|
1
|
Kondo T, Suga H, Takeuchi K, Fuse Y, Sato Y, Hirose T, Hideyuki H, Nagata Y, Saito R. Benchmark for Setting ACTH Cell Dosage in Clinical Regenerative Medicine for Post-Operative Hypopituitarism. Diseases 2025; 13:112. [PMID: 40277822 PMCID: PMC12025586 DOI: 10.3390/diseases13040112] [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: 02/28/2025] [Revised: 04/06/2025] [Accepted: 04/07/2025] [Indexed: 04/26/2025] Open
Abstract
BACKGROUND/OBJECTIVES Our objective is to develop hormone-producing pituitary cells that can function in the same manner as the human body and provide more effective treatments than current hormone replacement therapy. We have already established a technique for generating hypothalamic-pituitary organoids using feeder-free human pluripotent stem cells (hPSCs) and demonstrated their effectiveness in vivo through transplantation into hypopituitary mouse models. To prospectively determine the upper limit of transplanting adenohypophyseal cells into humans, we investigated the human maximum secretion capacity of adrenocorticotropic hormone (ACTH) and growth hormone (GH). METHODS We analyzed data from 28 patients with pituitary adenomas, among whom 16 evinced no abnormality of ACTH secretion and 12 showed no GH secretion on corticotropin-releasing hormone (CRH) and growth hormone-releasing hormone-2 (GHRP-2) stimulation testing. RESULTS The average ACTH peak value after CRH stimulation tests was 97.2 pg/mL, and the average GH peak value after GHRP-2 stimulation tests was 25.1 ng/mL. CONCLUSIONS These data will likely serve as benchmarks of ACTH and GH secretion when transplanting cultured cells into humans.
Collapse
Affiliation(s)
- Tatsuma Kondo
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
- Department of Endocrinology and Diabetes, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan
| | - Hidetaka Suga
- Department of Endocrinology and Diabetes, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan
| | - Kazuhito Takeuchi
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
| | - Yutaro Fuse
- Department of Artificial Intelligence Medicine, Graduate School of Medicine, Chiba University, Chiba 260-8722, Japan;
| | - Yoshiki Sato
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
| | - Toshiaki Hirose
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
| | - Harada Hideyuki
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
| | - Yuichi Nagata
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
| | - Ryuta Saito
- Department of Neurosurgery, Graduate School of Medicine, Nagoya University, Nagoya 468-0066, Japan; (T.K.); (Y.S.); (T.H.); (H.H.); (Y.N.); (R.S.)
| |
Collapse
|
|
2
|
Scherrer C, Loret C, Védrenne N, Buckley C, Lia AS, Kermene V, Sturtz F, Favreau F, Rovini A, Faye PA. From in vivo models to in vitro bioengineered neuromuscular junctions for the study of Charcot-Marie-Tooth disease. J Tissue Eng 2025; 16:20417314241310508. [PMID: 40078221 PMCID: PMC11898049 DOI: 10.1177/20417314241310508] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2024] [Accepted: 12/14/2024] [Indexed: 03/14/2025] Open
Abstract
Peripheral neuropathies are disorders affecting the peripheral nervous system. Among them, Charcot-Marie-Tooth disease is an inherited sensorimotor neuropathy for which no effective treatment exists yet. Research on Charcot-Marie-Tooth disease has been hampered by difficulties in accessing relevant cells, such as sensory and motor neurons, Schwann cells, and myocytes, which interact at the neuromuscular junction, the specialized synapses formed between nerves and skeletal muscles. This review first outlines the various in vivo models and methods used to study neuromuscular junction deficiencies in Charcot-Marie-Tooth disease. We then explore novel in vitro techniques and models, including complex hiPSC-derived cultures, which offer promising isogenic and reproducible neuromuscular junction models. The adaptability of in vitro culture methods, including cell origin, cell-type combinations, and choice of culture format, adds complexity and excitement to this rapidly evolving field. This review aims to recapitulate available tools for studying Charcot-Marie-Tooth disease to understand its pathophysiological mechanisms and test potential therapies.
Collapse
Affiliation(s)
- Camille Scherrer
- University of Limoges, NeurIT UR 20218, GEIST Institute, Limoges, France
| | - Camille Loret
- University of Limoges, NeurIT UR 20218, GEIST Institute, Limoges, France
| | - Nicolas Védrenne
- University of Limoges, Inserm U1248 Pharmacology & Transplantation, Limoges, France
| | - Colman Buckley
- University of Limoges, XLIM, CNRS UMR 7252, Limoges, France
| | - Anne-Sophie Lia
- University of Limoges, NeurIT UR 20218, GEIST Institute, Limoges, France
- Department of Biochemistry and Molecular Genetics, CHU Limoges, Limoges, France
- Department of Bioinformatics, CHU Limoges, Limoges, France
| | | | - Franck Sturtz
- University of Limoges, NeurIT UR 20218, GEIST Institute, Limoges, France
- Department of Biochemistry and Molecular Genetics, CHU Limoges, Limoges, France
| | - Frédéric Favreau
- University of Limoges, NeurIT UR 20218, GEIST Institute, Limoges, France
- Department of Biochemistry and Molecular Genetics, CHU Limoges, Limoges, France
| | - Amandine Rovini
- University of Limoges, NeurIT UR 20218, GEIST Institute, Limoges, France
| | - Pierre-Antoine Faye
- University of Limoges, NeurIT UR 20218, GEIST Institute, Limoges, France
- Department of Biochemistry and Molecular Genetics, CHU Limoges, Limoges, France
| |
Collapse
|
|
3
|
Caputo L, Stamenkovic C, Tierney MT, Falzarano MS, Bassel-Duby R, Ferlini A, Olson EN, Puri PL, Sacco A. Modulation of the JAK2-STAT3 pathway promotes expansion and maturation of human iPSCs-derived myogenic progenitor cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.12.09.624203. [PMID: 39713478 PMCID: PMC11661153 DOI: 10.1101/2024.12.09.624203] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/24/2024]
Abstract
Generation of in vitro induced pluripotent cells (hiPSCs)-derived skeletal muscle progenitor cells (SMPCs) holds great promise for regenerative medicine for skeletal muscle wasting diseases, as for example Duchenne Muscular Dystrophy (DMD). Multiple approaches, involving ectopic expression of key regulatory myogenic genes or small molecules cocktails, have been described by different groups to obtain SMPC towards cell-transplantation in vivo as a therapeutic approach to skeletal muscle diseases. However, hiPSCs-derived SMPC generated using transgene-free protocols are usually obtained in a low amount and resemble a more embryonal/fetal stage of differentiation. Here we demonstrate that modulation of the JAK2/STAT3 signaling pathway during an in vitro skeletal muscle differentiation protocol, increases the yield of PAX7+ and CD54+ SMPCs and drive them to a postnatal maturation stage, in both human ES and patient-derived iPSCs. Importantly, upon removal of the inhibition from the cultures, the obtained SMPCs are able to differentiate into multinucleated myotubes in vitro. These findings reveal that modulation of the JAK2/STAT3 signaling pathway is a potential therapeutic avenue to generate SMPCs in vitro with increase potential for cell-therapy approaches.
Collapse
Affiliation(s)
- Luca Caputo
- Sanford Burnham Prebys Medical Discovery Institute, Development, Aging and Regeneration Program, La Jolla, CA 92037, USA
| | - Cedomir Stamenkovic
- Sanford Burnham Prebys Medical Discovery Institute, Development, Aging and Regeneration Program, La Jolla, CA 92037, USA
- Graduate School of Biomedical Sciences, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA
| | - Matthew T. Tierney
- Sanford Burnham Prebys Medical Discovery Institute, Development, Aging and Regeneration Program, La Jolla, CA 92037, USA
- Graduate School of Biomedical Sciences, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA
| | | | - Rhonda Bassel-Duby
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
| | | | - Eric N. Olson
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
| | - Pier Lorenzo Puri
- Sanford Burnham Prebys Medical Discovery Institute, Development, Aging and Regeneration Program, La Jolla, CA 92037, USA
| | - Alessandra Sacco
- Sanford Burnham Prebys Medical Discovery Institute, Development, Aging and Regeneration Program, La Jolla, CA 92037, USA
| |
Collapse
|
|
4
|
Tey SR, Anderson RS, Yu CH, Robertson S, Kletzien H, Connor NP, Tanaka K, Ohkawa Y, Suzuki M. Cellular and transcriptomic changes by the supplementation of aged rat serum in human pluripotent stem cell-derived myogenic progenitors. Front Cell Dev Biol 2024; 12:1481491. [PMID: 39474351 PMCID: PMC11518775 DOI: 10.3389/fcell.2024.1481491] [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: 08/16/2024] [Accepted: 09/25/2024] [Indexed: 11/06/2024] Open
Abstract
Introduction The changing composition of non-cell autonomous circulating factors in blood as humans age is believed to play a role in muscle mass and strength loss. The mechanisms through which these circulating factors act in age-related skeletal muscle changes is not fully understood. In this study, we used human myogenic progenitors derived from human pluripotent stem cells to study non-cell autonomous roles of circulating factors during the process of myogenic differentiation. Methods Myogenic progenitors from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were supplemented with serum samples from aged or young Fischer 344 × Brown Norway F1-hybrid rats. The effect of aged or young serum supplementation on myogenic progenitor proliferation, myotube formation capacity, differentiation, and early transcriptomic profiles were analyzed. Results We found that aged rat serum supplementation significantly reduced cell proliferation and increased cell death in both ESC- and iPSC-derived myogenic progenitors. Next, we found that the supplementation of aged rat serum inhibited myotube formation and maturation during terminal differentiation from progenitors to skeletal myocytes when compared to the cells treated with young adult rat serum. Lastly, we identified that gene expression profiles were affected following serum supplementation in culture. Discussion Together, aged serum supplementation caused cellular and transcriptomic changes in human myogenic progenitors. The current data from our in vitro model possibly simulate non-cell autonomous contributions of blood composition to age-related processes in human skeletal muscle.
Collapse
Affiliation(s)
- Sin-Ruow Tey
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI, United States
| | - Ryan S. Anderson
- Department of Surgical Sciences, University of Wisconsin-Madison, Madison, WI, United States
| | - Clara H. Yu
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI, United States
| | - Samantha Robertson
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI, United States
| | - Heidi Kletzien
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, United States
- Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States
| | - Nadine P. Connor
- Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States
- Department of Communication Sciences and Disorders, University of Wisconsin-Madison, Madison, WI, United States
| | - Kaori Tanaka
- Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
| | - Yasuyuki Ohkawa
- Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
| | - Masatoshi Suzuki
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI, United States
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, United States
- Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison, Madison, WI, United States
| |
Collapse
|
|
5
|
Iwasaki N, Roldo M, Karali A, Blunn G. In vitro development of a muscle-tendon junction construct using decellularised extracellular matrix: Effect of cyclic tensile loading. BIOMATERIALS ADVANCES 2024; 161:213873. [PMID: 38692180 DOI: 10.1016/j.bioadv.2024.213873] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/03/2024] [Revised: 04/10/2024] [Accepted: 04/24/2024] [Indexed: 05/03/2024]
Abstract
The muscle tendon junction (MTJ) plays a crucial role in transmitting the force generated by muscles to the tendon and then to the bone. Injuries such as tears and strains frequently happen at the MTJ, where the regenerative process is limited due to poor vascularization and the complex structure of the tissue. Current solutions for a complete tear at the MTJ have not been successful and therefore, the development of a tissue-engineered MTJ may provide a more effective treatment. In this study, decellularised extracellular matrix (DECM) derived from sheep MTJ was used to provide a scaffold for the MTJ with the relevant mechanical properties and differentiation cues such as the relase of growth factors. Human mesenchymal stem cells (MSCs) were seeded on DECM and 10 % cyclic strain was applied using a bioreactor. MSCs cultured on DECM showed significantly higher gene and protein expression of MTJ markers such as collagen 22, paxillin and talin, than MSCs in 2D culture. Although collagen 22 protein expression was higher in the cells with strain than without strain, reduced gene expression of other MTJ markers was observed when the strain was applied. DECM combined with 10 % strain enhanced myogenic differentiation, while tenogenic differentiation was reduced when compared to static cultures of MSCs on DECM. For the first time, these results showed that DECM derived from the MTJ can induce MTJ marker gene and protein expression by MSCs, however, the effect of strain on the MTJ development in DECM culture needs further investigation.
