series of hiPSCs, we found abnormalities in primary cilia associated with NPHP1 deficiency in hiPSCs. Kidney organoids generated from the hiPSCs lacking NPHP1 formed renal cysts frequently in suspension culture with constant rotation. This cyst formation in patient-derived kidney organoids was rescued by overexpression of NPHP1. Transcriptome analysis on these kidney organoids revealed that loss of NPHP1 caused lower expression of genes related to primary cilia in epithelial cells and higher expression of genes related to the cell cycle. These findings suggested the relationship between abnormality in primary cilia induced by NPHP1 loss and abnormal proliferative characteristics in the formation of renal cysts. These findings demonstrated that hiPSC-based systematic disease modeling of juvenile nephronophthisis contributed to elucidating the molecular pathogenesis and developing new therapies.
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Affiliation(s)
- Yutaka Arai
- iPS Cell Advanced Characterization and Development Team, Bioresource Research Center, RIKEN, Tsukuba, Ibaraki, Japan
- Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences and Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan
| | - Hidenori Ito
- iPS Cell Advanced Characterization and Development Team, Bioresource Research Center, RIKEN, Tsukuba, Ibaraki, Japan
| | - Tomoya Shimizu
- iPS Cell Advanced Characterization and Development Team, Bioresource Research Center, RIKEN, Tsukuba, Ibaraki, Japan
- Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences and Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan
| | - Yuzuno Shimoda
- iPS Cell Advanced Characterization and Development Team, Bioresource Research Center, RIKEN, Tsukuba, Ibaraki, Japan
- School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Ibaraki, Japan
| | - Dan Song
- iPS Cell Advanced Characterization and Development Team, Bioresource Research Center, RIKEN, Tsukuba, Ibaraki, Japan
| | - Mami Matsuo-Takasaki
- iPS Cell Advanced Characterization and Development Team, Bioresource Research Center, RIKEN, Tsukuba, Ibaraki, Japan
| | - Tadayoshi Hayata
- Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences and Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan
| | - Yohei Hayashi
- iPS Cell Advanced Characterization and Development Team, Bioresource Research Center, RIKEN, Tsukuba, Ibaraki, Japan
- School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Ibaraki, Japan
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Shoji M, Ohashi T, Nagase S, Yuri H, Ichihashi K, Takagishi T, Nagata Y, Nomura Y, Fukunaka A, Kenjou S, Miyake H, Hara T, Yoshigai E, Fujitani Y, Sakurai H, Dos Santos HG, Fukada T, Kuzuhara T. Possible involvement of zinc transporter ZIP13 in myogenic differentiation. Sci Rep 2024; 14:8052. [PMID: 38609428 PMCID: PMC11014994 DOI: 10.1038/s41598-024-56912-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2022] [Accepted: 03/12/2024] [Indexed: 04/14/2024] Open
Abstract
Ehlers-Danlos syndrome spondylodysplastic type 3 (EDSSPD3, OMIM 612350) is an inherited recessive connective tissue disorder that is caused by loss of function of SLC39A13/ZIP13, a zinc transporter belonging to the Slc39a/ZIP family. We previously reported that patients with EDSSPD3 harboring a homozygous loss of function mutation (c.221G > A, p.G64D) in ZIP13 exon 2 (ZIP13G64D) suffer from impaired development of bone and connective tissues, and muscular hypotonia. However, whether ZIP13 participates in the early differentiation of these cell types remains unclear. In the present study, we investigated the role of ZIP13 in myogenic differentiation using a murine myoblast cell line (C2C12) as well as patient-derived induced pluripotent stem cells (iPSCs). We found that ZIP13 gene expression was upregulated by myogenic stimulation in C2C12 cells, and its knockdown disrupted myotubular differentiation. Myocytes differentiated from iPSCs derived from patients with EDSSPD3 (EDSSPD3-iPSCs) also exhibited incomplete myogenic differentiation. Such phenotypic abnormalities of EDSSPD3-iPSC-derived myocytes were corrected by genomic editing of the pathogenic ZIP13G64D mutation. Collectively, our findings suggest the possible involvement of ZIP13 in myogenic differentiation, and that EDSSPD3-iPSCs established herein may be a promising tool to study the molecular basis underlying the clinical features caused by loss of ZIP13 function.
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Affiliation(s)
- Masaki Shoji
- Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan.
| | - Takuto Ohashi
- Laboratory of Molecular and Cellular Physiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan
| | - Saki Nagase
- Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan
| | - Haato Yuri
- Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan
| | - Kenta Ichihashi
- Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan
| | - Teruhisa Takagishi
- Laboratory of Molecular and Cellular Physiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan
| | - Yuji Nagata
- Laboratory of Molecular and Cellular Physiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan
| | - Yuki Nomura
- Laboratory of Molecular and Cellular Physiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan
| | - Ayako Fukunaka
- Laboratory of Developmental Biology and Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi-City, Gunma, Japan
| | - Sae Kenjou
- Laboratory of Molecular and Cellular Physiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan
| | - Hatsuna Miyake
- Laboratory of Molecular and Cellular Physiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan
| | - Takafumi Hara
- Laboratory of Molecular and Cellular Physiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan
| | - Emi Yoshigai
- Laboratory of Molecular and Cellular Physiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan
| | - Yoshio Fujitani
- Laboratory of Developmental Biology and Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi-City, Gunma, Japan
| | - Hidetoshi Sakurai
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto-City, Kyoto, Japan
| | | | - Toshiyuki Fukada
- Laboratory of Molecular and Cellular Physiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan.
| | - Takashi Kuzuhara
- Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamahouji, Yamashirocho, Tokushima-City, Tokushima, 770-8514, Japan.
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9
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Bou Akar R, Lama C, Aubin D, Maruotti J, Onteniente B, Esteves de Lima J, Relaix F. Generation of highly pure pluripotent stem cell-derived myogenic progenitor cells and myotubes. Stem Cell Reports 2024; 19:84-99. [PMID: 38101399 PMCID: PMC10828960 DOI: 10.1016/j.stemcr.2023.11.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2023] [Revised: 11/14/2023] [Accepted: 11/15/2023] [Indexed: 12/17/2023] Open
Abstract
Driving efficient and pure skeletal muscle cell differentiation from pluripotent stem cells (PSCs) has been challenging. Here, we report an optimized protocol that generates skeletal muscle progenitor cells with high efficiency and purity in a short period of time. Human induced PSCs (hiPSCs) and murine embryonic stem cells (mESCs) were specified into the mesodermal myogenic fate using distinct and species-specific protocols. We used a specific maturation medium to promote the terminal differentiation of both human and mouse myoblast populations, and generated myotubes associated with a large pool of cell-cycle arrested PAX7+ cells. We also show that myotube maturation is modulated by dish-coating properties, cell density, and percentage of myogenic progenitor cells. Given the high efficiency in the generation of myogenic progenitors and differentiated myofibers, this protocol provides an attractive strategy for tissue engineering, modeling of muscle dystrophies, and evaluation of new therapeutic approaches in vitro.
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Affiliation(s)
- Reem Bou Akar
- University Paris Est Creteil, INSERM, EnvA, EFS, AP-HP, IMRB, 94010 Creteil, France
| | - Chéryane Lama
- University Paris Est Creteil, INSERM, EnvA, EFS, AP-HP, IMRB, 94010 Creteil, France
| | | | | | | | | | - Frédéric Relaix
- University Paris Est Creteil, INSERM, EnvA, EFS, AP-HP, IMRB, 94010 Creteil, France.
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10
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Ito Y, Yamagata M, Yamamoto T, Hirasaka K, Nikawa T, Sato T. The reciprocal regulation between mitochondrial-associated membranes and Notch signaling in skeletal muscle atrophy. eLife 2023; 12:RP89381. [PMID: 38099641 PMCID: PMC10723794 DOI: 10.7554/elife.89381] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2023] Open
Abstract
Skeletal muscle atrophy and the inhibition of muscle regeneration are known to occur as a natural consequence of aging, yet the underlying mechanisms that lead to these processes in atrophic myofibers remain largely unclear. Our research has revealed that the maintenance of proper mitochondrial-associated endoplasmic reticulum membranes (MAM) is vital for preventing skeletal muscle atrophy in microgravity environments. We discovered that the deletion of the mitochondrial fusion protein Mitofusin2 (MFN2), which serves as a tether for MAM, in human induced pluripotent stem (iPS) cells or the reduction of MAM in differentiated myotubes caused by microgravity interfered with myogenic differentiation process and an increased susceptibility to muscle atrophy, as well as the activation of the Notch signaling pathway. The atrophic phenotype of differentiated myotubes in microgravity and the regenerative capacity of Mfn2-deficient muscle stem cells in dystrophic mice were both ameliorated by treatment with the gamma-secretase inhibitor DAPT. Our findings demonstrate how the orchestration of mitochondrial morphology in differentiated myotubes and regenerating muscle stem cells plays a crucial role in regulating Notch signaling through the interaction of MAM.
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Affiliation(s)
- Yurika Ito
- Faculty of Medical Sciences, Fujita Health UniversityToyoakeJapan
| | - Mari Yamagata
- Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha UniversityKyotanabeJapan
| | - Takuya Yamamoto
- Center for iPS Cell Research and Application, Kyoto UniversityKyotoJapan
- Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto UniversityKyotoJapan
- Medical-risk Avoidance based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP)KyotoJapan
| | - Katsuya Hirasaka
- Organization for Marine Science and Technology, Nagasaki University Graduate SchoolNagasakiJapan
| | - Takeshi Nikawa
- Department of Nutritional Physiology, Institute of Medical Nutrition, Tokushima University Graduate SchoolTokushimaJapan
| | - Takahiko Sato
- Department of Ophthalmology, Kyoto Prefectural University of MedicineKyotoJapan
- Department of Anatomy, Faculty of Medicine, Fujita Health UniversityToyoakeJapan
- International Center for Cell and Gene Therapy, Fujita Health UniversityToyoakeJapan
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11
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Otomo J, Woltjen K, Sakurai H. Uniform transgene activation in Tet-On systems depends on sustained rtTA expression. iScience 2023; 26:107685. [PMID: 37701566 PMCID: PMC10494183 DOI: 10.1016/j.isci.2023.107685] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2023] [Revised: 06/13/2023] [Accepted: 08/17/2023] [Indexed: 09/14/2023] Open
Abstract
Application of the tetracycline-inducible gene expression system (Tet-On) in human induced pluripotent stem cells (hiPSCs) has become a fundamental transgenic tool owing to its regulatable gene expression. One of the major hurdles in hiPSC application is non-uniform transgene activation. Here, we report that the supplementation of reverse tetracycline transactivator (rtTA) in polyclonal hiPSCs populations can achieve the uniform transgene activation of Tet-On. Furthermore, the choice of antibiotic selection markers connected by an internal ribosomal entry site (IRES) can influence the expression of upstream transgenes. In particular, expression of the rtTA is more uniform in cell populations when linked to puromycin as compared to neomycin, obviating the need for sub-cloning or supplementation of rtTA. Finally, to expand the range of applications, we adopted our findings to tetracycline-inducible MyoD vector (Tet-MyoD). Our Tet-MyoD promises efficient, robust, and reproducible directed myogenic differentiation of hiPSCs.
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Affiliation(s)
- Jun Otomo
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
| | - Knut Woltjen
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
| | - Hidetoshi Sakurai
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
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12
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Jara TC, Park K, Vahmani P, Van Eenennaam AL, Smith LR, Denicol AC. Stem cell-based strategies and challenges for production of cultivated meat. NATURE FOOD 2023; 4:841-853. [PMID: 37845547 DOI: 10.1038/s43016-023-00857-z] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/01/2023] [Accepted: 09/05/2023] [Indexed: 10/18/2023]
Abstract
Cultivated meat scale-up and industrial production will require multiple stable cell lines from different species to recreate the organoleptic and nutritional properties of meat from livestock. In this Review, we explore the potential of stem cells to create the major cellular components of cultivated meat. By using developments in the fields of tissue engineering and biomedicine, we explore the advantages and disadvantages of strategies involving primary adult and pluripotent stem cells for generating cell sources that can be grown at scale. These myogenic, adipogenic or extracellular matrix-producing adult stem cells as well as embryonic or inducible pluripotent stem cells are discussed for their proliferative and differentiation capacity, necessary for cultivated meat. We examine the challenges for industrial scale-up, including differentiation and culture protocols, as well as genetic modification options for stem cell immortalization and controlled differentiation. Finally, we discuss stem cell-related safety and regulatory challenges for bringing cultivated meat to the marketplace.