Collapse
Affiliation(s)
- Nodoka Iwasaki
- School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, UK.
| | - Marta Roldo
- School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, UK
| | - Aikaterina Karali
- School of Mechanical and Design Engineering, University of Portsmouth, Portsmouth, UK
| | - Gordon Blunn
- School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, UK
| |
Collapse
|
|
6
|
Couturier N, Hörner SJ, Nürnberg E, Joazeiro C, Hafner M, Rudolf R. Aberrant evoked calcium signaling and nAChR cluster morphology in a SOD1 D90A hiPSC-derived neuromuscular model. Front Cell Dev Biol 2024; 12:1429759. [PMID: 38966427 PMCID: PMC11222430 DOI: 10.3389/fcell.2024.1429759] [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: 05/08/2024] [Accepted: 06/03/2024] [Indexed: 07/06/2024] Open
Abstract
Familial amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder that is due to mutations in one of several target genes, including SOD1. So far, clinical records, rodent studies, and in vitro models have yielded arguments for either a primary motor neuron disease, or a pleiotropic pathogenesis of ALS. While mouse models lack the human origin, in vitro models using human induced pluripotent stem cells (hiPSC) have been recently developed for addressing ALS pathogenesis. In spite of improvements regarding the generation of muscle cells from hiPSC, the degree of maturation of muscle cells resulting from these protocols has remained limited. To fill these shortcomings, we here present a new protocol for an enhanced myotube differentiation from hiPSC with the option of further maturation upon coculture with hiPSC-derived motor neurons. The described model is the first to yield a combination of key myogenic maturation features that are consistent sarcomeric organization in association with complex nAChR clusters in myotubes derived from control hiPSC. In this model, myotubes derived from hiPSC carrying the SOD1 D90A mutation had reduced expression of myogenic markers, lack of sarcomeres, morphologically different nAChR clusters, and an altered nAChR-dependent Ca2+ response compared to control myotubes. Notably, trophic support provided by control hiPSC-derived motor neurons reduced nAChR cluster differences between control and SOD1 D90A myotubes. In summary, a novel hiPSC-derived neuromuscular model yields evidence for both muscle-intrinsic and nerve-dependent aspects of neuromuscular dysfunction in SOD1-based ALS.
Collapse
Affiliation(s)
- Nathalie Couturier
- CeMOS, Mannheim University of Applied Sciences, Mannheim, Germany
- Interdisciplinary Center for Neurosciences, Heidelberg University, Heidelberg, Germany
| | - Sarah Janice Hörner
- CeMOS, Mannheim University of Applied Sciences, Mannheim, Germany
- Interdisciplinary Center for Neurosciences, Heidelberg University, Heidelberg, Germany
| | - Elina Nürnberg
- CeMOS, Mannheim University of Applied Sciences, Mannheim, Germany
| | - Claudio Joazeiro
- Center for Molecular Biology, Heidelberg University, Heidelberg, Germany
| | - Mathias Hafner
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany
- Institute of Medical Technology, Mannheim University of Applied Sciences and Heidelberg University, Mannheim, Germany
| | - Rüdiger Rudolf
- CeMOS, Mannheim University of Applied Sciences, Mannheim, Germany
- Interdisciplinary Center for Neurosciences, Heidelberg University, Heidelberg, Germany
- Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany
- Institute of Medical Technology, Mannheim University of Applied Sciences and Heidelberg University, Mannheim, Germany
| |
Collapse
|
|
7
|
Jiang Y, Zhou R, Wu Y, Kong G, Zeng J, Li X, Wang B, Gu C, Liao F, Qi F, Zhu Q, Gu L, Zheng C. In vitro modeling of skeletal muscle ischemia-reperfusion injury based on sphere differentiation culture from human pluripotent stem cells. Exp Cell Res 2024; 439:114111. [PMID: 38823471 DOI: 10.1016/j.yexcr.2024.114111] [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: 02/12/2024] [Revised: 04/14/2024] [Accepted: 05/29/2024] [Indexed: 06/03/2024]
Abstract
Skeletal muscle ischemia-reperfusion (IR) injury poses significant challenges due to its local and systemic complications. Traditional studies relying on two-dimensional (2D) cell culture or animal models often fall short of faithfully replicating the human in vivo environment, thereby impeding the translational process from animal research to clinical applications. Three-dimensional (3D) constructs, such as skeletal muscle spheroids with enhanced cell-cell interactions from human pluripotent stem cells (hPSCs) offer a promising alternative by partially mimicking human physiological cellular environment in vivo processes. This study aims to establish an innovative in vitro model, human skeletal muscle spheroids based on sphere differentiation from hPSCs, to investigate human skeletal muscle developmental processes and IR mechanisms within a controlled laboratory setting. By eticulously recapitulating embryonic myogenesis through paraxial mesodermal differentiation of neuro-mesodermal progenitors, we successfully established 3D skeletal muscle spheroids that mirror the dynamic colonization observed during human skeletal muscle development. Co-culturing human skeletal muscle spheroids with spinal cord spheroids facilitated the formation of neuromuscular junctions, providing functional relevance to skeletal muscle spheroids. Furthermore, through oxygen-glucose deprivation/re-oxygenation treatment, 3D skeletal muscle spheroids provide insights into the molecular events and pathogenesis of IR injury. The findings presented in this study significantly contribute to our understanding of skeletal muscle development and offer a robust platform for in vitro studies on skeletal muscle IR injury, holding potential applications in drug testing, therapeutic development, and personalized medicine within the realm of skeletal muscle-related pathologies.
Collapse
Affiliation(s)
- Yifei Jiang
- Department of Microsurgery, Orthopedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Guangdong Province Engineering Laboratory for Soft Tissue Biofabrication, Guangzhou, China; Guangdong Provincial Peripheral Nerve Tissue Engineering and Technology Research Center, Guangzhou, China; Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China
| | - Runtao Zhou
- Department of Microsurgery, Orthopedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Guangdong Province Engineering Laboratory for Soft Tissue Biofabrication, Guangzhou, China; Guangdong Provincial Peripheral Nerve Tissue Engineering and Technology Research Center, Guangzhou, China; Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China
| | - Yixun Wu
- Department of Microsurgery, Orthopedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Guangdong Province Engineering Laboratory for Soft Tissue Biofabrication, Guangzhou, China; Guangdong Provincial Peripheral Nerve Tissue Engineering and Technology Research Center, Guangzhou, China; Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China
| | - Ganggang Kong
- Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China; Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Jingguang Zeng
- Department of Microsurgery, Orthopedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Guangdong Province Engineering Laboratory for Soft Tissue Biofabrication, Guangzhou, China; Guangdong Provincial Peripheral Nerve Tissue Engineering and Technology Research Center, Guangzhou, China; Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China
| | - Xubo Li
- Department of Microsurgery, Orthopedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Guangdong Province Engineering Laboratory for Soft Tissue Biofabrication, Guangzhou, China; Guangdong Provincial Peripheral Nerve Tissue Engineering and Technology Research Center, Guangzhou, China; Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China
| | - Bo Wang
- Department of Microsurgery, Orthopedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Guangdong Province Engineering Laboratory for Soft Tissue Biofabrication, Guangzhou, China; Guangdong Provincial Peripheral Nerve Tissue Engineering and Technology Research Center, Guangzhou, China; Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China
| | - Cheng Gu
- Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China; Department of Joint Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Fawei Liao
- Department of Microsurgery, Orthopedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Guangdong Province Engineering Laboratory for Soft Tissue Biofabrication, Guangzhou, China; Guangdong Provincial Peripheral Nerve Tissue Engineering and Technology Research Center, Guangzhou, China; Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China
| | - Fangze Qi
- Department of Microsurgery, Orthopedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Guangdong Province Engineering Laboratory for Soft Tissue Biofabrication, Guangzhou, China; Guangdong Provincial Peripheral Nerve Tissue Engineering and Technology Research Center, Guangzhou, China; Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China
| | - Qintang Zhu
- Department of Microsurgery, Orthopedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Guangdong Province Engineering Laboratory for Soft Tissue Biofabrication, Guangzhou, China; Guangdong Provincial Peripheral Nerve Tissue Engineering and Technology Research Center, Guangzhou, China; Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China
| | - Liqiang Gu
- Department of Microsurgery, Orthopedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Guangdong Province Engineering Laboratory for Soft Tissue Biofabrication, Guangzhou, China; Guangdong Provincial Peripheral Nerve Tissue Engineering and Technology Research Center, Guangzhou, China; Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China
| | - Canbin Zheng
- Department of Microsurgery, Orthopedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Guangdong Province Engineering Laboratory for Soft Tissue Biofabrication, Guangzhou, China; Guangdong Provincial Peripheral Nerve Tissue Engineering and Technology Research Center, Guangzhou, China; Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China.
| |
Collapse
|
|
8
|
Josvai M, Polyak E, Kalluri M, Robertson S, Crone WC, Suzuki M. An engineered in vitro model of the human myotendinous junction. Acta Biomater 2024; 180:279-294. [PMID: 38604466 PMCID: PMC11088524 DOI: 10.1016/j.actbio.2024.04.007] [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: 11/21/2023] [Revised: 03/12/2024] [Accepted: 04/03/2024] [Indexed: 04/13/2024]
Abstract
The myotendinous junction (MTJ) is a vulnerable region at the interface of skeletal muscle and tendon that forms an integrated mechanical unit. This study presents a technique for the spatially restrictive co-culture of human embryonic stem cell (hESC)-derived skeletal myocytes and primary tenocytes for two-dimensional modeling of the MTJ. Micropatterned lanes of extracellular matrix and a 2-well culture chamber define the initial regions of occupation. On day 1, both lines occupy less than 20 % of the initially vacant interstitial zone, referred to henceforth as the junction. Myocyte-tenocyte interdigitations are observed by day 7. Immunocytochemistry reveals enhanced organization and alignment of patterned myocyte and tenocyte features, as well as differential expression of multiple MTJ markers. On day 24, electrically stimulated junction myocytes demonstrate negative contractile strains, while positive tensile strains are exhibited by mechanically passive tenocytes at the junction. Unpatterned tenocytes distal to the junction experience significantly decreased strains in comparison to cells at the interface. Unpatterned myocytes have impaired organization and uncoordinated contractile behavior. These findings suggest that this platform is capable of inducing myocyte-tenocyte junction formation and mechanical coupling similar to the native MTJ, showing transduction of force across the cell-cell interface. STATEMENT OF SIGNIFICANCE: The myotendinous junction (MTJ) is an integrated structure that transduces force across the muscle-tendon boundary, making the region vulnerable to strain injury. Despite the clinical relevance, previous in vitro models of the MTJ lack the structure and mechanical accuracy of the native tissue and have difficulty transmitting force across the cell-cell interface. This study demonstrates an in vitro model of the MTJ, using spatially restrictive cues to inform human myocyte-tenocyte interactions and architecture. The model expressed MTJ markers and developed anisotropic myocyte-tenocyte integrations that resemble the native tissue and allow for force transduction from contracting myocytes to passive tenocyte regions. As such, this study presents a system capable of investigating development, injury, and pathology in the human MTJ.