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Affiliation(s)
- T C Jara
- Department of Animal Science, University of California Davis, Davis, CA, USA
| | - K Park
- Department of Animal Science, University of California Davis, Davis, CA, USA
| | - P Vahmani
- Department of Animal Science, University of California Davis, Davis, CA, USA
| | - A L Van Eenennaam
- Department of Animal Science, University of California Davis, Davis, CA, USA
| | - L R Smith
- Department of Neurobiology, Physiology and Behavior, University of California Davis, Davis, CA, USA.
| | - A C Denicol
- Department of Animal Science, University of California Davis, Davis, CA, USA
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13
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Rashid MI, Ito T, Miya F, Shimojo D, Arimoto K, Onodera K, Okada R, Nagashima T, Yamamoto K, Khatun Z, Shimul RI, Niwa JI, Katsuno M, Sobue G, Okano H, Sakurai H, Shimizu K, Doyu M, Okada Y. Simple and efficient differentiation of human iPSCs into contractible skeletal muscles for muscular disease modeling. Sci Rep 2023; 13:8146. [PMID: 37231024 DOI: 10.1038/s41598-023-34445-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2022] [Accepted: 04/30/2023] [Indexed: 05/27/2023] Open
Abstract
Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patient pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) differentiated into affected cell types can potentially recapitulate disease pathology more accurately than existing disease models. Such successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection, and clonal variations must be overcome. Moreover, their functionality should be carefully examined. Here, we demonstrated that bulk MYOD1-hiPSCs established with puromycin selection rather than G418 selection showed rapid and highly efficient differentiation. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties of clonally established MYOD1-hiPSCs, suggesting that it is possible to minimize clonal variations. Moreover, disease-specific hiPSCs of spinal bulbar muscular atrophy (SBMA) could be efficiently differentiated via this method into skeletal muscle that showed disease phenotypes, suggesting the applicability of this method for disease analysis. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical stimulation, indicating their functionality. Thus, our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and may facilitate the generation of muscular disease models.
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Affiliation(s)
- Muhammad Irfanur Rashid
- Department of Neural iPSC Research, Institute for Medical Science of Aging, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
- Department of Neurology, Aichi Medical University School of Medicine, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
| | - Takuji Ito
- Department of Neural iPSC Research, Institute for Medical Science of Aging, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
- Department of Neurology, Aichi Medical University School of Medicine, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
- Japan Society for the Promotion of Science, 5-3-1 Kojimachi, Chiyoda-ku, Tokyo, 102-0083, Japan
| | - Fuyuki Miya
- Center for Medical Genetics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Daisuke Shimojo
- Department of Neurology, Aichi Medical University School of Medicine, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Kanae Arimoto
- Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8603, Japan
| | - Kazunari Onodera
- Department of Neural iPSC Research, Institute for Medical Science of Aging, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
- Department of Neurology, Aichi Medical University School of Medicine, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
- Department of Neurology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Aichi, 466-8650, Japan
| | - Rina Okada
- Department of Neural iPSC Research, Institute for Medical Science of Aging, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
- Department of Neurology, Aichi Medical University School of Medicine, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
- Japan Society for the Promotion of Science, 5-3-1 Kojimachi, Chiyoda-ku, Tokyo, 102-0083, Japan
| | - Takunori Nagashima
- Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8603, Japan
| | - Kazuki Yamamoto
- Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8603, Japan
| | - Zohora Khatun
- Department of Neural iPSC Research, Institute for Medical Science of Aging, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
- Department of Neurology, Aichi Medical University School of Medicine, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
| | - Rayhanul Islam Shimul
- Department of Neural iPSC Research, Institute for Medical Science of Aging, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
| | - Jun-Ichi Niwa
- Department of Neurology, Aichi Medical University School of Medicine, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
| | - Masahisa Katsuno
- Department of Neurology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Aichi, 466-8650, Japan
- Department of Clinical Research Education, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Aichi, 466-8650, Japan
| | - Gen Sobue
- Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
| | - Hideyuki Okano
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Hidetoshi Sakurai
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Kazunori Shimizu
- Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8603, Japan
| | - Manabu Doyu
- Department of Neurology, Aichi Medical University School of Medicine, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan
| | - Yohei Okada
- Department of Neural iPSC Research, Institute for Medical Science of Aging, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan.
- Department of Neurology, Aichi Medical University School of Medicine, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan.
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14
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Rossiaud L, Fragner P, Barbon E, Gardin A, Benabides M, Pellier E, Cosette J, El Kassar L, Giraud-Triboult K, Nissan X, Ronzitti G, Hoch L. Pathological modeling of glycogen storage disease type III with CRISPR/Cas9 edited human pluripotent stem cells. Front Cell Dev Biol 2023; 11:1163427. [PMID: 37250895 PMCID: PMC10213880 DOI: 10.3389/fcell.2023.1163427] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Accepted: 05/02/2023] [Indexed: 05/31/2023] Open
Abstract
Introduction: Glycogen storage disease type III (GSDIII) is a rare genetic disease caused by mutations in the AGL gene encoding the glycogen debranching enzyme (GDE). The deficiency of this enzyme, involved in cytosolic glycogen degradation, leads to pathological glycogen accumulation in liver, skeletal muscles and heart. Although the disease manifests with hypoglycemia and liver metabolism impairment, the progressive myopathy is the major disease burden in adult GSDIII patients, without any curative treatment currently available. Methods: Here, we combined the self-renewal and differentiation capabilities of human induced pluripotent stem cells (hiPSCs) with cutting edge CRISPR/Cas9 gene editing technology to establish a stable AGL knockout cell line and to explore glycogen metabolism in GSDIII. Results: Following skeletal muscle cells differentiation of the edited and control hiPSC lines, our study reports that the insertion of a frameshift mutation in AGL gene results in the loss of GDE expression and persistent glycogen accumulation under glucose starvation conditions. Phenotypically, we demonstrated that the edited skeletal muscle cells faithfully recapitulate the phenotype of differentiated skeletal muscle cells of hiPSCs derived from a GSDIII patient. We also demonstrated that treatment with recombinant AAV vectors expressing the human GDE cleared the accumulated glycogen. Discussion: This study describes the first skeletal muscle cell model of GSDIII derived from hiPSCs and establishes a platform to study the mechanisms that contribute to muscle impairments in GSDIII and to assess the therapeutic potential of pharmacological inducers of glycogen degradation or gene therapy approaches.
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Affiliation(s)
- Lucille Rossiaud
- CECS, I-Stem, Corbeil-Essonnes, France
- INSERM U861, I-Stem, Corbeil-Essonnes, France
- UEVE U861, I-Stem, Corbeil-Essonnes, France
- Genethon, Evry, France
- Université Paris-Saclay, Univ Evry, Inserm, Genethon, Integrare Research Unit UMR_S951, Evry, France
| | - Pascal Fragner
- CECS, I-Stem, Corbeil-Essonnes, France
- INSERM U861, I-Stem, Corbeil-Essonnes, France
- UEVE U861, I-Stem, Corbeil-Essonnes, France
| | - Elena Barbon
- Genethon, Evry, France
- Université Paris-Saclay, Univ Evry, Inserm, Genethon, Integrare Research Unit UMR_S951, Evry, France
| | - Antoine Gardin
- Genethon, Evry, France
- Université Paris-Saclay, Univ Evry, Inserm, Genethon, Integrare Research Unit UMR_S951, Evry, France
| | - Manon Benabides
- CECS, I-Stem, Corbeil-Essonnes, France
- INSERM U861, I-Stem, Corbeil-Essonnes, France
- UEVE U861, I-Stem, Corbeil-Essonnes, France
| | - Emilie Pellier
- CECS, I-Stem, Corbeil-Essonnes, France
- INSERM U861, I-Stem, Corbeil-Essonnes, France
- UEVE U861, I-Stem, Corbeil-Essonnes, France
| | | | - Lina El Kassar
- CECS, I-Stem, Corbeil-Essonnes, France
- INSERM U861, I-Stem, Corbeil-Essonnes, France
- UEVE U861, I-Stem, Corbeil-Essonnes, France
| | - Karine Giraud-Triboult
- CECS, I-Stem, Corbeil-Essonnes, France
- INSERM U861, I-Stem, Corbeil-Essonnes, France
- UEVE U861, I-Stem, Corbeil-Essonnes, France
| | - Xavier Nissan
- CECS, I-Stem, Corbeil-Essonnes, France
- INSERM U861, I-Stem, Corbeil-Essonnes, France
- UEVE U861, I-Stem, Corbeil-Essonnes, France
| | - Giuseppe Ronzitti
- Genethon, Evry, France
- Université Paris-Saclay, Univ Evry, Inserm, Genethon, Integrare Research Unit UMR_S951, Evry, France
| | - Lucile Hoch
- CECS, I-Stem, Corbeil-Essonnes, France
- INSERM U861, I-Stem, Corbeil-Essonnes, France
- UEVE U861, I-Stem, Corbeil-Essonnes, France
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15
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Ikenaka A, Kitagawa Y, Yoshida M, Lin CY, Niwa A, Nakahata T, Saito MK. SMN promotes mitochondrial metabolic maturation during myogenesis by regulating the MYOD-miRNA axis. Life Sci Alliance 2023; 6:e202201457. [PMID: 36604149 PMCID: PMC9834662 DOI: 10.26508/lsa.202201457] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2022] [Revised: 12/12/2022] [Accepted: 12/13/2022] [Indexed: 01/07/2023] Open
Abstract
Spinal muscular atrophy (SMA) is a congenital neuromuscular disease caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene. Although the primary cause of progressive muscle atrophy in SMA has classically been considered the degeneration of motor neurons, recent studies have indicated a skeletal muscle-specific pathological phenotype such as impaired mitochondrial function and enhanced cell death. Here, we found that the down-regulation of SMN causes mitochondrial dysfunction and subsequent cell death in in vitro models of skeletal myogenesis with both a murine C2C12 cell line and human induced pluripotent stem cells. During myogenesis, SMN binds to the upstream genomic regions of MYOD1 and microRNA (miR)-1 and miR-206. Accordingly, the loss of SMN down-regulates these miRs, whereas supplementation of the miRs recovers the mitochondrial function, cell survival, and myotube formation of SMN-deficient C2C12, indicating the SMN-miR axis is essential for myogenic metabolic maturation. In addition, the introduction of the miRs into ex vivo muscle stem cells derived from Δ7-SMA mice caused myotube formation and muscle contraction. In conclusion, our data revealed novel transcriptional roles of SMN during myogenesis, providing an alternative muscle-oriented therapeutic strategy for SMA patients.
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Affiliation(s)
- Akihiro Ikenaka
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Yohko Kitagawa
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Michiko Yoshida
- Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Chuang-Yu Lin
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
- Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - Akira Niwa
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Tatsutoshi Nakahata
- Drug Discovery Technology Development Office, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Megumu K Saito
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
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16
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Bomkamp C, Musgrove L, Marques DMC, Fernando GF, Ferreira FC, Specht EA. Differentiation and Maturation of Muscle and Fat Cells in Cultivated Seafood: Lessons from Developmental Biology. MARINE BIOTECHNOLOGY (NEW YORK, N.Y.) 2023; 25:1-29. [PMID: 36374393 PMCID: PMC9931865 DOI: 10.1007/s10126-022-10174-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/25/2022] [Accepted: 10/10/2022] [Indexed: 06/16/2023]
Abstract
Cultivated meat, also known as cultured or cell-based meat, is meat produced directly from cultured animal cells rather than from a whole animal. Cultivated meat and seafood have been proposed as a means of mitigating the substantial harms associated with current production methods, including damage to the environment, antibiotic resistance, food security challenges, poor animal welfare, and-in the case of seafood-overfishing and ecological damage associated with fishing and aquaculture. Because biomedical tissue engineering research, from which cultivated meat draws a great deal of inspiration, has thus far been conducted almost exclusively in mammals, cultivated seafood suffers from a lack of established protocols for producing complex tissues in vitro. At the same time, fish such as the zebrafish Danio rerio have been widely used as model organisms in developmental biology. Therefore, many of the mechanisms and signaling pathways involved in the formation of muscle, fat, and other relevant tissue are relatively well understood for this species. The same processes are understood to a lesser degree in aquatic invertebrates. This review discusses the differentiation and maturation of meat-relevant cell types in aquatic species and makes recommendations for future research aimed at recapitulating these processes to produce cultivated fish and shellfish.
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Affiliation(s)
- Claire Bomkamp
- Department of Science & Technology, The Good Food Institute, Washington, DC USA
| | - Lisa Musgrove
- University of the Sunshine Coast, Sippy Downs, Queensland Australia
| | - Diana M. C. Marques
- Department of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal
- Associate Laboratory i4HB—Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal
| | - Gonçalo F. Fernando
- Department of Science & Technology, The Good Food Institute, Washington, DC USA
| | - Frederico C. Ferreira
- Department of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal
- Associate Laboratory i4HB—Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal
| | - Elizabeth A. Specht
- Department of Science & Technology, The Good Food Institute, Washington, DC USA
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17
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Kawada R, Jonouchi T, Kagita A, Sato M, Hotta A, Sakurai H. Establishment of quantitative and consistent in vitro skeletal muscle pathological models of myotonic dystrophy type 1 using patient-derived iPSCs. Sci Rep 2023; 13:94. [PMID: 36631509 PMCID: PMC9834395 DOI: 10.1038/s41598-022-26614-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2022] [Accepted: 12/16/2022] [Indexed: 01/13/2023] Open
Abstract
Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats (CTGexp) in the dystrophia myotonica protein kinase (DMPK) gene, and the transcription products, expanded CUG repeats, sequester muscleblind like splicing regulator 1 (MBNL1), resulting in the nuclear MBNL1 aggregation in the DM1 cells. Loss of MBNL1 function is the pivotal mechanism underlying the pathogenesis of DM1. To develop therapeutics for DM1, proper human in vitro models based on the pathologic mechanism of DM1 are required. In this study, we established robust in vitro skeletal muscle cell models of DM1 with patient-derived induced pluripotent stem cells (iPSCs) using the MyoD1-induced system and iPSCs-derived muscle stem cell (iMuSC) differentiation system. Our newly established DM1 models enable simple quantitative evaluation of nuclear MBNL1 aggregation and the downstream splicing defects. Quantitative analyses using the MyoD1-induced myotubes showed that CTGexp-deleted DM1 skeletal myotubes exhibited a reversal of MBNL1-related pathologies, and antisense oligonucleotide treatment recovered these disease phenotypes in the DM1-iPSCs-derived myotubes. Furthermore, iMuSC-derived myotubes exhibited higher maturity than the MyoD1-induced myotubes, which enabled us to recapitulate the SERCA1 splicing defect in the DM1-iMuSC-derived myotubes. Our quantitative and reproducible in vitro models for DM1 established using human iPSCs are promising for drug discovery against DM1.
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Affiliation(s)
- Ryu Kawada
- grid.258799.80000 0004 0372 2033Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507 Japan ,grid.419836.10000 0001 2162 3360Discovery Research Laboratories, Taisho Pharmaceutical Co., Ltd., Saitama, 331-9530 Japan
| | - Tatsuya Jonouchi
- grid.258799.80000 0004 0372 2033Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507 Japan
| | - Akihiro Kagita
- grid.258799.80000 0004 0372 2033Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507 Japan
| | - Masae Sato
- grid.258799.80000 0004 0372 2033Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507 Japan
| | - Akitsu Hotta
- grid.258799.80000 0004 0372 2033Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507 Japan
| | - Hidetoshi Sakurai
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan.
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18
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Joung J, Ma S, Tay T, Geiger-Schuller KR, Kirchgatterer PC, Verdine VK, Guo B, Arias-Garcia MA, Allen WE, Singh A, Kuksenko O, Abudayyeh OO, Gootenberg JS, Fu Z, Macrae RK, Buenrostro JD, Regev A, Zhang F. A transcription factor atlas of directed differentiation. Cell 2023; 186:209-229.e26. [PMID: 36608654 PMCID: PMC10344468 DOI: 10.1016/j.cell.2022.11.026] [Citation(s) in RCA: 93] [Impact Index Per Article: 46.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Revised: 08/04/2022] [Accepted: 11/23/2022] [Indexed: 01/07/2023]
Abstract
Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.
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Affiliation(s)
- Julia Joung
- Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA
| | - Sai Ma
- Department of Biology, MIT, Cambridge, MA 02139, USA; Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Tristan Tay
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Kathryn R Geiger-Schuller
- Department of Biology, MIT, Cambridge, MA 02139, USA; Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Paul C Kirchgatterer
- Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA
| | - Vanessa K Verdine
- Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA
| | - Baolin Guo
- McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Mario A Arias-Garcia
- McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - William E Allen
- Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA; Society of Fellows, Harvard University, Cambridge, MA, USA
| | - Ankita Singh
- Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA
| | - Olena Kuksenko
- Department of Biology, MIT, Cambridge, MA 02139, USA; Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Omar O Abudayyeh
- Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA
| | - Jonathan S Gootenberg
- Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA
| | - Zhanyan Fu
- McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Rhiannon K Macrae
- Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA
| | - Jason D Buenrostro
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Aviv Regev
- Department of Biology, MIT, Cambridge, MA 02139, USA; Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Feng Zhang
- Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA.
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19
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Caron L, Testa S, Magdinier F. Induced Pluripotent Stem Cells for Modeling Physiological and Pathological Striated Muscle Complexity. J Neuromuscul Dis 2023; 10:761-776. [PMID: 37522215 PMCID: PMC10578229 DOI: 10.3233/jnd-230076] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/13/2023] [Indexed: 08/01/2023]
Abstract
Neuromuscular disorders (NMDs) are a large group of diseases associated with either alterations of skeletal muscle fibers, motor neurons or neuromuscular junctions. Most of these diseases is characterized with muscle weakness or wasting and greatly alter the life of patients. Animal models do not always recapitulate the phenotype of patients. The development of innovative and representative human preclinical models is thus strongly needed for modeling the wide diversity of NMDs, characterization of disease-associated variants, investigation of novel genes function, or the development of therapies. Over the last decade, the use of patient's derived induced pluripotent stem cells (hiPSC) has resulted in tremendous progress in biomedical research, including for NMDs. Skeletal muscle is a complex tissue with multinucleated muscle fibers supported by a dense extracellular matrix and multiple cell types including motor neurons required for the contractile activity. Major challenges need now to be tackled by the scientific community to increase maturation of muscle fibers in vitro, in particular for modeling adult-onset diseases affecting this tissue (neuromuscular disorders, cachexia, sarcopenia) and the evaluation of therapeutic strategies. In the near future, rapidly evolving bioengineering approaches applied to hiPSC will undoubtedly become highly instrumental for investigating muscle pathophysiology and the development of therapeutic strategies.
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Affiliation(s)
- Leslie Caron
- Aix-Marseille Univ-INSERM, MMG, Marseille, France
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20
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Oceguera-Yanez F, Avila-Robinson A, Woltjen K. Differentiation of pluripotent stem cells for modeling human skin development and potential applications. Front Cell Dev Biol 2022; 10:1030339. [PMID: 36506084 PMCID: PMC9728031 DOI: 10.3389/fcell.2022.1030339] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2022] [Accepted: 11/04/2022] [Indexed: 11/25/2022] Open
Abstract
The skin of mammals is a multilayered and multicellular tissue that forms an environmental barrier with key functions in protection, regulation, and sensation. While animal models have long served to study the basic functions of the skin in vivo, new insights are expected from in vitro models of human skin development. Human pluripotent stem cells (PSCs) have proven to be invaluable tools for studying human development in vitro. To understand the mechanisms regulating human skin homeostasis and injury repair at the molecular level, recent efforts aim to differentiate PSCs towards skin epidermal keratinocytes, dermal fibroblasts, and skin appendages such as hair follicles and sebaceous glands. Here, we present an overview of the literature describing strategies for human PSC differentiation towards the components of skin, with a particular focus on keratinocytes. We highlight fundamental advances in the field employing patient-derived human induced PSCs (iPSCs) and skin organoid generation. Importantly, PSCs allow researchers to model inherited skin diseases in the search for potential treatments. Skin differentiation from human PSCs holds the potential to clarify human skin biology.
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Affiliation(s)
- Fabian Oceguera-Yanez
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan,*Correspondence: Fabian Oceguera-Yanez, ; Knut Woltjen,
| | | | - Knut Woltjen
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan,*Correspondence: Fabian Oceguera-Yanez, ; Knut Woltjen,
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21
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Sanchez MM, Bagdasarian IA, Darch W, Morgan JT. Organotypic cultures as aging associated disease models. Aging (Albany NY) 2022; 14:9338-9383. [PMID: 36435511 PMCID: PMC9740367 DOI: 10.18632/aging.204361] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2022] [Accepted: 10/21/2022] [Indexed: 11/24/2022]
Abstract
Aging remains a primary risk factor for a host of diseases, including leading causes of death. Aging and associated diseases are inherently multifactorial, with numerous contributing factors and phenotypes at the molecular, cellular, tissue, and organismal scales. Despite the complexity of aging phenomena, models currently used in aging research possess limitations. Frequently used in vivo models often have important physiological differences, age at different rates, or are genetically engineered to match late disease phenotypes rather than early causes. Conversely, routinely used in vitro models lack the complex tissue-scale and systemic cues that are disrupted in aging. To fill in gaps between in vivo and traditional in vitro models, researchers have increasingly been turning to organotypic models, which provide increased physiological relevance with the accessibility and control of in vitro context. While powerful tools, the development of these models is a field of its own, and many aging researchers may be unaware of recent progress in organotypic models, or hesitant to include these models in their own work. In this review, we describe recent progress in tissue engineering applied to organotypic models, highlighting examples explicitly linked to aging and associated disease, as well as examples of models that are relevant to aging. We specifically highlight progress made in skin, gut, and skeletal muscle, and describe how recently demonstrated models have been used for aging studies or similar phenotypes. Throughout, this review emphasizes the accessibility of these models and aims to provide a resource for researchers seeking to leverage these powerful tools.
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Affiliation(s)
- Martina M. Sanchez
- Department of Bioengineering, University of California, Riverside, CA 92521, USA
| | | | - William Darch
- Department of Bioengineering, University of California, Riverside, CA 92521, USA
| | - Joshua T. Morgan
- Department of Bioengineering, University of California, Riverside, CA 92521, USA
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22
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Kamiya D, Takenaka-Ninagawa N, Motoike S, Kajiya M, Akaboshi T, Zhao C, Shibata M, Senda S, Toyooka Y, Sakurai H, Kurihara H, Ikeya M. Induction of functional xeno-free MSCs from human iPSCs via a neural crest cell lineage. NPJ Regen Med 2022; 7:47. [PMID: 36109564 PMCID: PMC9477888 DOI: 10.1038/s41536-022-00241-8] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Accepted: 08/08/2022] [Indexed: 11/09/2022] Open
Abstract
AbstractMesenchymal stem/stromal cells (MSCs) are adult multipotent stem cells. Here, we induced MSCs from human induced pluripotent stem cells (iPSCs) via a neural crest cell (NCC) lineage under xeno-free conditions and evaluated their in vivo functions. We modified a previous MSC induction method to work under xeno-free conditions. Bovine serum albumin-containing NCC induction medium and fetal bovine serum-containing MSC induction medium were replaced with xeno-free medium. Through our optimized method, iPSCs differentiated into MSCs with high efficiency. To evaluate their in vivo activities, we transplanted the xeno-free-induced MSCs (XF-iMSCs) into mouse models for bone and skeletal muscle regeneration and confirmed their regenerative potency. These XF-iMSCs mainly promoted the regeneration of surrounding host cells, suggesting that they secrete soluble factors into affected regions. We also found that the peroxidasin and IGF2 secreted by the XF-iMSCs partially contributed to myotube differentiation. These results suggest that XF-iMSCs are important for future applications in regenerative medicine.