Collapse
Affiliation(s)
- Mitchell Josvai
- Department of Biomedical Engineering, University of Wisconsin-Madison, Engineering Centers Building, 2126, 1550 Engineering Dr, Madison WI 53706, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, 330 N Orchard St, Madison, WI 53715, USA
| | - Erzsebet Polyak
- Department of Comparative Biosciences, University of Wisconsin-Madison, Veterinary Medicine Bldg, 2015 Linden Dr, Madison, WI 53706, USA
| | - Meghana Kalluri
- Department of Biomedical Engineering, University of Wisconsin-Madison, Engineering Centers Building, 2126, 1550 Engineering Dr, Madison WI 53706, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, 330 N Orchard St, Madison, WI 53715, USA
| | - Samantha Robertson
- Department of Comparative Biosciences, University of Wisconsin-Madison, Veterinary Medicine Bldg, 2015 Linden Dr, Madison, WI 53706, USA
| | - Wendy C Crone
- Department of Biomedical Engineering, University of Wisconsin-Madison, Engineering Centers Building, 2126, 1550 Engineering Dr, Madison WI 53706, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, 330 N Orchard St, Madison, WI 53715, USA; The Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison, 1111 Highland Ave, Madison, WI 53705, USA; Department of Nuclear Engineering and Engineering Physics, University of Wisconsin-Madison, 1500 Engineering Drive, Madison, WI 53706, USA; Department of Mechanical Engineering, University of Wisconsin-Madison, 1513 University Avenue, Madison, WI 53706, USA.
| | - Masatoshi Suzuki
- Department of Biomedical Engineering, University of Wisconsin-Madison, Engineering Centers Building, 2126, 1550 Engineering Dr, Madison WI 53706, USA; Department of Comparative Biosciences, University of Wisconsin-Madison, Veterinary Medicine Bldg, 2015 Linden Dr, Madison, WI 53706, USA; The Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison, 1111 Highland Ave, Madison, WI 53705, USA.
| |
Collapse
|
|
9
|
Sakai‐Takemura F, Saito F, Nogami K, Maruyama Y, Elhussieny A, Matsumura K, Takeda S, Aoki Y, Miyagoe‐Suzuki Y. Antioxidants restore store-operated Ca 2+ entry in patient-iPSC-derived myotubes with tubular aggregate myopathy-associated Ile484ArgfsX21 STIM1 mutation via upregulation of binding immunoglobulin protein. FASEB Bioadv 2023; 5:453-469. [PMID: 37936920 PMCID: PMC10626159 DOI: 10.1096/fba.2023-00069] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2023] [Revised: 09/27/2023] [Accepted: 10/11/2023] [Indexed: 11/09/2023] Open
Abstract
Store-operated Ca2+ entry (SOCE) is indispensable for intracellular Ca2+ homeostasis in skeletal muscle, and constitutive activation of SOCE causes tubular aggregate myopathy (TAM). To understand the pathogenesis of TAM, we induced pluripotent stem cells (iPSCs) from a TAM patient with a rare mutation (c.1450_1451insGA; p. Ile484ArgfsX21) in the STIM1 gene. This frameshift mutation produces a truncated STIM1 with a disrupted C-terminal inhibitory domain (CTID) and was reported to diminish SOCE. Myotubes induced from the patient's-iPSCs (TAM myotubes) showed severely impaired SOCE, but antioxidants greatly restored SOCE partly via upregulation of an endoplasmic reticulum (ER) chaperone, BiP (GRP78), in the TAM myotubes. Our observation suggests that antioxidants are promising tools for treatment of TAM caused by reduced SOCE.
Collapse
Affiliation(s)
- Fusako Sakai‐Takemura
- Department of Molecular TherapyNational Institute of Neuroscience, National Center of Neurology and PsychiatryTokyoJapan
| | - Fumiaki Saito
- Department of Neurology, School of MedicineTeikyo UniversityTokyoJapan
| | - Ken'ichiro Nogami
- Department of Molecular TherapyNational Institute of Neuroscience, National Center of Neurology and PsychiatryTokyoJapan
- Department of Neurology, Neurological Institute, Graduate School of Medical ScienceKyushu UniversityFukuokaJapan
| | - Yusuke Maruyama
- Department of Molecular TherapyNational Institute of Neuroscience, National Center of Neurology and PsychiatryTokyoJapan
- Department of Gene Regulation, Faculty of Pharmaceutical ScienceTokyo University of ScienceChibaJapan
| | - Ahmed Elhussieny
- Department of Molecular TherapyNational Institute of Neuroscience, National Center of Neurology and PsychiatryTokyoJapan
- Department of Neurology, Faculty of MedicineMinia UniversityMiniaEgypt
| | | | - Shin'ichi Takeda
- Department of Molecular TherapyNational Institute of Neuroscience, National Center of Neurology and PsychiatryTokyoJapan
| | - Yoshitsugu Aoki
- Department of Molecular TherapyNational Institute of Neuroscience, National Center of Neurology and PsychiatryTokyoJapan
| | - Yuko Miyagoe‐Suzuki
- Department of Molecular TherapyNational Institute of Neuroscience, National Center of Neurology and PsychiatryTokyoJapan
| |
Collapse
|
|
10
|
Yun J, Robertson S, Kim C, Suzuki M, Murphy WL, Gopalan P. Aligned skeletal muscle assembly on a biofunctionalized plant leaf scaffold. Acta Biomater 2023; 171:327-335. [PMID: 37730079 PMCID: PMC10913149 DOI: 10.1016/j.actbio.2023.09.016] [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: 03/17/2023] [Revised: 08/07/2023] [Accepted: 09/11/2023] [Indexed: 09/22/2023]
Abstract
Decellularized plant scaffolds have drawn attention as alternative tissue culture platforms due to their wide accessibility, biocompatibility, and diversity of innate microstructures. Particularly, in this work, monocot leaves with innate uniaxial micropatterned topography were utilized to promote cell alignment and elongation. The leaf scaffold was biofunctionalized with poly(PEGMEMA-r-VDM-r-GMA) copolymer that prevented non-specific protein adsorption and was modified with cell adhesive RGD peptide to enable cell adhesion and growth in serum-free media. The biofunctionalized leaf supported the adhesion, growth, and alignment of various human cells including embryonic stem cells (hESC) derived muscle cells. The hESC-derived myogenic progenitor cells cultured on the biofunctionalized leaf scaffold adopted a parallel orientation and were elongated along the leaf topography. These cells showed significant early myogenic differentiation and muscle-like bundled myotube formation. The aligned cells formed compact myotube assemblies and showed uniaxial muscle contraction under chemical stimulation, a critical requirement for developing functional skeletal muscle tissue. Polymer-functionalized plant leaf scaffolds offer a novel human cell culture platform and have potential in human tissue engineering applications that require parallel alignment of cells. STATEMENT OF SIGNIFICANCE: Plant scaffolds are plentiful sources in nature and present a prefabricated construct to present topographical cues to cells. Their feature width is ideal for human cell alignment and elongation, especially for muscle cells. However, plant scaffolds lack proteins that support mammalian cell culture. We have developed a polymer coated leaf scaffold that enables cell adhesion and growth in serum-free media. Human muscle cells cultured on the biofunctionalized leaf, aligned along the natural parallel micro-patterned leaf topography, and formed muscle-like bundled myotube assemblies. These assemblies showed uniaxial muscular contraction, a critical requirement for developing functional skeletal muscle tissue. The biodiversity of the plant materials offers a novel human cell culture platform with potential in human tissue engineering.
Collapse
Affiliation(s)
- Junsu Yun
- Department of Materials Science and Engineering, University of Wisconsin-Madison, Madison, WI 53705, United States
| | - Samantha Robertson
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53705, United States
| | - Chanul Kim
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53075, United States
| | - Masatoshi Suzuki
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53705, United States.
| | - William L Murphy
- Department of Materials Science and Engineering, University of Wisconsin-Madison, Madison, WI 53705, United States; Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53075, United States; Department of Orthopedics and Rehabilitation, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, United States.
| | - Padma Gopalan
- Department of Materials Science and Engineering, University of Wisconsin-Madison, Madison, WI 53705, United States; Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53075, United States.
| |
Collapse
|
|
11
|
Kulus M, Jankowski M, Kranc W, Golkar Narenji A, Farzaneh M, Dzięgiel P, Zabel M, Antosik P, Bukowska D, Mozdziak P, Kempisty B. Bioreactors, scaffolds and microcarriers and in vitro meat production-current obstacles and potential solutions. Front Nutr 2023; 10:1225233. [PMID: 37743926 PMCID: PMC10513094 DOI: 10.3389/fnut.2023.1225233] [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: 05/18/2023] [Accepted: 08/21/2023] [Indexed: 09/26/2023] Open
Abstract
In vitro meat production presents a potential viable alternative for meat consumption, which could provide the consumer with a product indistinguishable from the original, with very similar nutritional and culinary values. Indeed, the alternative products currently accessible often lack comparable nutritional value or culinary attributes to their animal-derived counterparts. This creates challenges for their global acceptance, particularly in countries where meat consumption holds cultural significance. However, while cultured meat research has been progressing rapidly in recent years, some significant obstacles still need to be overcome before its possible commercialization. Hence, this review summarizes the most current knowledge regarding the history of cultured meat, the currently used cell sources and methods used for the purpose of in vitro meat production, with particular focus on the role of bioreactors, scaffolds and microcarriers in overcoming the current obstacles. The authors put the potential microcarrier and scaffold-based solutions in a context, discussing the ways in which they can impact the way forward for the technology, including the use of considering the potential practical and societal barriers to implementing it as a viable food source worldwide.
Collapse
Affiliation(s)
- Magdalena Kulus
- Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Toruń, Toruń, Poland
| | - Maurycy Jankowski
- Department of Computer Science and Statistics, Poznan University of Medical Sciences, Poznan, Poland
- Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland
| | - Wiesława Kranc
- Department of Anatomy, Poznan University of Medical Sciences, Poznań, Poland
| | - Afsaneh Golkar Narenji
- Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC, United States
| | - Maryam Farzaneh
- Fertility, Infertility and Perinatology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Piotr Dzięgiel
- Division of Histology and Embryology, Department of Human Morphology and Embryology, Wroclaw Medical University, Wroclaw, Poland
| | - Maciej Zabel
- Division of Histology and Embryology, Department of Human Morphology and Embryology, Wroclaw Medical University, Wroclaw, Poland
- Division of Anatomy and Histology, University of Zielona Góra, Zielona Góra, Poland
| | - Paweł Antosik
- Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Toruń, Toruń, Poland
| | - Dorota Bukowska
- Department of Basic and Preclinical Sciences, Institute of Veterinary Medicine, Nicolaus Copernicus University in Toruń, Toruń, Poland
| | - Paul Mozdziak
- Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC, United States
- Physiology Graduate Faculty, North Carolina State University, Raleigh, NC, United States
| | - Bartosz Kempisty
- Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Toruń, Toruń, Poland
- Physiology Graduate Faculty, North Carolina State University, Raleigh, NC, United States
- Division of Anatomy, Department of Human Morphology and Embryology, Wroclaw Medical University, Wroclaw, Poland
- Department of Obstetrics and Gynecology, University Hospital and Masaryk University, Brno, Czechia
| |
Collapse
|
|
12
|
Zhao S, Chen J, Wu L, Tao X, Yaqub N, Chang J. Induced Pluripotent Stem Cells for Tissue-Engineered Skeletal Muscles. Int J Mol Sci 2023; 24:11520. [PMID: 37511279 PMCID: PMC10380861 DOI: 10.3390/ijms241411520] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2023] [Revised: 07/10/2023] [Accepted: 07/13/2023] [Indexed: 07/30/2023] Open
Abstract
Skeletal muscle, which comprises a significant portion of the body, is responsible for vital functions such as movement, metabolism, and overall health. However, severe injuries often result in volumetric muscle loss (VML) and compromise the regenerative capacity of the muscle. Tissue-engineered muscles offer a potential solution to address lost or damaged muscle tissue, thereby restoring muscle function and improving patients' quality of life. Induced pluripotent stem cells (iPSCs) have emerged as a valuable cell source for muscle tissue engineering due to their pluripotency and self-renewal capacity, enabling the construction of tissue-engineered artificial skeletal muscles with applications in transplantation, disease modelling, and bio-hybrid robots. Next-generation iPSC-based models have the potential to revolutionize drug discovery by offering personalized muscle cells for testing, reducing reliance on animal models. This review provides a comprehensive overview of iPSCs in tissue-engineered artificial skeletal muscles, highlighting the advancements, applications, advantages, and challenges for clinical translation. We also discussed overcoming limitations and considerations in differentiation protocols, characterization methods, large-scale production, and translational regulations. By tackling these challenges, iPSCs can unlock transformative advancements in muscle tissue engineering and therapeutic interventions for the future.