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23
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Carraro E, Rossi L, Maghin E, Canton M, Piccoli M. 3D in vitro Models of Pathological Skeletal Muscle: Which Cells and Scaffolds to Elect? Front Bioeng Biotechnol 2022; 10:941623. [PMID: 35898644 PMCID: PMC9313593 DOI: 10.3389/fbioe.2022.941623] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2022] [Accepted: 06/21/2022] [Indexed: 12/29/2022] Open
Abstract
Skeletal muscle is a fundamental tissue of the human body with great plasticity and adaptation to diseases and injuries. Recreating this tissue in vitro helps not only to deepen its functionality, but also to simulate pathophysiological processes. In this review we discuss the generation of human skeletal muscle three-dimensional (3D) models obtained through tissue engineering approaches. First, we present an overview of the most severe myopathies and the two key players involved: the variety of cells composing skeletal muscle tissue and the different components of its extracellular matrix. Then, we discuss the peculiar characteristics among diverse in vitro models with a specific focus on cell sources, scaffold composition and formulations, and fabrication techniques. To conclude, we highlight the efficacy of 3D models in mimicking patient-specific myopathies, deepening muscle disease mechanisms or investigating possible therapeutic effects.
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Affiliation(s)
- Eugenia Carraro
- Istituto di Ricerca Pediatrica Città della Speranza, Padova, Italy
- Department of Biomedical Sciences, University of Padova, Padova, Italy
| | - Lucia Rossi
- Istituto di Ricerca Pediatrica Città della Speranza, Padova, Italy
| | - Edoardo Maghin
- Istituto di Ricerca Pediatrica Città della Speranza, Padova, Italy
| | - Marcella Canton
- Istituto di Ricerca Pediatrica Città della Speranza, Padova, Italy
- Department of Biomedical Sciences, University of Padova, Padova, Italy
| | - Martina Piccoli
- Istituto di Ricerca Pediatrica Città della Speranza, Padova, Italy
- *Correspondence: Martina Piccoli,
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24
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Generation of human myogenic progenitors from pluripotent stem cells for in vivo regeneration. Cell Mol Life Sci 2022; 79:406. [PMID: 35802202 PMCID: PMC9270264 DOI: 10.1007/s00018-022-04434-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2022] [Revised: 05/31/2022] [Accepted: 06/15/2022] [Indexed: 11/29/2022]
Abstract
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.
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25
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Fujiwara K, Yamamoto R, Kubota T, Tazumi A, Sabuta T, Takahashi MP, Sakurai H. Mature Myotubes Generated From Human-Induced Pluripotent Stem Cells Without Forced Gene Expression. Front Cell Dev Biol 2022; 10:886879. [PMID: 35706901 PMCID: PMC9189389 DOI: 10.3389/fcell.2022.886879] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Accepted: 04/11/2022] [Indexed: 11/17/2022] Open
Abstract
Human-induced pluripotent stem cells (hiPSCs) are a promising tool for disease modeling and drug screening. To apply them to skeletal muscle disorders, it is necessary to establish mature myotubes because the onset of many skeletal muscle disorders is after birth. However, to make mature myotubes, the forced expression of specific genes should be avoided, as otherwise dysregulation of the intracellular networks may occur. Here, we achieved this goal by purifying hiPSC-derived muscle stem cells (iMuSC) by Pax7-fluorescence monitoring and antibody sorting. The resulting myotubes displayed spontaneous self-contraction, aligned sarcomeres, and a triad structure. Notably, the phenotype of sodium channels was changed to the mature type in the course of the differentiation, and a characteristic current pattern was observed. Moreover, the protocol resulted in highly efficient differentiation and high homogeneity and is applicable to drug screening.
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Affiliation(s)
- Kei Fujiwara
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Risa Yamamoto
- Clinical Neurophysiology, Department of Clinical Laboratory and Biomedical Sciences, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Tomoya Kubota
- Clinical Neurophysiology, Department of Clinical Laboratory and Biomedical Sciences, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Atsutoshi Tazumi
- Laboratory for Pharmacology, Pharmaceutical Research Center, Asahi Kasei Pharma Corporation, Shizuoka, Japan
| | - Tomoka Sabuta
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Masanori P Takahashi
- Clinical Neurophysiology, Department of Clinical Laboratory and Biomedical Sciences, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Hidetoshi Sakurai
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
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26
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Nalbandian M, Zhao M, Kato H, Jonouchi T, Nakajima-Koyama M, Yamamoto T, Sakurai H. Single-cell RNA-seq reveals heterogeneity in hiPSC-derived muscle progenitors and E2F family as a key regulator of proliferation. Life Sci Alliance 2022; 5:5/8/e202101312. [PMID: 35459735 PMCID: PMC9034463 DOI: 10.26508/lsa.202101312] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2021] [Revised: 03/28/2022] [Accepted: 03/30/2022] [Indexed: 11/25/2022] Open
Abstract
This study identified and characterized four different populations of muscle progenitor cells derived from human induced pluripotent stem cells. Human pluripotent stem cell-derived muscle progenitor cells (hiPSC-MuPCs) resemble fetal-stage muscle progenitor cells and possess in vivo regeneration capacity. However, the heterogeneity of hiPSC-MuPCs is unknown, which could impact the regenerative potential of these cells. Here, we established an hiPSC-MuPC atlas by performing single-cell RNA sequencing of hiPSC-MuPC cultures. Bioinformatic analysis revealed four cell clusters for hiPSC-MuPCs: myocytes, committed, cycling, and noncycling progenitors. Using FGFR4 as a marker for noncycling progenitors and cycling cells and CD36 as a marker for committed and myocyte cells, we found that FGFR4+ cells possess a higher regenerative capacity than CD36+ cells. We also identified the family of E2F transcription factors are key regulators of hiPSC-MuPC proliferation. Our study provides insights on the purification of hiPSC-MuPCs with higher regenerative potential and increases the understanding of the transcriptional regulation of hiPSC-MuPCs.
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Affiliation(s)
- Minas Nalbandian
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Mingming Zhao
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Hiroki Kato
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.,Asahi Kasei Co., Ltd., Tokyo, Japan
| | - Tatsuya Jonouchi
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - May Nakajima-Koyama
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Takuya Yamamoto
- Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.,Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan.,Medical-risk Avoidance Based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan
| | - Hidetoshi Sakurai
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
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27
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Iberite F, Gruppioni E, Ricotti L. Skeletal muscle differentiation of human iPSCs meets bioengineering strategies: perspectives and challenges. NPJ Regen Med 2022; 7:23. [PMID: 35393412 PMCID: PMC8991236 DOI: 10.1038/s41536-022-00216-9] [Citation(s) in RCA: 42] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2021] [Accepted: 03/01/2022] [Indexed: 12/31/2022] Open
Abstract
Although skeletal muscle repairs itself following small injuries, genetic diseases or severe damages may hamper its ability to do so. Induced pluripotent stem cells (iPSCs) can generate myogenic progenitors, but their use in combination with bioengineering strategies to modulate their phenotype has not been sufficiently investigated. This review highlights the potential of this combination aimed at pushing the boundaries of skeletal muscle tissue engineering. First, the overall organization and the key steps in the myogenic process occurring in vivo are described. Second, transgenic and non-transgenic approaches for the myogenic induction of human iPSCs are compared. Third, technologies to provide cells with biophysical stimuli, biomaterial cues, and biofabrication strategies are discussed in terms of recreating a biomimetic environment and thus helping to engineer a myogenic phenotype. The embryonic development process and the pro-myogenic role of the muscle-resident cell populations in co-cultures are also described, highlighting the possible clinical applications of iPSCs in the skeletal muscle tissue engineering field.
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Affiliation(s)
- Federica Iberite
- The BioRobotics Institute, Scuola Superiore Sant'Anna, 56127, Pisa (PI), Italy. .,Department of Excellence in Robotics & AI, Scuola Superiore Sant'Anna, 56127, Pisa (PI), Italy.
| | - Emanuele Gruppioni
- Centro Protesi INAIL, Istituto Nazionale per l'Assicurazione contro gli Infortuni sul Lavoro, 40054, Vigorso di Budrio (BO), Italy
| | - Leonardo Ricotti
- The BioRobotics Institute, Scuola Superiore Sant'Anna, 56127, Pisa (PI), Italy.,Department of Excellence in Robotics & AI, Scuola Superiore Sant'Anna, 56127, Pisa (PI), Italy
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28
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Smith AST, Luttrell SM, Dupont JB, Gray K, Lih D, Fleming JW, Cunningham NJ, Jepson S, Hesson J, Mathieu J, Maves L, Berry BJ, Fisher EC, Sniadecki NJ, Geisse NA, Mack DL. High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing. J Tissue Eng 2022; 13:20417314221122127. [PMID: 36082311 PMCID: PMC9445471 DOI: 10.1177/20417314221122127] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2022] [Accepted: 08/03/2022] [Indexed: 12/03/2022] Open
Abstract
Engineered muscle tissues represent powerful tools for examining tissue level contractile properties of skeletal muscle. However, limitations in the throughput associated with standard analysis methods limit their utility for longitudinal study, high throughput drug screens, and disease modeling. Here we present a method for integrating 3D engineered skeletal muscles with a magnetic sensing system to facilitate non-invasive, longitudinal analysis of developing contraction kinetics. Using this platform, we show that engineered skeletal muscle tissues derived from both induced pluripotent stem cell and primary sources undergo improvements in contractile output over time in culture. We demonstrate how magnetic sensing of contractility can be employed for simultaneous assessment of multiple tissues subjected to different doses of known skeletal muscle inotropes as well as the stratification of healthy versus diseased functional profiles in normal and dystrophic muscle cells. Based on these data, this combined culture system and magnet-based contractility platform greatly broadens the potential for 3D engineered skeletal muscle tissues to impact the translation of novel therapies from the lab to the clinic.
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Affiliation(s)
- Alec ST Smith
- Department of Physiology and Biophysics, University of Washington, Seattle, WA, USA
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA
| | | | - Jean-Baptiste Dupont
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA
- Nantes Université, INSERM, TARGET, Nantes, France
| | - Kevin Gray
- Curi Bio Inc., 3000 Western Avenue, Seattle, WA, USA
| | - Daniel Lih
- Curi Bio Inc., 3000 Western Avenue, Seattle, WA, USA
| | | | | | - Sofia Jepson
- Department of Bioengineering, University of Washington, Seattle, WA, USA
| | - Jennifer Hesson
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA
- Department of Comparative Medicine, University of Washington, Seattle, WA, USA
| | - Julie Mathieu
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA
- Department of Comparative Medicine, University of Washington, Seattle, WA, USA
| | - Lisa Maves
- Seattle Children’s Research Institute, Seattle, WA, USA
| | | | | | - Nathan J Sniadecki
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA
- Department of Bioengineering, University of Washington, Seattle, WA, USA
- Department of Mechanical Engineering, University of Washington, Seattle, WA, USA
| | | | - David L Mack
- Department of Physiology and Biophysics, University of Washington, Seattle, WA, USA
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA
- Department of Bioengineering, University of Washington, Seattle, WA, USA
- Department of Rehabilitation Medicine, University of Washington, Seattle, WA, USA
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29
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Jalal S, Dastidar S, Tedesco FS. Advanced models of human skeletal muscle differentiation, development and disease: Three-dimensional cultures, organoids and beyond. Curr Opin Cell Biol 2021; 73:92-104. [PMID: 34384976 PMCID: PMC8692266 DOI: 10.1016/j.ceb.2021.06.004] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2021] [Accepted: 06/23/2021] [Indexed: 02/08/2023]
Abstract
Advanced in vitro models of human skeletal muscle tissue are increasingly needed to model complex developmental dynamics and disease mechanisms not recapitulated in animal models or in conventional monolayer cell cultures. There has been impressive progress towards creating such models by using tissue engineering approaches to recapitulate a range of physical and biochemical components of native human skeletal muscle tissue. In this review, we discuss recent studies focussed on developing complex in vitro models of human skeletal muscle beyond monolayer cell cultures, involving skeletal myogenic differentiation from human primary myoblasts or pluripotent stem cells, often in the presence of structural scaffolding support. We conclude with our outlook on the future of advanced skeletal muscle three-dimensional cultures (e.g. organoids and biofabrication) to produce physiologically and clinically relevant platforms for disease modelling and therapy development in musculoskeletal and neuromuscular disorders.
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Affiliation(s)
- Salma Jalal
- Department of Cell and Developmental Biology, University College London, WC1E 6DE London, United Kingdom
| | - Sumitava Dastidar
- Department of Cell and Developmental Biology, University College London, WC1E 6DE London, United Kingdom
| | - Francesco Saverio Tedesco
- Department of Cell and Developmental Biology, University College London, WC1E 6DE London, United Kingdom; The Francis Crick Institute, 1 Midland Road, London NW1 1AT, United Kingdom; Dubowitz Neuromuscular Centre, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, United Kingdom; Department of Paediatric Neurology, Great Ormond Street Hospital for Children, WC1N 3JH London, United Kingdom.
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30
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Uchimura T, Sakurai H. Orai1-STIM1 Regulates Increased Ca 2+ Mobilization, Leading to Contractile Duchenne Muscular Dystrophy Phenotypes in Patient-Derived Induced Pluripotent Stem Cells. Biomedicines 2021; 9:biomedicines9111589. [PMID: 34829817 PMCID: PMC8615222 DOI: 10.3390/biomedicines9111589] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2021] [Revised: 10/24/2021] [Accepted: 10/26/2021] [Indexed: 11/21/2022] Open
Abstract
Ca2+ overload is one of the factors leading to Duchenne muscular dystrophy (DMD) pathogenesis. However, the molecular targets of dystrophin deficiency-dependent Ca2+ overload and the correlation between Ca2+ overload and contractile DMD phenotypes in in vitro human models remain largely elusive. In this study, we utilized DMD patient-derived induced pluripotent stem cells (iPSCs) to differentiate myotubes using doxycycline-inducible MyoD overexpression, and searched for a target molecule that mediates dystrophin deficiency-dependent Ca2+ overload using commercially available chemicals and siRNAs. We found that several store-operated Ca2+ channel (SOC) inhibitors effectively prevented Ca2+ overload and identified that STIM1–Orai1 is a molecular target of SOCs. These findings were further confirmed by demonstrating that STIM1–Orai1 inhibitors, CM4620, AnCoA4, and GSK797A, prevented Ca2+ overload in dystrophic myotubes. Finally, we evaluated CM4620, AnCoA4, and GSK7975A activities using a previously reported model recapitulating a muscle fatigue-like decline in contractile performance in DMD. All three chemicals ameliorated the decline in contractile performance, indicating that modulating STIM1–Orai1-mediated Ca2+ overload is effective in rescuing contractile phenotypes. In conclusion, SOCs are major contributors to dystrophin deficiency-dependent Ca2+ overload through STIM1–Orai1 as molecular mediators. Modulating STIM1–Orai1 activity was effective in ameliorating the decline in contractile performance in DMD.
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Affiliation(s)
- Tomoya Uchimura
- Center for iPSC Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
- Takeda-CiRA Joint Program, Fujisawa 251-8555, Japan
- Correspondence: (T.U.); (H.S.)
| | - Hidetoshi Sakurai
- Center for iPSC Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
- Takeda-CiRA Joint Program, Fujisawa 251-8555, Japan
- Correspondence: (T.U.); (H.S.)
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31
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Fralish Z, Lotz EM, Chavez T, Khodabukus A, Bursac N. Neuromuscular Development and Disease: Learning From in vitro and in vivo Models. Front Cell Dev Biol 2021; 9:764732. [PMID: 34778273 PMCID: PMC8579029 DOI: 10.3389/fcell.2021.764732] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2021] [Accepted: 10/06/2021] [Indexed: 01/02/2023] Open
Abstract
The neuromuscular junction (NMJ) is a specialized cholinergic synaptic interface between a motor neuron and a skeletal muscle fiber that translates presynaptic electrical impulses into motor function. NMJ formation and maintenance require tightly regulated signaling and cellular communication among motor neurons, myogenic cells, and Schwann cells. Neuromuscular diseases (NMDs) can result in loss of NMJ function and motor input leading to paralysis or even death. Although small animal models have been instrumental in advancing our understanding of the NMJ structure and function, the complexities of studying this multi-tissue system in vivo and poor clinical outcomes of candidate therapies developed in small animal models has driven the need for in vitro models of functional human NMJ to complement animal studies. In this review, we discuss prevailing models of NMDs and highlight the current progress and ongoing challenges in developing human iPSC-derived (hiPSC) 3D cell culture models of functional NMJs. We first review in vivo development of motor neurons, skeletal muscle, Schwann cells, and the NMJ alongside current methods for directing the differentiation of relevant cell types from hiPSCs. We further compare the efficacy of modeling NMDs in animals and human cell culture systems in the context of five NMDs: amyotrophic lateral sclerosis, myasthenia gravis, Duchenne muscular dystrophy, myotonic dystrophy, and Pompe disease. Finally, we discuss further work necessary for hiPSC-derived NMJ models to function as effective personalized NMD platforms.
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Affiliation(s)
| | | | | | | | - Nenad Bursac
- Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC, United States
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32
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Taniguchi-Ikeda M, Koyanagi-Aoi M, Maruyama T, Takaori T, Hosoya A, Tezuka H, Nagase S, Ishihara T, Kadoshima T, Muguruma K, Ishigaki K, Sakurai H, Mizoguchi A, Novitch BG, Toda T, Watanabe M, Aoi T. Restoration of the defect in radial glial fiber migration and cortical plate organization in a brain organoid model of Fukuyama muscular dystrophy. iScience 2021; 24:103140. [PMID: 34632335 PMCID: PMC8487058 DOI: 10.1016/j.isci.2021.103140] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2021] [Revised: 08/11/2021] [Accepted: 09/14/2021] [Indexed: 01/05/2023] Open
Abstract
Fukuyama congenital muscular dystrophy (FCMD) is a severe, intractable genetic disease that affects the skeletal muscle, eyes, and brain and is attributed to a defect in alpha dystroglycan (αDG) O-mannosyl glycosylation. We previously established disease models of FCMD; however, they did not fully recapitulate the phenotypes observed in human patients. In this study, we generated induced pluripotent stem cells (iPSCs) from a human FCMD patient and differentiated these cells into three-dimensional brain organoids and skeletal muscle. The brain organoids successfully mimicked patient phenotypes not reliably reproduced by existing models, including decreased αDG glycosylation and abnormal radial glial (RG) fiber migration. The basic polycyclic compound Mannan-007 (Mn007) restored αDG glycosylation in the brain and muscle models tested and partially rescued the abnormal RG fiber migration observed in cortical organoids. Therefore, our study underscores the importance of αDG O-mannosyl glycans for normal RG fiber architecture and proper neuronal migration in corticogenesis.
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Affiliation(s)
- Mariko Taniguchi-Ikeda
- Department of Clinical Genetics, Fujita Health University Hospital, 1-98 Dengakugakubo, Kutsukake-chou, Toyoake, Aichi 470-1192, Japan
- Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan
- Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan
| | - Michiyo Koyanagi-Aoi
- Division of Advanced Medical Science, Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Hyogo 650-0017, Japan
- Department of iPS Cell Applications, Graduate School of Medicine, Kobe University, Kobe, Hyogo 650-0017, Japan
- Center for Human Resource Development for Regenerative Medicine, Kobe University Hospital, Kobe, Hyogo 650-0017, Japan
| | - Tatsuo Maruyama
- Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Hyogo 657-8501, Japan
| | - Toru Takaori
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan
| | - Akiko Hosoya
- Division of Advanced Medical Science, Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Hyogo 650-0017, Japan
- Department of iPS Cell Applications, Graduate School of Medicine, Kobe University, Kobe, Hyogo 650-0017, Japan
- Center for Human Resource Development for Regenerative Medicine, Kobe University Hospital, Kobe, Hyogo 650-0017, Japan
| | - Hiroyuki Tezuka
- Department of Cellular Function Analysis, Research Promotion and Support Headquarters, Fujita Health University, Toyoake, Aichi 470-1192, Japan
| | | | - Takuma Ishihara
- Innovative and Clinical Research Promotion Center, Gifu University Hospital, Yanagido, Gifu 501-1194, Japan
| | | | - Keiko Muguruma
- Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan
- Department of iPS Cell Applied Medicine, Graduate School of Medicine, Kansai Medical University, Hirakata, Osaka 573-1010, Japan
| | - Keiko Ishigaki
- Department of Pediatrics, Tokyo Women’s Medical University, School of Medicine, Shinjuku-ku, Tokyo 162-8666, Japan
| | - Hidetoshi Sakurai
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
| | - Akira Mizoguchi
- Department of Personalized Cancer Immunotherapy, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Bennett G. Novitch
- Department of Neurobiology, David Geffen School of Medicine at the University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA, USA
- Intellectual and Developmental Disabilities Research Center, UCLA, Los Angeles, CA 90095, USA
| | - Tatsushi Toda
- Department of Neurology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Momoko Watanabe
- Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, Irvine, CA 92697, USA
- Sue & Bill Gross Stem Cell Research Center, School of Medicine, University of California, Irvine, Irvine, CA 92697, USA
| | - Takashi Aoi
- Division of Advanced Medical Science, Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Hyogo 650-0017, Japan
- Department of iPS Cell Applications, Graduate School of Medicine, Kobe University, Kobe, Hyogo 650-0017, Japan
- Center for Human Resource Development for Regenerative Medicine, Kobe University Hospital, Kobe, Hyogo 650-0017, Japan
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33
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Esteves de Lima J, Relaix F. Master regulators of skeletal muscle lineage development and pluripotent stem cells differentiation. CELL REGENERATION 2021; 10:31. [PMID: 34595600 PMCID: PMC8484369 DOI: 10.1186/s13619-021-00093-5] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/03/2021] [Accepted: 08/24/2021] [Indexed: 12/16/2022]
Abstract
In vertebrates, the skeletal muscles of the body and their associated stem cells originate from muscle progenitor cells, during development. The specification of the muscles of the trunk, head and limbs, relies on the activity of distinct genetic hierarchies. The major regulators of trunk and limb muscle specification are the paired-homeobox transcription factors PAX3 and PAX7. Distinct gene regulatory networks drive the formation of the different muscles of the head. Despite the redeployment of diverse upstream regulators of muscle progenitor differentiation, the commitment towards the myogenic fate requires the expression of the early myogenic regulatory factors MYF5, MRF4, MYOD and the late differentiation marker MYOG. The expression of these genes is activated by muscle progenitors throughout development, in several waves of myogenic differentiation, constituting the embryonic, fetal and postnatal phases of muscle growth. In order to achieve myogenic cell commitment while maintaining an undifferentiated pool of muscle progenitors, several signaling pathways regulate the switch between proliferation and differentiation of myoblasts. The identification of the gene regulatory networks operating during myogenesis is crucial for the development of in vitro protocols to differentiate pluripotent stem cells into myoblasts required for regenerative medicine.
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Affiliation(s)
| | - Frédéric Relaix
- Univ Paris Est Creteil, INSERM, EnvA, EFS, AP-HP, IMRB, 94010, Creteil, France.
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34
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Contractile Activity of Myotubes Derived from Human Induced Pluripotent Stem Cells: A Model of Duchenne Muscular Dystrophy. Cells 2021; 10:cells10102556. [PMID: 34685536 PMCID: PMC8534131 DOI: 10.3390/cells10102556] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2021] [Revised: 09/16/2021] [Accepted: 09/25/2021] [Indexed: 01/10/2023] Open
Abstract
Duchenne muscular dystrophy (DMD) is a genetic disorder that results from deficiency of the dystrophin protein. In recent years, DMD pathological models have been created using induced pluripotent stem (iPS) cells derived from DMD patients. In addition, gene therapy using CRISPR-Cas9 technology to repair the dystrophin gene has been proposed as a new treatment method for DMD. However, it is not known whether the contractile function of myotubes derived from gene-repaired iPS cells can be restored. We therefore investigated the maturation of myotubes in electrical pulse stimulation culture and examined the effect of gene repair by observing the contractile behaviour of myotubes. The contraction activity of myotubes derived from dystrophin-gene repaired iPS cells was improved by electrical pulse stimulation culture. The iPS cell method used in this study for evaluating muscle contractile activity is a useful technique for analysing the mechanism of hereditary muscular disease pathogenesis and for evaluating the efficacy of new drugs and gene therapy.