Collapse
Affiliation(s)
- Shudong Zhao
- Division of Surgery and Interventional Science, University College London, London NW3 2QG, UK
| | - Jishizhan Chen
- Division of Surgery and Interventional Science, University College London, London NW3 2QG, UK
| | - Lei Wu
- Division of Surgery and Interventional Science, University College London, London NW3 2QG, UK
| | - Xin Tao
- Department of iPS Cell Applications, Kobe University, Kobe 657-8501, Japan
| | - Naheem Yaqub
- Division of Surgery and Interventional Science, University College London, London NW3 2QG, UK
| | - Jinke Chang
- Division of Surgery and Interventional Science, University College London, London NW3 2QG, UK
| |
Collapse
|
|
13
|
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.
Collapse
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.
| |
Collapse
|
|
14
|
Reilly M, Robertson S, Suzuki M. Sphere-Based Expansion of Myogenic Progenitors from Human Pluripotent Stem Cells. Methods Mol Biol 2023; 2640:159-174. [PMID: 36995594 DOI: 10.1007/978-1-0716-3036-5_12] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/31/2023]
Abstract
The protocol presented here is to derive, maintain, and differentiate human pluripotent stem cells into skeletal muscle progenitor/stem cells (myogenic progenitors) using a sphere-based culture approach. This sphere-based culture is an attractive method for maintaining progenitor cells due to their longevity and the presence of cell-cell interactions and molecules. Large numbers of cells can be expanded in culture using this method, which represents a valuable source for cell-based tissue modeling and regenerative medicine.
Collapse
Affiliation(s)
- Megan Reilly
- Department of Comparative Biosciences, The Stem Cell and Regenerative Medicine Center, University of Wisconsin, Madison, WI, USA
| | - Samantha Robertson
- Department of Comparative Biosciences, The Stem Cell and Regenerative Medicine Center, University of Wisconsin, Madison, WI, USA
| | - Masatoshi Suzuki
- Department of Comparative Biosciences, The Stem Cell and Regenerative Medicine Center, University of Wisconsin, Madison, WI, USA.
| |
Collapse
|
|
15
|
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.
Collapse
Affiliation(s)
- Leslie Caron
- Aix-Marseille Univ-INSERM, MMG, Marseille, France
| | | | | |
Collapse
|
|
16
|
Takahashi J, Mizutani T, Sugihara HY, Nagata S, Kato S, Hiraguri Y, Takeoka S, Tsuchiya M, Kuno R, Kakinuma S, Watanabe M, Okamoto R. Suspension culture in a rotating bioreactor for efficient generation of human intestinal organoids. CELL REPORTS METHODS 2022; 2:100337. [PMID: 36452871 PMCID: PMC9701612 DOI: 10.1016/j.crmeth.2022.100337] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/21/2022] [Revised: 09/21/2022] [Accepted: 10/20/2022] [Indexed: 06/17/2023]
Abstract
Human intestinal organoids (HIOs) derived from human pluripotent stem cells (hPSCs) hold great promise for translational medical applications. A common method to obtain HIOs has been to harvest floating hindgut spheroids arising from hPSCs. As this technique is elegant but burdensome due to the complex protocol and line-to-line variability, a more feasible method is desired. Here, we establish a robust differentiation method into suspension-cultured HIOs (s-HIOs) by seeding dissociated cells on a spheroid-forming plate. This protocol realizes the reliable generation of size-controllable spheroids. Under optimized conditions in a rotating bioreactor, the generated spheroids quickly grow and mature into large s-HIOs with supporting mesenchyme. Upon mesenteric transplantation, s-HIOs further mature and develop complex tissue architecture in vivo. This method demonstrates that intestinal tissue can be generated from iPSC-derived HIOs via suspension induction and bioreactor maturation, establishing a reliable culture platform with wide applications in regenerative medicine.
Collapse
Affiliation(s)
- Junichi Takahashi
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Tomohiro Mizutani
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Hady Yuki Sugihara
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Sayaka Nagata
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Shu Kato
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Yui Hiraguri
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Sayaka Takeoka
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Mao Tsuchiya
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Reiko Kuno
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Sei Kakinuma
- Department of Clinical and Diagnostic Laboratory Science, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Mamoru Watanabe
- Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Ryuichi Okamoto
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| |
Collapse
|
|
17
|
Disease Modeling of Pituitary Adenoma Using Human Pluripotent Stem Cells. Cancers (Basel) 2022; 14:cancers14153660. [PMID: 35954322 PMCID: PMC9367606 DOI: 10.3390/cancers14153660] [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: 06/25/2022] [Revised: 07/22/2022] [Accepted: 07/23/2022] [Indexed: 11/17/2022] Open
Abstract
Simple Summary Pituitary adenoma pathophysiology has been studied mainly using murine cell lines, animal models, and pituitary tumor samples. However, the lack of human pituitary cell line is a significant limiting factor in studying the molecular mechanisms of human pituitary tumors. Recently, pituitary induction methods from human-induced pluripotent stem cells (hiPSCs) have been established. These methods can induce human pituitary hormone-producing cells that retain physiological properties. hiPSCs in which tumor-causing gene mutations are introduced using genome-editing techniques, such as CRISPR/Cas9 systems, provide great opportunities to establish in vitro human pituitary adenoma disease models. The models will be a novel platform to discover novel drugs and investigate tumorigenesis and pathophysiology. The purpose of this review is to provide an overview of the applications of iPSCs for pituitary and neoplastic disorder research and genome-editing technologies to create strategies for developing pituitary adenoma models using iPSCs. Abstract Pituitary adenomas are characterized by abnormal growth in the pituitary gland. Surgical excision is the first-line treatment for functional (hormone-producing) pituitary adenomas, except for prolactin-producing adenomas; however, complete excision is technically challenging, and many patients require long-term medication after the treatment. In addition, the pathophysiology of pituitary adenomas, such as tumorigenesis, has not been fully understood. Pituitary adenoma pathophysiology has mainly been studied using animal models and animal tumor-derived cell lines. Nevertheless, experimental studies on human pituitary adenomas are difficult because of the significant differences among species and the lack of reliable cell lines. Recently, several methods have been established to differentiate pituitary cells from human pluripotent stem cells (hPSCs). The induced pituitary hormone-producing cells retain the physiological properties already lost in tumor-derived cell lines. Moreover, CRISPR/Cas9 systems have expedited the introduction of causative gene mutations in various malignant tumors into hPSCs. Therefore, hPSC-derived pituitary cells have great potential as a novel platform for studying the pathophysiology of human-specific pituitary adenomas and developing novel drugs. This review presents an overview of the recent progresses in hPSC applications for pituitary research, functional pituitary adenoma pathogenesis, and genome-editing techniques for introducing causative mutations. We also discuss future applications of hPSCs for studying pituitary adenomas.
Collapse
|
|
18
|
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.
Collapse
|
|
19
|
Marchioretti C, Zuccaro E, Pandey UB, Rosati J, Basso M, Pennuto M. Skeletal Muscle Pathogenesis in Polyglutamine Diseases. Cells 2022; 11:2105. [PMID: 35805189 PMCID: PMC9265456 DOI: 10.3390/cells11132105] [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: 05/19/2022] [Revised: 06/24/2022] [Accepted: 06/28/2022] [Indexed: 01/27/2023] Open
Abstract
Polyglutamine diseases are characterized by selective dysfunction and degeneration of specific types of neurons in the central nervous system. In addition, nonneuronal cells can also be affected as a consequence of primary degeneration or due to neuronal dysfunction. Skeletal muscle is a primary site of toxicity of polyglutamine-expanded androgen receptor, but it is also affected in other polyglutamine diseases, more likely due to neuronal dysfunction and death. Nonetheless, pathological processes occurring in skeletal muscle atrophy impact the entire body metabolism, thus actively contributing to the inexorable progression towards the late and final stages of disease. Skeletal muscle atrophy is well recapitulated in animal models of polyglutamine disease. In this review, we discuss the impact and relevance of skeletal muscle in patients affected by polyglutamine diseases and we review evidence obtained in animal models and patient-derived cells modeling skeletal muscle.
Collapse
Affiliation(s)
- Caterina Marchioretti
- Department of Biomedical Sciences (DBS), University of Padova, 35131 Padova, Italy; (C.M.); (E.Z.)
- Veneto Institute of Molecular Medicine (VIMM), 35129 Padova, Italy
| | - Emanuela Zuccaro
- Department of Biomedical Sciences (DBS), University of Padova, 35131 Padova, Italy; (C.M.); (E.Z.)
- Veneto Institute of Molecular Medicine (VIMM), 35129 Padova, Italy
| | - Udai Bhan Pandey
- Department of Pediatrics, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15100, USA;
| | - Jessica Rosati
- Cellular Reprogramming Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71100 Foggia, Italy;
| | - Manuela Basso
- Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, 38100 Trento, Italy;
| | - Maria Pennuto
- Department of Biomedical Sciences (DBS), University of Padova, 35131 Padova, Italy; (C.M.); (E.Z.)
- Veneto Institute of Molecular Medicine (VIMM), 35129 Padova, Italy
| |
Collapse
|
|
20
|
Patel N, Chong K, Baydur A. Methods and Applications in Respiratory Physiology: Respiratory Mechanics, Drive and Muscle Function in Neuromuscular and Chest Wall Disorders. Front Physiol 2022; 13:838414. [PMID: 35774289 PMCID: PMC9237333 DOI: 10.3389/fphys.2022.838414] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2021] [Accepted: 05/10/2022] [Indexed: 11/13/2022] Open
Abstract
Individuals with neuromuscular and chest wall disorders experience respiratory muscle weakness, reduced lung volume and increases in respiratory elastance and resistance which lead to increase in work of breathing, impaired gas exchange and respiratory pump failure. Recently developed methods to assess respiratory muscle weakness, mechanics and movement supplement traditionally employed spirometry and methods to evaluate gas exchange. These include recording postural change in vital capacity, respiratory pressures (mouth and sniff), electromyography and ultrasound evaluation of diaphragmatic thickness and excursions. In this review, we highlight key aspects of the pathophysiology of these conditions as they impact the patient and describe measures to evaluate respiratory dysfunction. We discuss potential areas of physiologic investigation in the evaluation of respiratory aspects of these disorders.
Collapse
|
|
21
|
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.
Collapse
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
| |
Collapse
|
|
22
|
Cell Surface Proteins for Enrichment and In Vitro Characterization of Human Pluripotent Stem Cell-Derived Myogenic Progenitors. Stem Cells Int 2022; 2022:2735414. [PMID: 35251185 PMCID: PMC8894063 DOI: 10.1155/2022/2735414] [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: 10/15/2021] [Revised: 01/04/2022] [Accepted: 01/12/2022] [Indexed: 11/17/2022] Open
Abstract
Human myogenic progenitors can be derived from pluripotent stem cells (PSCs) for use in modeling natural and pathological myogenesis, as well as treating muscle diseases. Transgene-free methods of deriving myogenic progenitors from different PSC lines often produce mixed populations that are heterogeneous in myogenic differentiation potential, yet detailed and accurate characterization of human PSC-derived myogenic progenitors remains elusive in the field. The isolation and purification of human PSC-derived myogenic progenitors is thus an important methodological consideration when we investigate the properties and behaviors of these cells in culture. We previously reported a transgene-free, serum-free floating sphere culture method for the derivation of myogenic progenitors from human PSCs. In this study, we first performed comprehensive cell surface protein profiling of the sphere culture cells through the screening of 255 antibodies. Next, we used magnetic activated cell sorting and enriched the cells according to the expression of specific surface markers. The ability of muscle differentiation in the resulting cells was characterized by immunofluorescent labeling and quantification of positively stained cells. Our results revealed that myotube-forming cells resided in the differentiated cultures of CD29+, CD56+, CD271+, and CD15– fractions, while thick and multinucleated myotubes were identified in the differentiated cultures from CD9+ and CD146+ fractions. We found that PAX7 localization to the nucleus correlates with myotube-forming ability in these sorted populations. We also demonstrated that cells in unsorted, CD271+, and CD15– fractions responded differently to cryopreservation and prolonged culture expansion. Lastly, we showed that CD271 expression is essential for terminal differentiation of human PSC-derived myogenic progenitors. Taken together, these cell surface proteins are not only useful markers to identify unique cellular populations in human PSC-derived myogenic progenitors but also functionally important molecules that can provide valuable insight into human myogenesis.
Collapse
|
|
23
|
Singh S, Singh T, Kunja C, Dhoat NS, Dhania NK. Gene-editing, immunological and iPSCs based therapeutics for muscular dystrophy. Eur J Pharmacol 2021; 912:174568. [PMID: 34656607 DOI: 10.1016/j.ejphar.2021.174568] [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: 06/24/2021] [Revised: 09/25/2021] [Accepted: 10/11/2021] [Indexed: 10/20/2022]
Abstract
Muscular dystrophy is a well-known genetically heterogeneous group of rare muscle disorders. This progressive disease causes the breakdown of skeletal muscles over time and leads to grave weakness. This breakdown is caused by a diverse pattern of mutations in dystrophin and dystrophin associated protein complex. These mutations lead to the production of altered proteins in response to which, the body stimulates production of various cytokines and immune cells, particularly reactive oxygen species and NFκB. Immune cells display/exhibit a dual role by inducing muscle damage and muscle repair. Various anti-oxidants, anti-inflammatory and glucocorticoid drugs serve as potent therapeutics for muscular dystrophy. Along with the above mentioned therapeutics, induced pluripotent stem cells also serve as a novel approach paving a way for personalized treatment. These pluripotent stem cells allow regeneration of large numbers of regenerative myogenic progenitors that can be administered in muscular dystrophy patients which assist in the recovery of lost muscle fibers. In this review, we have summarized gene-editing, immunological and induced pluripotent stem cell based therapeutics for muscular dystrophy treatment.
Collapse
Affiliation(s)
- Shagun Singh
- Department of Human Genetics and Molecular Medicine, School of Health Sciences, Central University of Punjab, Bathinda-151001, Punjab, India
| | - Tejpal Singh
- Department of Human Genetics and Molecular Medicine, School of Health Sciences, Central University of Punjab, Bathinda-151001, Punjab, India
| | - Chaitanya Kunja
- Department of Human Genetics and Molecular Medicine, School of Health Sciences, Central University of Punjab, Bathinda-151001, Punjab, India
| | - Navdeep S Dhoat
- Department of Pediatrics Surgery, All India Institute of Medical Sciences, Bathinda, 151001, Punjab, India
| | - Narender K Dhania
- Department of Human Genetics and Molecular Medicine, School of Health Sciences, Central University of Punjab, Bathinda-151001, Punjab, India.
| |
Collapse
|
|
24
|
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.
Collapse
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.
| |
Collapse
|
|
25
|
Yan L, Rodríguez-delaRosa A, Pourquié O. Human muscle production in vitro from pluripotent stem cells: Basic and clinical applications. Semin Cell Dev Biol 2021; 119:39-48. [PMID: 33941447 PMCID: PMC8530835 DOI: 10.1016/j.semcdb.2021.04.017] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2021] [Accepted: 04/19/2021] [Indexed: 10/21/2022]
Abstract
Human pluripotent stem cells (PSCs), which have the capacity to self-renew and differentiate into multiple cell types, offer tremendous therapeutic potential and invaluable flexibility as research tools. Recently, remarkable progress has been made in directing myogenic differentiation of human PSCs. The differentiation strategies, which were inspired by our knowledge of myogenesis in vivo, have provided an important platform for the study of human muscle development and modeling of muscular diseases, as well as a promising source of cells for cell therapy to treat muscular dystrophies. In this review, we summarize the current state of skeletal muscle generation from human PSCs, including transgene-based and transgene-free differentiation protocols, and 3D muscle tissue production through bioengineering approaches. We also highlight their basic and clinical applications, which facilitate the study of human muscle biology and deliver new hope for muscular disease treatment.
Collapse
Affiliation(s)
- Lu Yan
- Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA; Department of Genetics, Harvard Medical School, Boston, MA, USA; Harvard Stem Cell Institute, Boston, MA, USA
| | - Alejandra Rodríguez-delaRosa
- Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA; Department of Genetics, Harvard Medical School, Boston, MA, USA; Harvard Stem Cell Institute, Boston, MA, USA
| | - Olivier Pourquié
- Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA; Department of Genetics, Harvard Medical School, Boston, MA, USA; Harvard Stem Cell Institute, Boston, MA, USA.
| |
Collapse
|
|
26
|
Saburina IN, Kosheleva NV, Kopylov AT, Lipina TV, Krasina ME, Zurina IM, Gorkun AA, Girina SS, Pulin AA, Kaysheva AL, Morozov SG. Proteomic and electron microscopy study of myogenic differentiation of alveolar mucosa multipotent mesenchymal stromal cells in three-dimensional culture. Proteomics 2021; 22:e2000304. [PMID: 34674377 DOI: 10.1002/pmic.202000304] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 08/24/2021] [Accepted: 10/08/2021] [Indexed: 12/15/2022]
Abstract
Myocyte differentiation is featured by adaptation processes, including mitochondria repopulation and cytoskeleton re-organization. The difference between monolayer and spheroid cultured cells at the proteomic level is uncertain. We cultivated alveolar mucosa multipotent mesenchymal stromal cells in spheroids in a myogenic way for the proper conditioning of ECM architecture and cell morphology, which induced spontaneous myogenic differentiation of cells within spheroids. Electron microscopy analysis was used for the morphometry of mitochondria biogenesis, and proteomic was used complementary to unveil events underlying differences between two-dimensional/three-dimensional myoblasts differentiation. The prevalence of elongated mitochondria with an average area of 0.097 μm2 was attributed to monolayer cells 7 days after the passage. The population of small mitochondria with a round shape and area of 0.049 μm2 (p < 0.05) was observed in spheroid cells cultured under three-dimensional conditions. Cells in spheroids were quantitatively enriched in proteins of mitochondria biogenesis (DNM1L, IDH2, SSBP1), respiratory chain (ACO2, ATP5I, COX5A), extracellular proteins (COL12A1, COL6A1, COL6A2), and cytoskeleton (MYL6, MYL12B, MYH10). Most of the Rab-related transducers were inhibited in spheroid culture. The proteomic assay demonstrated delicate mechanisms of mitochondria autophagy and repopulation, cytoskeleton assembling, and biogenesis. Differences in the ultrastructure of mitochondria indicate active biogenesis under three-dimensional conditions.
Collapse
Affiliation(s)
- Irina N Saburina
- FSBSI Institute of General Pathology and Pathophysiology, Moscow, Russian Federation
| | - Nastasia V Kosheleva
- FSBSI Institute of General Pathology and Pathophysiology, Moscow, Russian Federation.,Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow, Russian Federation.,World-Class Research Center "Digital Biodesign and Personalized Healthcare", Sechenov University, Moscow, Russia
| | - Arthur T Kopylov
- FSBSI Institute of General Pathology and Pathophysiology, Moscow, Russian Federation.,World-Class Research Center "Digital Biodesign and Personalized Healthcare", Sechenov University, Moscow, Russia.,Department of Proteomic Research, Institute of Biomedical Chemistry, Moscow, Russian Federation
| | - Tatiana V Lipina
- Faculty of Biology, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Marina E Krasina
- Faculty of Biology, Lomonosov Moscow State University, Moscow, Russian Federation
| | - Irina M Zurina
- FSBSI Institute of General Pathology and Pathophysiology, Moscow, Russian Federation.,Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow, Russian Federation
| | - Anastasiya A Gorkun
- FSBSI Institute of General Pathology and Pathophysiology, Moscow, Russian Federation.,Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow, Russian Federation
| | - Svetlana S Girina
- FSBSI Institute of General Pathology and Pathophysiology, Moscow, Russian Federation
| | - Andrey A Pulin
- Pirogov National Medical Surgical Center, Moscow, Russian Federation
| | - Anna L Kaysheva
- Department of Proteomic Research, Institute of Biomedical Chemistry, Moscow, Russian Federation
| | - Sergey G Morozov
- FSBSI Institute of General Pathology and Pathophysiology, Moscow, Russian Federation
| |
Collapse
|
|
27
|
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.
Collapse
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
| |
Collapse
|
|
28
|
Lynch EM, Robertson S, FitzGibbons C, Reilly M, Switalski C, Eckardt A, Tey SR, Hayakawa K, Suzuki M. Transcriptome analysis using patient iPSC-derived skeletal myocytes: Bet1L as a new molecule possibly linked to neuromuscular junction degeneration in ALS. Exp Neurol 2021; 345:113815. [PMID: 34310943 DOI: 10.1016/j.expneurol.2021.113815] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2021] [Revised: 06/23/2021] [Accepted: 07/20/2021] [Indexed: 02/07/2023]
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease in which patients gradually become paralyzed due to loss of motor function. Many genetically inheritable mutations have been linked to ALS; however, the majority of ALS patients are considered sporadic. Therefore, there is a need for a common therapy that is effective for all ALS patients. Although there is evidence of the disease beginning in the periphery at the neuromuscular junction (NMJ), the specific processes involved in skeletal muscle and at the NMJ are still largely unknown. To study common disease mechanisms in ALS skeletal muscle, we performed RNA sequencing of skeletal myocytes differentiated from induced pluripotent stem cells (iPSCs) derived from familial ALS (with C9ORF72, SOD1, or TARDBP mutations) and sporadic ALS patients. Compared to healthy control lines, the myocytes from all ALS lines showed downregulation of four genes: BET1L, DCX, GPC3, and HNRNPK. We next measured the expression levels of these four genes in hind limb muscle samples from a rat model of familial ALS (SOD1G93A transgenic) and found that only the Bet1L gene, which encodes Bet1 Golgi Vesicular Membrane Trafficking Protein Like, was commonly downregulated. Bet1L protein appeared to be localized to the basal lamina of the NMJ, with decreased expression over time in SOD1G93A transgenic rats. Importantly, the expression levels began to decrease early in the disease process. Our results indicate that loss of Bet1L at the NMJ could be of interest for better understanding ALS disease progression.