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35
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Hirota A, AlMusawi S, Nateri AS, Ordóñez-Morán P, Imajo M. Biomaterials for intestinal organoid technology and personalized disease modeling. Acta Biomater 2021; 132:272-287. [PMID: 34023456 DOI: 10.1016/j.actbio.2021.05.010] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 04/08/2021] [Accepted: 05/07/2021] [Indexed: 12/20/2022]
Abstract
Recent advances in intestinal organoid technologies have paved the way for in vitro recapitulation of the homeostatic renewal of adult tissues, tissue or organ morphogenesis during development, and pathogenesis of many disorders. In vitro modelling of individual patient diseases using organoid systems have been considered key in establishing rational design of personalized treatment strategies and in improving therapeutic outcomes. In addition, the transplantation of organoids into diseased tissues represents a novel approach to treat currently incurable diseases. Emerging evidence from intensive studies suggests that organoid systems' development and functional maturation depends on the presence of an extracellular matrix with suitable biophysical properties, where advanced synthetic hydrogels open new avenues for theoretical control of organoid phenotypes and potential applications of organoids in therapeutic purposes. In this review, we discuss the status, applications, challenges and perspectives of intestinal organoid systems emphasising on hydrogels and their properties suitable for intestinal organoid culture. We provide an overview of hydrogels used for intestinal organoid culture and key factors regulating their biological activity. The comparison of different hydrogels would be a theoretical basis for establishing design principles of synthetic niches directing intestinal cell fates and functions. STATEMENT OF SIGNIFICANCE: Intestinal organoid is an in vitro recapitulation of the gut, which self-organizes from intestinal stem cells and maintains many features of the native tissue. Since the development of this technology, intestinal organoid systems have made significant contribution to rapid progress in intestinal biology. Prevailing methodology for organoid culture, however, depends on animal-derived matrices and suffers from variability and potential risk for contamination of pathogens, limiting their therapeutic application. Synthetic scaffold matrices, hydrogels, might provide solutions to these issues and deepen our understanding on how intestinal cells sense and respond to key biophysical properties of the surrounding matrices. This review provides an overview of developing intestinal models and biomaterials, thereby leading to better understanding of current intestinal organoid systems for both biologists and materials scientists.
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Affiliation(s)
- Akira Hirota
- Institute for Chemical Reaction Design and Discovery (WPI-ICReDD), Hokkaido University, N15, W7, Kita-ku, Sapporo 060-8638, Japan
| | - Shaikha AlMusawi
- Cancer Genetic and Stem Cell group, Translational Medical Sciences, School of Medicine, Biodiscovery Institute, Centre for Cancer Sciences, University of Nottingham, NG7 2RD, Nottingham, United Kingdom; Stem Cell biology and Cancer group, Translational Medical Sciences, School of Medicine, Biodiscovery Institute, Centre for Cancer Sciences, University of Nottingham, NG7 2RD, Nottingham, United Kingdom
| | - Abdolrahman S Nateri
- Cancer Genetic and Stem Cell group, Translational Medical Sciences, School of Medicine, Biodiscovery Institute, Centre for Cancer Sciences, University of Nottingham, NG7 2RD, Nottingham, United Kingdom
| | - Paloma Ordóñez-Morán
- Stem Cell biology and Cancer group, Translational Medical Sciences, School of Medicine, Biodiscovery Institute, Centre for Cancer Sciences, University of Nottingham, NG7 2RD, Nottingham, United Kingdom.
| | - Masamichi Imajo
- Institute for Chemical Reaction Design and Discovery (WPI-ICReDD), Hokkaido University, N15, W7, Kita-ku, Sapporo 060-8638, Japan.
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36
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Tan GW, Kondo T, Imamura K, Suga M, Enami T, Nagahashi A, Tsukita K, Inoue I, Kawaguchi J, Shu T, Inoue H. Simple derivation of skeletal muscle from human pluripotent stem cells using temperature-sensitive Sendai virus vector. J Cell Mol Med 2021; 25:9586-9596. [PMID: 34510713 PMCID: PMC8505837 DOI: 10.1111/jcmm.16899] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2021] [Revised: 08/04/2021] [Accepted: 08/23/2021] [Indexed: 01/24/2023] Open
Abstract
Human pluripotent stem cells have the potential to differentiate into various cell types including skeletal muscles (SkM), and they are applied to regenerative medicine or in vitro modelling for intractable diseases. A simple differentiation method is required for SkM cells to accelerate neuromuscular disease studies. Here, we established a simple method to convert human pluripotent stem cells into SkM cells by using temperature‐sensitive Sendai virus (SeV) vector encoding myoblast determination protein 1 (SeV‐Myod1), a myogenic master transcription factor. SeV‐Myod1 treatment converted human embryonic stem cells (ESCs) into SkM cells, which expressed SkM markers including myosin heavy chain (MHC). We then removed the SeV vector by temporal treatment at a high temperature of 38℃, which also accelerated mesodermal differentiation, and found that SkM cells exhibited fibre‐like morphology. Finally, after removal of the residual human ESCs by pluripotent stem cell‐targeting delivery of cytotoxic compound, we generated SkM cells with 80% MHC positivity and responsiveness to electrical stimulation. This simple method for myogenic differentiation was applicable to human‐induced pluripotent stem cells and will be beneficial for investigations of disease mechanisms and drug discovery in the future.
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Affiliation(s)
- Ghee Wan Tan
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Takayuki Kondo
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.,iPSC-based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan.,Medical-risk Avoidance based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan
| | - Keiko Imamura
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.,iPSC-based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan.,Medical-risk Avoidance based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan
| | - Mika Suga
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.,iPSC-based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan
| | - Takako Enami
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.,Medical-risk Avoidance based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan
| | - Ayako Nagahashi
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.,Medical-risk Avoidance based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan
| | - Kayoko Tsukita
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.,iPSC-based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan
| | - Ikuyo Inoue
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.,Medical-risk Avoidance based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan
| | | | | | - Haruhisa Inoue
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.,iPSC-based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan.,Medical-risk Avoidance based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan
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37
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Perspectives on hiPSC-Derived Muscle Cells as Drug Discovery Models for Muscular Dystrophies. Int J Mol Sci 2021; 22:ijms22179630. [PMID: 34502539 PMCID: PMC8431796 DOI: 10.3390/ijms22179630] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2021] [Revised: 08/28/2021] [Accepted: 08/30/2021] [Indexed: 12/29/2022] Open
Abstract
Muscular dystrophies are a heterogeneous group of inherited diseases characterized by the progressive degeneration and weakness of skeletal muscles, leading to disability and, often, premature death. To date, no effective therapies are available to halt or reverse the pathogenic process, and meaningful treatments are urgently needed. From this perspective, it is particularly important to establish reliable in vitro models of human muscle that allow the recapitulation of disease features as well as the screening of genetic and pharmacological therapies. We herein review and discuss advances in the development of in vitro muscle models obtained from human induced pluripotent stem cells, which appear to be capable of reproducing the lack of myofiber proteins as well as other specific pathological hallmarks, such as inflammation, fibrosis, and reduced muscle regenerative potential. In addition, these platforms have been used to assess genetic correction strategies such as gene silencing, gene transfer and genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as well as to evaluate novel small molecules aimed at ameliorating muscle degeneration. Furthermore, we discuss the challenges related to in vitro drug testing and provide a critical view of potential therapeutic developments to foster the future clinical translation of preclinical muscular dystrophy studies.
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38
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Schneider MR, Oelgeschlaeger M, Burgdorf T, van Meer P, Theunissen P, Kienhuis AS, Piersma AH, Vandebriel RJ. Applicability of organ-on-chip systems in toxicology and pharmacology. Crit Rev Toxicol 2021; 51:540-554. [PMID: 34463591 DOI: 10.1080/10408444.2021.1953439] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Organ-on-chip (OoC) systems are microfabricated cell culture devices designed to model functional units of human organs by harboring an in vitro generated organ surrogate. In the present study, we reviewed issues and opportunities related to the application of OoC in the safety and efficacy assessment of chemicals and pharmaceuticals, as well as the steps needed to achieve this goal. The relative complexity of OoC over simple in vitro assays provides advantages and disadvantages in the context of compound testing. The broader biological domain of OoC potentially enhances their predictive value, whereas their complexity present issues with throughput, standardization and transferability. Using OoCs for regulatory purposes requires detailed and standardized protocols, providing reproducible results in an interlaboratory setting. The extent to which interlaboratory standardization of OoC is feasible and necessary for regulatory application is a matter of debate. The focus of applying OoCs in safety assessment is currently directed to characterization (the biology represented in the test) and qualification (the performance of the test). To this aim, OoCs are evaluated on a limited scale, especially in the pharmaceutical industry, with restricted sets of reference substances. Given the low throughput of OoC, it is questionable whether formal validation, in which many reference substances are extensively tested in different laboratories, is feasible for OoCs. Rather, initiatives such as open technology platforms, and collaboration between OoC developers and risk assessors may prove an expedient strategy to build confidence in OoCs for application in safety and efficacy assessment.
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Affiliation(s)
- Marlon R Schneider
- German Centre for the Protection of Laboratory Animals (Bf3R), German Federal Institute for Risk Assessment (BfR), Berlin, Germany
| | - Michael Oelgeschlaeger
- German Centre for the Protection of Laboratory Animals (Bf3R), German Federal Institute for Risk Assessment (BfR), Berlin, Germany
| | - Tanja Burgdorf
- German Centre for the Protection of Laboratory Animals (Bf3R), German Federal Institute for Risk Assessment (BfR), Berlin, Germany
| | - Peter van Meer
- Section on Pharmacology, Toxicology and Kinetics, Medicines Evaluation Board, Utrecht, The Netherlands.,Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
| | - Peter Theunissen
- Section on Pharmacology, Toxicology and Kinetics, Medicines Evaluation Board, Utrecht, The Netherlands
| | - Anne S Kienhuis
- Laboratory for Health Protection, National Institute of Public Health and the Environment, Bilthoven, The Netherlands
| | - Aldert H Piersma
- Laboratory for Health Protection, National Institute of Public Health and the Environment, Bilthoven, The Netherlands
| | - Rob J Vandebriel
- Laboratory for Health Protection, National Institute of Public Health and the Environment, Bilthoven, The Netherlands
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39
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Vila OF, Chavez M, Ma SP, Yeager K, Zholudeva LV, Colón-Mercado JM, Qu Y, Nash TR, Lai C, Feliciano CM, Carter M, Kamm RD, Judge LM, Conklin BR, Ward ME, McDevitt TC, Vunjak-Novakovic G. Bioengineered optogenetic model of human neuromuscular junction. Biomaterials 2021; 276:121033. [PMID: 34403849 DOI: 10.1016/j.biomaterials.2021.121033] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2021] [Revised: 07/09/2021] [Accepted: 07/15/2021] [Indexed: 12/28/2022]
Abstract
Functional human tissues engineered from patient-specific induced pluripotent stem cells (hiPSCs) hold great promise for investigating the progression, mechanisms, and treatment of musculoskeletal diseases in a controlled and systematic manner. For example, bioengineered models of innervated human skeletal muscle could be used to identify novel therapeutic targets and treatments for patients with complex central and peripheral nervous system disorders. There is a need to develop standardized and objective quantitative methods for engineering and using these complex tissues, in order increase their robustness, reproducibility, and predictiveness across users. Here we describe a standardized method for engineering an isogenic, patient specific human neuromuscular junction (NMJ) that allows for automated quantification of NMJ function to diagnose disease using a small sample of blood serum and evaluate new therapeutic modalities. By combining tissue engineering, optogenetics, microfabrication, optoelectronics and video processing, we created a novel platform for the precise investigation of the development and degeneration of human NMJ. We demonstrate the utility of this platform for the detection and diagnosis of myasthenia gravis, an antibody-mediated autoimmune disease that disrupts the NMJ function.