Collapse
Affiliation(s)
- Eileen M Lynch
- Department of Comparative Biosciences, University of Wisconsin-Madison, WI, USA
| | - Samantha Robertson
- Department of Comparative Biosciences, University of Wisconsin-Madison, WI, USA
| | - Claire FitzGibbons
- Department of Comparative Biosciences, University of Wisconsin-Madison, WI, USA
| | - Megan Reilly
- Department of Comparative Biosciences, University of Wisconsin-Madison, WI, USA
| | - Colton Switalski
- Department of Comparative Biosciences, University of Wisconsin-Madison, WI, USA
| | - Adam Eckardt
- Department of Comparative Biosciences, University of Wisconsin-Madison, WI, USA
| | - Sin-Ruow Tey
- Department of Comparative Biosciences, University of Wisconsin-Madison, WI, USA
| | - Koji Hayakawa
- Department of Toxicology, Faculty of Veterinary Medicine, Okayama University of Science, Ehime, Japan
| | - Masatoshi Suzuki
- Department of Comparative Biosciences, University of Wisconsin-Madison, WI, USA; Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison, WI, USA.
| |
Collapse
|
|
29
|
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.
Collapse
Affiliation(s)
- Takahiko Sato
- Department of Anatomy, Fujita Health University, Toyoake, Japan.,AMED-CREST, AMED, Otemachi, Chiyoda, Tokyo, Japan
| |
Collapse
|
|
30
|
Reiss J, Robertson S, Suzuki M. Cell Sources for Cultivated Meat: Applications and Considerations throughout the Production Workflow. Int J Mol Sci 2021; 22:7513. [PMID: 34299132 PMCID: PMC8307620 DOI: 10.3390/ijms22147513] [Citation(s) in RCA: 67] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2021] [Revised: 07/08/2021] [Accepted: 07/09/2021] [Indexed: 12/11/2022] Open
Abstract
Cellular agriculture is an emerging scientific discipline that leverages the existing principles behind stem cell biology, tissue engineering, and animal sciences to create agricultural products from cells in vitro. Cultivated meat, also known as clean meat or cultured meat, is a prominent subfield of cellular agriculture that possesses promising potential to alleviate the negative externalities associated with conventional meat production by producing meat in vitro instead of from slaughter. A core consideration when producing cultivated meat is cell sourcing. Specifically, developing livestock cell sources that possess the necessary proliferative capacity and differentiation potential for cultivated meat production is a key technical component that must be optimized to enable scale-up for commercial production of cultivated meat. There are several possible approaches to develop cell sources for cultivated meat production, each possessing certain advantages and disadvantages. This review will discuss the current cell sources used for cultivated meat production and remaining challenges that need to be overcome to achieve scale-up of cultivated meat for commercial production. We will also discuss cell-focused considerations in other components of the cultivated meat production workflow, namely, culture medium composition, bioreactor expansion, and biomaterial tissue scaffolding.
Collapse
Affiliation(s)
- Jacob Reiss
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA; (J.R.); (S.R.)
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Samantha Robertson
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA; (J.R.); (S.R.)
| | - Masatoshi Suzuki
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA; (J.R.); (S.R.)
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
- Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison, Madison, WI 53706, USA
| |
Collapse
|
|
31
|
Viral vector gene delivery of the novel chaperone protein SRCP1 to modify insoluble protein in in vitro and in vivo models of ALS. Gene Ther 2021:10.1038/s41434-021-00276-4. [PMID: 34239068 PMCID: PMC8741877 DOI: 10.1038/s41434-021-00276-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2020] [Revised: 06/17/2021] [Accepted: 06/25/2021] [Indexed: 01/11/2023]
Abstract
Protein misfolding and aggregation are shared features of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and protein quality control disruption contributes to neuronal toxicity. Therefore, reducing protein aggregation could hold therapeutic potential. We previously identified a novel chaperone protein, serine-rich chaperone protein 1 (SRCP1), that effectively prevents protein aggregation in cell culture and zebrafish models of Huntington's disease. Here we tested whether this benefit extends to aggregated proteins found in ALS. We used viral-mediated expression of SRCP1 in in vitro and in vivo models of ALS. We found that SRCP1 reduced insoluble SOD1 protein levels in HEK293T cells overexpressing either the A4V or G93R mutant SOD1. However, the reduction of insoluble protein was not observed in either mutant C9orf72 or SOD1 ALS iPSC-derived motor neurons infected with a lentivirus expressing SRCP1. SOD1-G93A ALS mice injected with AAV-SRCP1 showed a small but significant reduction in insoluble and soluble SOD1 in both the brain and spinal cord, but SRCP1 expression did not improve mouse survival. These data indicate that SRCP1 likely reduces insoluble protein burden in a protein and/or context-dependent manner indicating a need for additional insight into SRCP1 function and therapeutic potential.
Collapse
|
|
32
|
Lutz AK, Pfaender S, Incearap B, Ioannidis V, Ottonelli I, Föhr KJ, Cammerer J, Zoller M, Higelin J, Giona F, Stetter M, Stoecker N, Alami NO, Schön M, Orth M, Liebau S, Barbi G, Grabrucker AM, Delorme R, Fauler M, Mayer B, Jesse S, Roselli F, Ludolph AC, Bourgeron T, Verpelli C, Demestre M, Boeckers TM. Autism-associated SHANK3 mutations impair maturation of neuromuscular junctions and striated muscles. Sci Transl Med 2021; 12:12/547/eaaz3267. [PMID: 32522805 DOI: 10.1126/scitranslmed.aaz3267] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2019] [Revised: 12/09/2019] [Accepted: 04/07/2020] [Indexed: 12/12/2022]
Abstract
Heterozygous mutations of the gene encoding the postsynaptic protein SHANK3 are associated with syndromic forms of autism spectrum disorders (ASDs). One of the earliest clinical symptoms in SHANK3-associated ASD is neonatal skeletal muscle hypotonia. This symptom can be critical for the early diagnosis of affected children; however, the mechanism mediating hypotonia in ASD is not completely understood. Here, we used a combination of patient-derived human induced pluripotent stem cells (hiPSCs), Shank3Δ11(-/-) mice, and Phelan-McDermid syndrome (PMDS) muscle biopsies from patients of different ages to analyze the role of SHANK3 on motor unit development. Our results suggest that the hypotonia in SHANK3 deficiency might be caused by dysfunctions in all elements of the voluntary motor system: motoneurons, neuromuscular junctions (NMJs), and striated muscles. We found that SHANK3 localizes in Z-discs in the skeletal muscle sarcomere and co-immunoprecipitates with α-ACTININ. SHANK3 deficiency lead to shortened Z-discs and severe impairment of acetylcholine receptor clustering in hiPSC-derived myotubes and in muscle from Shank3Δ11(-/-) mice and patients with PMDS, indicating a crucial role for SHANK3 in the maturation of NMJs and striated muscle. Functional motor defects in Shank3Δ11(-/-) mice could be rescued with the troponin activator Tirasemtiv that sensitizes muscle fibers to calcium. Our observations give insight into the function of SHANK3 besides the central nervous system and imply potential treatment strategies for SHANK3-associated ASD.
Collapse
Affiliation(s)
- Anne-Kathrin Lutz
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany
| | - Stefanie Pfaender
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany
| | - Berra Incearap
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany
| | - Valentin Ioannidis
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany
| | - Ilaria Ottonelli
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany
| | - Karl J Föhr
- Department of Anesthesiology, Ulm University Hospital, 89081 Ulm, Germany
| | - Judith Cammerer
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany
| | - Marvin Zoller
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany
| | - Julia Higelin
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany
| | - Federica Giona
- CNR Neuroscience Institute, University of Milan, 20129 Milan, Italy.,BIOMETRA University of Milan, 20129 Milan, Italy
| | - Maximilian Stetter
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany
| | - Nicole Stoecker
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany
| | | | - Michael Schön
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany
| | | | - Stefan Liebau
- Institute of Neuroanatomy and Developmental Biology, Eberhard Karls University Tübingen, 72074 Tübingen, Germany
| | - Gotthold Barbi
- Institute for Human Genetics, Ulm University Hospital, 89081 Ulm, Germany
| | - Andreas M Grabrucker
- Cellular Neurobiology and Neuro-Nanotechnology Lab, Department of Biological Sciences, University of Limerick, V94PH61 Limerick, Ireland.,Bernal Institute, University of Limerick, V94T9PX Limerick, Ireland.,Health Research Institute (HRI), University of Limerick, V94T9PX Limerick, Ireland
| | - Richard Delorme
- Child and Adolescent Psychiatry Department, APHP, Robert-Debré Hospital, 750197 Paris, France
| | - Michael Fauler
- Institute of General Physiology, Ulm University, 89081 Ulm, Germany
| | - Benjamin Mayer
- Institute of Epidemiology and Medical Biometry, Ulm University, 89075 Ulm, Germany
| | | | | | | | - Thomas Bourgeron
- Génétique Humaine et Fonctions Cognitives, Université Paris Diderot, Institut Pasteur, 75015 Paris, France
| | - Chiara Verpelli
- CNR Neuroscience Institute, University of Milan, 20129 Milan, Italy.,BIOMETRA University of Milan, 20129 Milan, Italy
| | - Maria Demestre
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany.
| | - Tobias M Boeckers
- Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany. .,DZNE, Ulm Site, 89081 Ulm, Germany
| |
Collapse
|
|
33
|
Alarcin E, Bal-Öztürk A, Avci H, Ghorbanpoor H, Dogan Guzel F, Akpek A, Yesiltas G, Canak-Ipek T, Avci-Adali M. Current Strategies for the Regeneration of Skeletal Muscle Tissue. Int J Mol Sci 2021; 22:5929. [PMID: 34072959 PMCID: PMC8198586 DOI: 10.3390/ijms22115929] [Citation(s) in RCA: 37] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2021] [Revised: 05/21/2021] [Accepted: 05/26/2021] [Indexed: 12/11/2022] Open
Abstract
Traumatic injuries, tumor resections, and degenerative diseases can damage skeletal muscle and lead to functional impairment and severe disability. Skeletal muscle regeneration is a complex process that depends on various cell types, signaling molecules, architectural cues, and physicochemical properties to be successful. To promote muscle repair and regeneration, various strategies for skeletal muscle tissue engineering have been developed in the last decades. However, there is still a high demand for the development of new methods and materials that promote skeletal muscle repair and functional regeneration to bring approaches closer to therapies in the clinic that structurally and functionally repair muscle. The combination of stem cells, biomaterials, and biomolecules is used to induce skeletal muscle regeneration. In this review, we provide an overview of different cell types used to treat skeletal muscle injury, highlight current strategies in biomaterial-based approaches, the importance of topography for the successful creation of functional striated muscle fibers, and discuss novel methods for muscle regeneration and challenges for their future clinical implementation.