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Affiliation(s)
- Olaia F Vila
- Columbia University, 622 W 168th St, New York, NY, 10032, USA; Gladstone Institutes, 1650 Owens St, San Francisco, CA, 94158, USA.
| | - Miguel Chavez
- Columbia University, 622 W 168th St, New York, NY, 10032, USA
| | - Stephen P Ma
- Columbia University, 622 W 168th St, New York, NY, 10032, USA
| | - Keith Yeager
- Columbia University, 622 W 168th St, New York, NY, 10032, USA
| | | | | | - Yihuai Qu
- Columbia University, 622 W 168th St, New York, NY, 10032, USA
| | - Trevor R Nash
- Columbia University, 622 W 168th St, New York, NY, 10032, USA
| | - Carmen Lai
- Gladstone Institutes, 1650 Owens St, San Francisco, CA, 94158, USA
| | - Carissa M Feliciano
- Gladstone Institutes, 1650 Owens St, San Francisco, CA, 94158, USA; Department of Pediatrics, UCSF, 550 16th St, Floor 5, San Francisco, CA, 94143, USA
| | - Matthew Carter
- Gladstone Institutes, 1650 Owens St, San Francisco, CA, 94158, USA
| | - Roger D Kamm
- Department of Mechanical Engineering and Biological Engineering, Massachusetts Institute of Technology, Cambridge MA, 02139, USA
| | - Luke M Judge
- Gladstone Institutes, 1650 Owens St, San Francisco, CA, 94158, USA; Department of Pediatrics, UCSF, 550 16th St, Floor 5, San Francisco, CA, 94143, USA
| | - Bruce R Conklin
- Gladstone Institutes, 1650 Owens St, San Francisco, CA, 94158, USA
| | - Michael E Ward
- National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, 20892, USA
| | - Todd C McDevitt
- Gladstone Institutes, 1650 Owens St, San Francisco, CA, 94158, USA
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Luttrell SM, Smith AST, Mack DL. Creating stem cell-derived neuromuscular junctions in vitro. Muscle Nerve 2021; 64:388-403. [PMID: 34328673 PMCID: PMC9292444 DOI: 10.1002/mus.27360] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2019] [Revised: 05/28/2021] [Accepted: 06/21/2021] [Indexed: 12/14/2022]
Abstract
Recent development of novel therapies has improved mobility and quality of life for people suffering from inheritable neuromuscular disorders. Despite this progress, the majority of neuromuscular disorders are still incurable, in part due to a lack of predictive models of neuromuscular junction (NMJ) breakdown. Improvement of predictive models of a human NMJ would be transformative in terms of expanding our understanding of the mechanisms that underpin development, maintenance, and disease, and as a testbed with which to evaluate novel therapeutics. Induced pluripotent stem cells (iPSCs) are emerging as a clinically relevant and non‐invasive cell source to create human NMJs to study synaptic development and maturation, as well as disease modeling and drug discovery. This review will highlight the recent advances and remaining challenges to generating an NMJ capable of eliciting contraction of stem cell‐derived skeletal muscle in vitro. We explore the advantages and shortcomings of traditional NMJ culturing platforms, as well as the pioneering technologies and novel, biomimetic culturing systems currently in use to guide development and maturation of the neuromuscular synapse and extracellular microenvironment. Then, we will explore how this NMJ‐in‐a‐dish can be used to study normal assembly and function of the efferent portion of the neuromuscular arc, and how neuromuscular disease‐causing mutations disrupt structure, signaling, and function.
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Affiliation(s)
- Shawn M Luttrell
- Department of Rehabilitation Medicine, University of Washington, Seattle, Washington, USA.,Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA
| | - Alec S T Smith
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA.,Department of Physiology and Biophysics, University of Washington, Seattle, Washington, USA
| | - David L Mack
- Department of Rehabilitation Medicine, University of Washington, Seattle, Washington, USA.,Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA.,Department of Physiology and Biophysics, University of Washington, Seattle, Washington, USA
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41
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Liu Y, Yamane J, Tanaka A, Fujibuchi W, Yamashita JK. AMPK activation reverts mouse epiblast stem cells to naive state. iScience 2021; 24:102783. [PMID: 34308289 PMCID: PMC8283141 DOI: 10.1016/j.isci.2021.102783] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2020] [Revised: 05/01/2021] [Accepted: 06/23/2021] [Indexed: 12/25/2022] Open
Abstract
Despite increasing knowledge on primed and naive pluripotency, the cell signaling that regulates the pluripotency type in stem cells remains not fully understood. Here we show that AMP kinase (AMPK) activators can induce the reversion of primed mouse epiblast stem cells (mEpiSCs) to the naive pluripotent state. The addition of AMPK activators alone or together with leukemia inhibitory factor to primed mEpiSCs induced the appearance of naive-like cells. After passaging in naive culture conditions, the colony morphology, protein expression, and global gene expression profiles indicated the naive state, as did germline transmission ability. Loss-of-function and gain-of-function studies suggested that p38 is a critical downstream target in AMPK activation. Finally, single-cell RNA sequencing analysis revealed that the reversion process through AMPK signaling passes an intermediate naive-like population. In conclusion, the AMPK pathway is a critical driving force in the reversion of primed to naive pluripotency.
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Affiliation(s)
- Yajing Liu
- The Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Junko Yamane
- The Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Akito Tanaka
- The Department of Animal Research Facility, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Wataru Fujibuchi
- The Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Jun K. Yamashita
- The Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
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42
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Sato T. Induction of Skeletal Muscle Progenitors and Stem Cells from human induced Pluripotent Stem Cells. J Neuromuscul Dis 2021; 7:395-405. [PMID: 32538862 PMCID: PMC7592659 DOI: 10.3233/jnd-200497] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Induced pluripotent stem cells (iPSCs) have the potential to differentiate into various types of cells and tissues including skeletal muscle. The approach to convert these stem cells into skeletal muscle cells offers hope for patients afflicted with skeletal muscle diseases such as Duchenne muscular dystrophy (DMD). Several methods have been reported to induce myogenic differentiation with iPSCs derived from myogenic patients. An important point for generating skeletal muscle cells from iPSCs is to understand in vivo myogenic induction in development and regeneration. Current protocols of myogenic induction utilize techniques with overexpression of myogenic transcription factors such as Myod1(MyoD), Pax3, Pax7, and others, using recombinant proteins or small molecules to induce mesodermal cells followed by myogenic progenitors, and adult muscle stem cells. This review summarizes the current approaches used for myogenic induction and highlights recent improvements.
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Affiliation(s)
- Takahiko Sato
- Department of Anatomy, Fujita Health University, Toyoake, Japan.,AMED-CREST, AMED, Otemachi, Chiyoda, Tokyo, Japan
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43
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Myogenic Differentiation of iPS Cells Shows Different Efficiency in Simultaneous Comparison of Protocols. Cells 2021; 10:cells10071671. [PMID: 34359837 PMCID: PMC8307201 DOI: 10.3390/cells10071671] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2021] [Revised: 06/17/2021] [Accepted: 06/28/2021] [Indexed: 11/17/2022] Open
Abstract
Induced pluripotent stem (iPS) cells constitute a perfect tool to study human embryo development processes such as myogenesis, thanks to their ability to differentiate into three germ layers. Currently, many protocols to obtain myogenic cells have been described in the literature. They differ in many aspects, such as media components, including signaling modulators, feeder layer constituents, and duration of culture. In our study, we compared three different myogenic differentiation protocols to verify, side by side, their efficiency. Protocol I was based on embryonic bodies differentiation induction, ITS addition, and selection with adhesion to collagen I type. Protocol II was based on strong myogenic induction at the embryonic bodies step with BIO, forskolin, and bFGF, whereas cells in Protocol III were cultured in monolayers in three special media, leading to WNT activation and TGF-β and BMP signaling inhibition. Myogenic induction was confirmed by the hierarchical expression of myogenic regulatory factors MYF5, MYOD, MYF6 and MYOG, as well as the expression of myotubes markers MYH3 and MYH2, in each protocol. Our results revealed that Protocol III is the most efficient in obtaining myogenic cells. Furthermore, our results indicated that CD56 is not a specific marker for the evaluation of myogenic differentiation.
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Uchimura T, Asano T, Nakata T, Hotta A, Sakurai H. A muscle fatigue-like contractile decline was recapitulated using skeletal myotubes from Duchenne muscular dystrophy patient-derived iPSCs. CELL REPORTS MEDICINE 2021; 2:100298. [PMID: 34195678 PMCID: PMC8233665 DOI: 10.1016/j.xcrm.2021.100298] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/22/2020] [Revised: 01/28/2021] [Accepted: 05/10/2021] [Indexed: 02/07/2023]
Abstract
Duchenne muscular dystrophy (DMD) is a muscle degenerating disease caused by dystrophin deficiency, for which therapeutic options are limited. To facilitate drug development, it is desirable to develop in vitro disease models that enable the evaluation of DMD declines in contractile performance. Here, we show MYOD1-induced differentiation of hiPSCs into functional skeletal myotubes in vitro with collagen gel and electrical field stimulation (EFS). Long-term EFS training (0.5 Hz, 20 V, 2 ms, continuous for 2 weeks) mimicking muscle overuse recapitulates declines in contractile performance in dystrophic myotubes. A screening of clinically relevant drugs using this model detects three compounds that ameliorate this decline. Furthermore, we validate the feasibility of adapting the model to a 96-well culture system using optogenetic technology for large-scale screening. Our results support a disease model using patient-derived iPSCs that allows for the recapitulation of the contractile pathogenesis of DMD and a screening strategy for drug development.
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Affiliation(s)
- Tomoya Uchimura
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.,Takeda-CiRA Joint Program, Fujisawa, Kanagawa 251-8555, Japan
| | - Toshifumi Asano
- Department of Cell Biology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo 113-8510, Japan.,The Center for Brain Integration Research, Tokyo Medical and Dental University, Tokyo 113-8510, Japan
| | - Takao Nakata
- Department of Cell Biology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo 113-8510, Japan.,The Center for Brain Integration Research, Tokyo Medical and Dental University, Tokyo 113-8510, Japan
| | - Akitsu Hotta
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.,Takeda-CiRA Joint Program, Fujisawa, Kanagawa 251-8555, Japan
| | - Hidetoshi Sakurai
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.,Takeda-CiRA Joint Program, Fujisawa, Kanagawa 251-8555, Japan
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45
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Jeong J, Choi KH, Kim SH, Lee DK, Oh JN, Lee M, Choe GC, Lee CK. Combination of cell signaling molecules can facilitate MYOD1-mediated myogenic transdifferentiation of pig fibroblasts. J Anim Sci Biotechnol 2021; 12:64. [PMID: 33980301 PMCID: PMC8117598 DOI: 10.1186/s40104-021-00583-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Accepted: 03/11/2021] [Indexed: 02/06/2023] Open
Abstract
Background Myogenic transdifferentiation can be accomplished through ectopic MYOD1 expression, which is facilitated by various signaling pathways associated with myogenesis. In this study, we attempted to transdifferentiate pig embryonic fibroblasts (PEFs) myogenically into skeletal muscle through overexpression of the pig MYOD1 gene and modulation of the FGF, TGF-β, WNT, and cAMP signaling pathways. Results The MYOD1 overexpression vector was constructed based on comparative sequence analysis, demonstrating that pig MYOD1 has evolutionarily conserved domains across various species. Although forced MYOD1 expression through these vectors triggered the expression of endogenous muscle markers, transdifferentiated muscle cells from fibroblasts were not observed. Therefore, various signaling molecules, including FGF2, SB431542, CHIR99021, and forskolin, along with MYOD1 overexpression were applied to enhance the myogenic reprogramming. The modified conditions led to the derivation of myotubes and activation of muscle markers in PEFs, as determined by qPCR and immunostaining. Notably, a sarcomere-like structure was observed, indicating that terminally differentiated skeletal muscle could be obtained from transdifferentiated cells. Conclusions In summary, we established a protocol for reprogramming MYOD1-overexpressing PEFs into the mature skeletal muscle using signaling molecules. Our myogenic reprogramming can be used as a cell source for muscle disease models in regenerative medicine and the production of cultured meat in cellular agriculture.