Collapse
Affiliation(s)
- Emine Alarcin
- Department of Pharmaceutical Technology, Faculty of Pharmacy, Marmara University, 34854 Istanbul, Turkey;
| | - Ayca Bal-Öztürk
- Department of Analytical Chemistry, Faculty of Pharmacy, Istinye University, 34010 Istanbul, Turkey;
- Department of Stem Cell and Tissue Engineering, Institute of Health Sciences, Istinye University, 34010 Istanbul, Turkey
| | - Hüseyin Avci
- Department of Metallurgical and Materials Engineering, Eskisehir Osmangazi University, 26040 Eskisehir, Turkey;
- Cellular Therapy and Stem Cell Research Center, Eskisehir Osmangazi University, 26040 Eskisehir, Turkey
- AvciBio Research Group, Eskisehir Osmangazi University, 26040 Eskisehir, Turkey;
- Translational Medicine Research and Clinical Center, Eskisehir Osmangazi University, 26040 Eskisehir, Turkey
| | - Hamed Ghorbanpoor
- AvciBio Research Group, Eskisehir Osmangazi University, 26040 Eskisehir, Turkey;
- Department of Biomedical Engineering, Ankara Yildirim Beyazit University, 06010 Ankara, Turkey;
- Department of Biomedical Engineering, Eskisehir Osmangazi University, 26040 Eskisehir, Turkey
| | - Fatma Dogan Guzel
- Department of Biomedical Engineering, Ankara Yildirim Beyazit University, 06010 Ankara, Turkey;
| | - Ali Akpek
- Department of Bioengineering, Gebze Technical University, 41400 Gebze, Turkey; (A.A.); (G.Y.)
| | - Gözde Yesiltas
- Department of Bioengineering, Gebze Technical University, 41400 Gebze, Turkey; (A.A.); (G.Y.)
| | - Tuba Canak-Ipek
- Department of Thoracic and Cardiovascular Surgery, University Hospital Tuebingen, Calwerstraße 7/1, 72076 Tuebingen, Germany;
| | - Meltem Avci-Adali
- Department of Thoracic and Cardiovascular Surgery, University Hospital Tuebingen, Calwerstraße 7/1, 72076 Tuebingen, Germany;
| |
Collapse
|
|
34
|
Selmin G, Gagliano O, De Coppi P, Serena E, Urciuolo A, Elvassore N. MYOD modified mRNA drives direct on-chip programming of human pluripotent stem cells into skeletal myocytes. Biochem Biophys Res Commun 2021; 560:139-145. [PMID: 33989905 DOI: 10.1016/j.bbrc.2021.04.129] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2021] [Accepted: 04/28/2021] [Indexed: 10/21/2022]
Abstract
Drug screening and disease modelling for skeletal muscle related pathologies would strongly benefit from the integration of myogenic cells derived from human pluripotent stem cells within miniaturized cell culture devices, such as microfluidic platform. Here, we identified the optimal culture conditions that allow direct differentiation of human pluripotent stem cells in myogenic cells within microfluidic devices. Myogenic cells are efficiently derived from both human embryonic (hESC) or induced pluripotent stem cells (hiPSC) in eleven days by combining small molecules and non-integrating modified mRNA (mmRNA) encoding for the master myogenic transcription factor MYOD. Our work opens new perspective for the development of patient-specific platforms in which a one-step myogenic differentiation could be used to generate skeletal muscle on-a-chip.
Collapse
Affiliation(s)
- Giulia Selmin
- Great Ormond Street Institute of Child Health, University College London, WC1N1EH, London, UK
| | - Onelia Gagliano
- Venetian Institute of Molecular Medicine (VIMM), 35129, Padova, Italy; Industrial Engineering Department, University of Padova, 35131, Padova, Italy
| | - Paolo De Coppi
- Great Ormond Street Institute of Child Health, University College London, WC1N1EH, London, UK
| | - Elena Serena
- Venetian Institute of Molecular Medicine (VIMM), 35129, Padova, Italy
| | - Anna Urciuolo
- Great Ormond Street Institute of Child Health, University College London, WC1N1EH, London, UK; Molecular Medicine Department, University of Padova, Italy
| | - Nicola Elvassore
- Great Ormond Street Institute of Child Health, University College London, WC1N1EH, London, UK; Venetian Institute of Molecular Medicine (VIMM), 35129, Padova, Italy; Industrial Engineering Department, University of Padova, 35131, Padova, Italy.
| |
Collapse
|
|
35
|
Guo X, Badu-Mensah A, Thomas MC, McAleer CW, Hickman JJ. Characterization of Functional Human Skeletal Myotubes and Neuromuscular Junction Derived-From the Same Induced Pluripotent Stem Cell Source. Bioengineering (Basel) 2020; 7:E133. [PMID: 33105732 PMCID: PMC7712960 DOI: 10.3390/bioengineering7040133] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2020] [Revised: 09/25/2020] [Accepted: 10/15/2020] [Indexed: 01/16/2023] Open
Abstract
In vitro generation of functional neuromuscular junctions (NMJs) utilizing the same induced pluripotent stem cell (iPSC) source for muscle and motoneurons would be of great value for disease modeling and tissue engineering. Although, differentiation and characterization of iPSC-derived motoneurons are well established, and iPSC-derived skeletal muscle (iPSC-SKM) has been reported, there is a general lack of systemic and functional characterization of the iPSC-SKM. This study performed a systematic characterization of iPSC-SKM differentiated using a serum-free, small molecule-directed protocol. Morphologically, the iPSC-SKM demonstrated the expression and appropriate distribution of acetylcholine, ryanodine and dihydropyridine receptors. Fiber type analysis revealed a mixture of human fast (Type IIX, IIA) and slow (Type I) muscle types and the absence of animal Type IIB fibers. Functionally, the iPSC-SKMs contracted synchronously upon electrical stimulation, with the contraction force comparable to myofibers derived from primary myoblasts. Most importantly, when co-cultured with human iPSC-derived motoneurons from the same iPSC source, the myofibers contracted in response to motoneuron stimulation indicating the formation of functional NMJs. By demonstrating comparable structural and functional capacity to primary myoblast-derived myofibers, this defined, iPSC-SKM system, as well as the personal NMJ system, has applications for patient-specific drug testing and investigation of muscle physiology and disease.
Collapse
Affiliation(s)
- Xiufang Guo
- NanoScience Technology Center, University of Central Florida, 12424 Research Parkway, Suite 400, Orlando, FL 32826, USA; (X.G.); (A.B.-M.); (M.C.T.)
| | - Agnes Badu-Mensah
- NanoScience Technology Center, University of Central Florida, 12424 Research Parkway, Suite 400, Orlando, FL 32826, USA; (X.G.); (A.B.-M.); (M.C.T.)
- Biomolecular Science Center, Burnett School of Biomedical Sciences, University of Central Florida, Orlando, FL 32826, USA
| | - Michael C. Thomas
- NanoScience Technology Center, University of Central Florida, 12424 Research Parkway, Suite 400, Orlando, FL 32826, USA; (X.G.); (A.B.-M.); (M.C.T.)
| | | | - James J. Hickman
- NanoScience Technology Center, University of Central Florida, 12424 Research Parkway, Suite 400, Orlando, FL 32826, USA; (X.G.); (A.B.-M.); (M.C.T.)
- Biomolecular Science Center, Burnett School of Biomedical Sciences, University of Central Florida, Orlando, FL 32826, USA
- Hesperos Inc., 12501 Research Pkwy, Suite 100, Orlando, FL 32826, USA;
| |
Collapse
|
|
36
|
Adami R, Bottai D. Spinal Muscular Atrophy Modeling and Treatment Advances by Induced Pluripotent Stem Cells Studies. Stem Cell Rev Rep 2020; 15:795-813. [PMID: 31863335 DOI: 10.1007/s12015-019-09910-6] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Spinal Muscular Atrophy (SMA) is a neurodegenerative disease characterized by specific and predominantly lower motor neuron (MN) loss. SMA is the main reason for infant death, while about one in 40 children born is a healthy carrier. SMA is caused by decreased levels of production of a ubiquitously expressed gene: the survival motor neuron (SMN). All SMA patients present mutations of the telomeric SMN1 gene, but many copies of a centromeric, partially functional paralog gene, SMN2, can somewhat compensate for the SMN1 deficiency, scaling inversely with phenotypic harshness. Because the study of neural tissue in and from patients presents too many challenges and is very often not feasible; the use of animal models, such as the mouse, had a pivotal impact in our understanding of SMA pathology but could not portray totally satisfactorily the elaborate regulatory mechanisms that are present in higher animals, particularly in humans. And while recent therapeutic achievements have been substantial, especially for very young infants, some issues should be considered for the treatment of older patients. An alternative way to study SMA, and other neurological pathologies, is the use of induced pluripotent stem cells (iPSCs) derived from patients. In this work, we will present a wide analysis of the uses of iPSCs in SMA pathology, starting from basic science to their possible roles as therapeutic tools.
Collapse
Affiliation(s)
- Raffaella Adami
- Department of Health Sciences, University of Milan, via A. di Rudinì 8, 20142, Milan, Italy
| | - Daniele Bottai
- Department of Health Sciences, University of Milan, via A. di Rudinì 8, 20142, Milan, Italy.
| |
Collapse
|
|
37
|
Mazaleyrat K, Badja C, Broucqsault N, Chevalier R, Laberthonnière C, Dion C, Baldasseroni L, El-Yazidi C, Thomas M, Bachelier R, Altié A, Nguyen K, Lévy N, Robin JD, Magdinier F. Multilineage Differentiation for Formation of Innervated Skeletal Muscle Fibers from Healthy and Diseased Human Pluripotent Stem Cells. Cells 2020; 9:cells9061531. [PMID: 32585982 PMCID: PMC7349825 DOI: 10.3390/cells9061531] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2020] [Revised: 06/16/2020] [Accepted: 06/16/2020] [Indexed: 12/19/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) obtained by reprogramming primary somatic cells have revolutionized the fields of cell biology and disease modeling. However, the number protocols for generating mature muscle fibers with sarcolemmal organization using iPSCs remain limited, and partly mimic the complexity of mature skeletal muscle. Methods: We used a novel combination of small molecules added in a precise sequence for the simultaneous codifferentiation of human iPSCs into skeletal muscle cells and motor neurons. Results: We show that the presence of both cell types reduces the production time for millimeter-long multinucleated muscle fibers with sarcolemmal organization. Muscle fiber contractions are visible in 19–21 days, and can be maintained over long period thanks to the production of innervated multinucleated mature skeletal muscle fibers with autonomous cell regeneration of PAX7-positive cells and extracellular matrix synthesis. The sequential addition of specific molecules recapitulates key steps of human peripheral neurogenesis and myogenesis. Furthermore, this organoid-like culture can be used for functional evaluation and drug screening. Conclusion: Our protocol, which is applicable to hiPSCs from healthy individuals, was validated in Duchenne Muscular Dystrophy, Myotonic Dystrophy, Facio-Scapulo-Humeral Dystrophy and type 2A Limb-Girdle Muscular Dystrophy, opening new paths for the exploration of muscle differentiation, disease modeling and drug discovery.