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Affiliation(s)
- Jinsol Jeong
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, 08826, South Korea
| | - Kwang-Hwan Choi
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, 08826, South Korea.,Present address: Research and Development Center, Space F corporation, Hwasung-si, Gyeonggi-do, 18471, South Korea
| | - Seung-Hun Kim
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, 08826, South Korea
| | - Dong-Kyung Lee
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, 08826, South Korea.,Present address: Research and Development Center, Space F corporation, Hwasung-si, Gyeonggi-do, 18471, South Korea
| | - Jong-Nam Oh
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, 08826, South Korea
| | - Mingyun Lee
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, 08826, South Korea
| | - Gyung Cheol Choe
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, 08826, South Korea
| | - Chang-Kyu Lee
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, 08826, South Korea. .,Institute of Green Bio Science and Technology, Seoul National University, Pyeong Chang, Kangwon-do, 25354, South Korea.
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Sasamata M, Shimojo D, Fuse H, Nishi Y, Sakurai H, Nakahata T, Yamagishi Y, Sasaki-Iwaoka H. Establishment of a Robust Platform for Induced Pluripotent Stem Cell Research Using Maholo LabDroid. SLAS Technol 2021; 26:441-453. [PMID: 33775154 DOI: 10.1177/24726303211000690] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Induced pluripotent stem cells (iPSCs) are attractive for use in early drug discovery because they can differentiate into any cell type. Maintenance cultures and differentiation processes for iPSCs, however, require a high level of technical expertise. To overcome this problem, technological developments such as enhanced automation are necessary to replace manual operation. In addition, a robot system with the flexibility and expandability to carry out maintenance culture and each of the required differentiation processes would also be important. In this study, we established a platform to enable the multiple processes required for iPSC experiments using the Maholo LabDroid, which is a humanoid robotic system with excellent reproducibility and flexibility. The accuracy and robustness of Maholo LabDroid enabled us to cultivate undifferentiated iPSCs for 63 days while maintaining their ability to differentiate into the three embryonic germ layers. Maholo LabDroid maintained and harvested iPSCs in six-well plates, then seeded them into 96-well plates, induced differentiation, and implemented immunocytochemistry. As a result, Maholo LabDroid was confirmed to be able to perform the processes required for myogenic differentiation of iPSCs isolated from a patient with muscular disease and achieved a high differentiation rate with a coefficient of variation (CV) <10% in the first trial. Furthermore, the expandability and flexibility of Maholo LabDroid allowed us to experiment with multiple cell lines simultaneously.
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Affiliation(s)
- Miho Sasamata
- Drug Discovery Research, Astellas Pharma Inc., Tsukuba-shi, Ibaraki, Japan
| | - Daisuke Shimojo
- Drug Discovery Research, Astellas Pharma Inc., Tsukuba-shi, Ibaraki, Japan
| | - Hiromitsu Fuse
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto, Japan
| | - Yohei Nishi
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto, Japan
| | - Hidetoshi Sakurai
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto, Japan
| | - Tatsutoshi Nakahata
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto, Japan
| | - Yukiko Yamagishi
- Drug Discovery Research, Astellas Pharma Inc., Tsukuba-shi, Ibaraki, Japan.,Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto, Japan
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47
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Costantini M, Testa S, Fornetti E, Fuoco C, Sanchez Riera C, Nie M, Bernardini S, Rainer A, Baldi J, Zoccali C, Biagini R, Castagnoli L, Vitiello L, Blaauw B, Seliktar D, Święszkowski W, Garstecki P, Takeuchi S, Cesareni G, Cannata S, Gargioli C. Biofabricating murine and human myo-substitutes for rapid volumetric muscle loss restoration. EMBO Mol Med 2021; 13:e12778. [PMID: 33587336 PMCID: PMC7933978 DOI: 10.15252/emmm.202012778] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2020] [Revised: 01/05/2021] [Accepted: 01/12/2021] [Indexed: 12/31/2022] Open
Abstract
The importance of skeletal muscle tissue is undoubted being the controller of several vital functions including respiration and all voluntary locomotion activities. However, its regenerative capability is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. Here, we propose a biofabrication approach to rapidly restore skeletal muscle mass, 3D histoarchitecture, and functionality. By recapitulating muscle anisotropic organization at the microscale level, we demonstrate to efficiently guide cell differentiation and myobundle formation both in vitro and in vivo. Of note, upon implantation, the biofabricated myo-substitutes support the formation of new blood vessels and neuromuscular junctions-pivotal aspects for cell survival and muscle contractile functionalities-together with an advanced muscle mass and force recovery. Altogether, these data represent a solid base for further testing the myo-substitutes in large animal size and a promising platform to be eventually translated into clinical scenarios.
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Affiliation(s)
- Marco Costantini
- Institute of Physical ChemistryPolish Academy of SciencesWarsawPoland
| | - Stefano Testa
- Department of BiologyRome University Tor VergataRomeItaly
| | | | - Claudia Fuoco
- Department of BiologyRome University Tor VergataRomeItaly
| | | | - Minghao Nie
- Department of Mechano‐InformaticsGraduate School of Information Science and TechnologyThe University of TokyoTokyoJapan
| | | | - Alberto Rainer
- Department of EngineeringUniversità Campus Bio‐Medico di RomaRomeItaly
- Institute of Nanotechnology (NANOTEC)National Research CouncilLecceItaly
| | - Jacopo Baldi
- IRCCS Regina Elena National Cancer InstituteRomeItaly
| | | | | | | | | | - Bert Blaauw
- Department of Biomedical Science and Venetian Institute of Molecular MedicineUniversity of PadovaPadovaItaly
| | - Dror Seliktar
- Department of Biomedical EngineeringTechion InstituteHaifaIsrael
| | - Wojciech Święszkowski
- Faculty of Materials Science and EngineeringWarsaw University of TechnologyWarsawPoland
| | - Piotr Garstecki
- Institute of Physical ChemistryPolish Academy of SciencesWarsawPoland
| | - Shoji Takeuchi
- Department of Mechano‐InformaticsGraduate School of Information Science and TechnologyThe University of TokyoTokyoJapan
- Institute of Industrial ScienceThe University of TokyoTokyoJapan
| | - Gianni Cesareni
- Department of BiologyRome University Tor VergataRomeItaly
- IRCCS Fondazione Santa LuciaRomeItaly
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48
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Iwasaki H, Ichihara Y, Morino K, Lemecha M, Sugawara L, Sawano T, Miake J, Sakurai H, Nishi E, Maegawa H, Imamura T. MicroRNA-494-3p inhibits formation of fast oxidative muscle fibres by targeting E1A-binding protein p300 in human-induced pluripotent stem cells. Sci Rep 2021; 11:1161. [PMID: 33441918 PMCID: PMC7806978 DOI: 10.1038/s41598-020-80742-y] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2019] [Accepted: 12/17/2020] [Indexed: 01/29/2023] Open
Abstract
MYOD-induced microRNA-494-3p expression inhibits fast oxidative myotube formation by downregulating myosin heavy chain 2 (MYH2) in human induced pluripotent stem cells (hiPSCs) during skeletal myogenesis. However, the molecular mechanisms regulating MYH2 expression via miR-494-3p remain unknown. Here, using bioinformatic analyses, we show that miR-494-3p potentially targets the transcript of the E1A-binding protein p300 at its 3'-untranslated region (UTR). Myogenesis in hiPSCs with the Tet/ON-myogenic differentiation 1 (MYOD1) gene (MyoD-hiPSCs) was induced by culturing them in doxycycline-supplemented differentiation medium for 7 days. p300 protein expression decreased after transient induction of miR-494-3p during myogenesis. miR-494-3p mimics decreased the levels of p300 and its downstream targets MYOD and MYH2 and myotube formation efficiency. p300 knockdown decreased myotube formation efficiency, MYH2 expression, and basal oxygen consumption rate. The binding of miR-494-3p to the wild type p300 3'-UTR, but not the mutated site, was confirmed using luciferase assay. Overexpression of p300 rescued the miR-494-3p mimic-induced phenotype in MyoD-hiPSCs. Moreover, miR-494-3p mimic reduced the levels of p300, MYOD, and MYH2 in skeletal muscles in mice. Thus, miR-494-3p might modulate MYH2 expression and fast oxidative myotube formation by directly regulating p300 levels during skeletal myogenesis in MyoD-hiPSCs and murine skeletal muscle tissues.
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Affiliation(s)
- Hirotaka Iwasaki
- Department of Pharmacology, Shiga University of Medical Science, Otsu, Japan
| | - Yoshinori Ichihara
- Division of Pharmacology, Faculty of Medicine, Tottori University, Yonago, Japan
| | - Katsutaro Morino
- Division of Endocrinology and Metabolism, Department of Medicine, Shiga University of Medical Science, Tsukinowa, Seta, Otsu, Shiga, 520-2192, Japan.
| | - Mengistu Lemecha
- Division of Endocrinology and Metabolism, Department of Medicine, Shiga University of Medical Science, Tsukinowa, Seta, Otsu, Shiga, 520-2192, Japan
- Department of Molecular and Cellular Biology, City of Hope, Los Angeles, USA
| | - Lucia Sugawara
- Division of Endocrinology and Metabolism, Department of Medicine, Shiga University of Medical Science, Tsukinowa, Seta, Otsu, Shiga, 520-2192, Japan
| | - Tatsuya Sawano
- Division of Pharmacology, Faculty of Medicine, Tottori University, Yonago, Japan
| | - Junichiro Miake
- Division of Pharmacology, Faculty of Medicine, Tottori University, Yonago, Japan
| | - Hidetoshi Sakurai
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Eiichiro Nishi
- Department of Pharmacology, Shiga University of Medical Science, Otsu, Japan
| | - Hiroshi Maegawa
- Division of Endocrinology and Metabolism, Department of Medicine, Shiga University of Medical Science, Tsukinowa, Seta, Otsu, Shiga, 520-2192, Japan
| | - Takeshi Imamura
- Division of Pharmacology, Faculty of Medicine, Tottori University, Yonago, Japan
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49
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Insulin/Glucose-Responsive Cells Derived from Induced Pluripotent Stem Cells: Disease Modeling and Treatment of Diabetes. Cells 2020; 9:cells9112465. [PMID: 33198288 PMCID: PMC7696367 DOI: 10.3390/cells9112465] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2020] [Revised: 11/03/2020] [Accepted: 11/09/2020] [Indexed: 12/21/2022] Open
Abstract
Type 2 diabetes, characterized by dysfunction of pancreatic β-cells and insulin resistance in peripheral organs, accounts for more than 90% of all diabetes. Despite current developments of new drugs and strategies to prevent/treat diabetes, there is no ideal therapy targeting all aspects of the disease. Restoration, however, of insulin-producing β-cells, as well as insulin-responsive cells, would be a logical strategy for the treatment of diabetes. In recent years, generation of transplantable cells derived from stem cells in vitro has emerged as an important research area. Pluripotent stem cells, either embryonic or induced, are alternative and feasible sources of insulin-secreting and glucose-responsive cells. This notwithstanding, consistent generation of robust glucose/insulin-responsive cells remains challenging. In this review, we describe basic concepts of the generation of induced pluripotent stem cells and subsequent differentiation of these into pancreatic β-like cells, myotubes, as well as adipocyte- and hepatocyte-like cells. Use of these for modeling of human disease is now feasible, while development of replacement therapies requires continued efforts.
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50
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Yahata N, Boda H, Hata R. Elimination of Mutant mtDNA by an Optimized mpTALEN Restores Differentiation Capacities of Heteroplasmic MELAS-iPSCs. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2020; 20:54-68. [PMID: 33376755 PMCID: PMC7744650 DOI: 10.1016/j.omtm.2020.10.017] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Accepted: 10/19/2020] [Indexed: 01/20/2023]
Abstract
Various mitochondrial diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with heteroplasmic mutations in mitochondrial DNA (mtDNA). Herein, we refined a previously generated G13513A mtDNA-targeted platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate mtDNA heteroplasmy in MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying codons in their expression vectors could suppress the reduction in the mtDNA copy number, which contributed to the rapid recovery of mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover, MELAS-iPSCs with a high proportion of G13513A mutant mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally, drug-inducible, myogenic differentiation 1 (MYOD)-transfected MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into myosin heavy chain-positive myocytes, with or without mutant mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling.
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Affiliation(s)
- Naoki Yahata
- Department of Anatomy I, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
| | - Hiroko Boda
- Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
| | - Ryuji Hata
- Department of Anatomy I, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
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