Collapse
Affiliation(s)
- Kilian Mazaleyrat
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
| | - Cherif Badja
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
| | - Natacha Broucqsault
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
| | - Raphaël Chevalier
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
| | - Camille Laberthonnière
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
| | - Camille Dion
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
| | - Lyla Baldasseroni
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
| | - Claire El-Yazidi
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
| | - Morgane Thomas
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
| | - Richard Bachelier
- Aix-Marseille University, INSERM, INRA, C2VN, 13385 Marseille, France; (R.B.); (A.A.)
| | - Alexandre Altié
- Aix-Marseille University, INSERM, INRA, C2VN, 13385 Marseille, France; (R.B.); (A.A.)
| | - Karine Nguyen
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
- APHM, Département de Génétique Médicale, Hôpital de la Timone Enfants, 13385 Marseille, France
| | - Nicolas Lévy
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
- APHM, Département de Génétique Médicale, Hôpital de la Timone Enfants, 13385 Marseille, France
| | - Jérôme D. Robin
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
| | - Frédérique Magdinier
- Aix-Marseille University, INSERM, MMG, Marseille Medical Genetics, 13385 Marseille, France; (K.M.); (C.B.); (N.B.); (R.C.); (C.L.); (C.D.); (L.B.); (C.E.-Y.); (M.T.); (K.N.); (N.L.); (J.D.R.)
- Correspondence:
| |
Collapse
|
|
38
|
He R, Li H, Wang L, Li Y, Zhang Y, Chen M, Zhu Y, Zhang C. Engraftment of human induced pluripotent stem cell-derived myogenic progenitors restores dystrophin in mice with duchenne muscular dystrophy. Biol Res 2020; 53:22. [PMID: 32430065 PMCID: PMC7238630 DOI: 10.1186/s40659-020-00288-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2020] [Accepted: 04/28/2020] [Indexed: 02/07/2023] Open
Abstract
Background Duchenne muscular dystrophy (DMD) is a devastating genetic muscular disorder with no effective treatment that is caused by the loss of dystrophin. Human induced pluripotent stem cells (hiPSCs) offer a promising unlimited resource for cell-based therapies of muscular dystrophy. However, their clinical applications are hindered by inefficient myogenic differentiation, and moreover, the engraftment of non-transgene hiPSC-derived myogenic progenitors has not been examined in the mdx mouse model of DMD. Methods We investigated the muscle regenerative potential of myogenic progenitors derived from hiPSCs in mdx mice. The hiPSCs were transfected with enhanced green fluorescent protein (EGFP) vector and defined as EGFP hiPSCs. Myogenic differentiation was performed on EGFP hiPSCs with supplementary of basic fibroblast growth factor, forskolin, 6-bromoindirubin-3′-oxime as well as horse serum. EGFP hiPSCs-derived myogenic progenitors were engrafted into mdx mice via both intramuscular and intravenous injection. The restoration of dystrophin expression, the ratio of central nuclear myofibers, and the transplanted cells-derived satellite cells were accessed after intramuscular and systemic transplantation. Results We report that abundant myogenic progenitors can be generated from hiPSCs after treatment with these three small molecules, with consequent terminal differentiation giving rise to mature myotubes in vitro. Upon intramuscular or systemic transplantation into mdx mice, these myogenic progenitors engrafted and contributed to human-derived myofiber regeneration in host muscles, restored dystrophin expression, ameliorated pathological lesions, and seeded the satellite cell compartment in dystrophic muscles. Conclusions This study demonstrates the muscle regeneration potential of myogenic progenitors derived from hiPSCs using non-transgenic induction methods. Engraftment of hiPSC-derived myogenic progenitors could be a potential future therapeutic strategy to treat DMD in a clinical setting.
Collapse
Affiliation(s)
- Ruojie He
- Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.,Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases, National Key Clinical Department and Key Discipline of Neurology, Guangzhou, Guangdong, China
| | - Huan Li
- Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.,Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases, National Key Clinical Department and Key Discipline of Neurology, Guangzhou, Guangdong, China
| | - Liang Wang
- Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.,Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases, National Key Clinical Department and Key Discipline of Neurology, Guangzhou, Guangdong, China
| | - Yaqin Li
- Department of Neurology, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, Guangdong, China
| | - Yu Zhang
- Department of Neurology, The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong, China
| | - Menglong Chen
- Department of Neurology, The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong, China
| | - Yuling Zhu
- Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China. .,Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases, National Key Clinical Department and Key Discipline of Neurology, Guangzhou, Guangdong, China.
| | - Cheng Zhang
- Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China. .,Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases, National Key Clinical Department and Key Discipline of Neurology, Guangzhou, Guangdong, China.
| |
Collapse
|
|
39
|
Sakai-Takemura F, Nogami K, Elhussieny A, Kawabata K, Maruyama Y, Hashimoto N, Takeda S, Miyagoe-Suzuki Y. Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions. Commun Biol 2020; 3:182. [PMID: 32313117 PMCID: PMC7171165 DOI: 10.1038/s42003-020-0904-6] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2019] [Accepted: 03/20/2020] [Indexed: 11/17/2022] Open
Abstract
Understanding the signaling pathways that regulate proliferation and differentiation of muscle progenitors is essential for successful cell transplantation for treatment of Duchenne muscular dystrophy. Here, we report that a γ-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine tertial butyl ester), which inhibits the release of NICD (Notch intercellular domain), promotes the fusion of human muscle progenitors in vitro and improves their engraftment in the tibialis anterior muscle of immune-deficient mice. Gene expression analysis revealed that DAPT severely down-regulates PTGER2, which encodes prostaglandin (PG) E2 receptor 2 (EP2), in human muscle progenitors in the differentiation condition. Functional analysis suggested that Notch signaling inhibits differentiation and promotes self-renewal of human muscle progenitors via PGE2/EP2 signaling in a cAMP/PKA-independent manner.
Collapse
Affiliation(s)
- Fusako Sakai-Takemura
- Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
| | - Ken'ichiro Nogami
- Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.,Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Ahmed Elhussieny
- Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.,Department of Neurology, Faculty of Medicine, Minia University, Minia, Egypt
| | - Kota Kawabata
- Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
| | - Yusuke Maruyama
- Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
| | - Naohiro Hashimoto
- Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Aichi, Japan
| | - Shin'ichi Takeda
- Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
| | - Yuko Miyagoe-Suzuki
- Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.
| |
Collapse
|
|
40
|
Motor Neuron Generation from iPSCs from Identical Twins Discordant for Amyotrophic Lateral Sclerosis. Cells 2020; 9:cells9030571. [PMID: 32121108 PMCID: PMC7140469 DOI: 10.3390/cells9030571] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2020] [Revised: 02/25/2020] [Accepted: 02/26/2020] [Indexed: 12/13/2022] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder characterized by the loss of the upper and lower motor neurons. Approximately 10% of cases are caused by specific mutations in known genes, with the remaining cases having no known genetic link. As such, sporadic cases have been more difficult to model experimentally. Here, we describe the generation and differentiation of ALS induced pluripotent stem cells reprogrammed from discordant identical twins. Whole genome sequencing revealed no relevant mutations in known ALS-causing genes that differ between the twins. As protein aggregation is found in all ALS patients and is thought to contribute to motor neuron death, we sought to characterize the aggregation phenotype of the sporadic ALS induced pluripotent stem cells (iPSCs). Motor neurons from both twins had high levels of insoluble proteins that commonly aggregate in ALS that did not robustly change in response to exogenous glutamate. In contrast, established genetic ALS iPSC lines demonstrated insolubility in a protein- and genotype-dependent manner. Moreover, whereas the genetic ALS lines failed to induce autophagy after glutamate stress, motor neurons from both twins and independent controls did activate this protective pathway. Together, these data indicate that our unique model of sporadic ALS may provide key insights into disease pathology and highlight potential differences between sporadic and familial ALS.
Collapse
|
|
41
|
Hosoyama T. Possible application of muscle specific conditional mouse-derived induced pluripotent stem cells for muscle research. Biochem Biophys Rep 2020; 21:100744. [PMID: 32025579 PMCID: PMC6995992 DOI: 10.1016/j.bbrep.2020.100744] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2019] [Revised: 12/12/2019] [Accepted: 01/28/2020] [Indexed: 11/17/2022] Open
Abstract
The Cre-driver mouse line, which allows for in vivo regulation of target gene(s) in specific cells, is an indispensable tool for recent muscle research. In this study, I aimed to explore new applications of muscle specific Cre-driver mouse line in muscle research. For this purpose, I generated an iPS cells from a myofiber specific conditional mouse with tamoxifen inducible GFP expression, and then I checked whether homologous recombination was induced in the iPS-derived myogenic cells by tamoxifen administration. Fibroblasts were isolated from the tails of Myf6CE/wt::CAG-EGFP mice, which expressed GFP specifically in Myf6 lineages by tamoxifen injection, and then iPS cells was generated by transfection with a vector based on sendai-virus and containing OSKM genes. Muscle specific conditional mouse-derived iPS cells (mCM-iPSCs) were successfully differentiated to myogenic cells, such as Pax7+ muscle progenitors, MyoD+ myoblasts, and MHC+ myotubes, under myogenic differentiation conditions. Using this model, I examined whether homologous recombination was induced in mCM-iPSC-derived myotubes by 4-hydroxytamoxifen (4OH-TAM) administration. As a result, multinucleated myotubes showed GFP expression, while no GFP signals were detected in both Pax7+ muscle progenitor and non-myogenic cells. These results indicated that homologous recombination could be induced in mCM-iPSC–derived myotubes by tamoxifen administration, and that this system operated normally even in reprogrammed cells. Also, I evidenced that GFP reporter was expressed in myoblasts in addition to multinucleated myotubes when tamoxifen-pulse was applied at an early phase of myogenesis. Taken together, Myf6CE/wt::CAG-EGFP mouse-derived iPS cells reproduced at least in part Myf6 expression during mouse myogenesis. This study demonstrated a novel application of muscle specific conditional mouse in addition to in vivo application, and mCM-iPSCs could also be used in in vitro investigations with muscle specific conditional knock-out mouse.
iPS cells can be generated from muscle specific conditional mouse line. mCM-iPS cells reproduce early muscle development in vitro. mCM-iPS cells are available for lineage tracing of Myf6+ muscle progenitors.
Collapse
Affiliation(s)
- Tohru Hosoyama
- Department of Regenerative Medicine, National Center for Geriatrics and Gerontology, 7-430 Morioka-cho, Obu, Aichi, 474-8511, Japan
| |
Collapse
|
|
42
|
Fusto A, Moyle LA, Gilbert PM, Pegoraro E. Cored in the act: the use of models to understand core myopathies. Dis Model Mech 2019; 12:dmm041368. [PMID: 31874912 PMCID: PMC6955215 DOI: 10.1242/dmm.041368] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The core myopathies are a group of congenital myopathies with variable clinical expression - ranging from early-onset skeletal-muscle weakness to later-onset disease of variable severity - that are identified by characteristic 'core-like' lesions in myofibers and the presence of hypothonia and slowly or rather non-progressive muscle weakness. The genetic causes are diverse; central core disease is most often caused by mutations in ryanodine receptor 1 (RYR1), whereas multi-minicore disease is linked to pathogenic variants of several genes, including selenoprotein N (SELENON), RYR1 and titin (TTN). Understanding the mechanisms that drive core development and muscle weakness remains challenging due to the diversity of the excitation-contraction coupling (ECC) proteins involved and the differential effects of mutations across proteins. Because of this, the use of representative models expressing a mature ECC apparatus is crucial. Animal models have facilitated the identification of disease progression mechanisms for some mutations and have provided evidence to help explain genotype-phenotype correlations. However, many unanswered questions remain about the common and divergent pathological mechanisms that drive disease progression, and these mechanisms need to be understood in order to identify therapeutic targets. Several new transgenic animals have been described recently, expanding the spectrum of core myopathy models, including mice with patient-specific mutations. Furthermore, recent developments in 3D tissue engineering are expected to enable the study of core myopathy disease progression and the effects of potential therapeutic interventions in the context of human cells. In this Review, we summarize the current landscape of core myopathy models, and assess the hurdles and opportunities of future modeling strategies.
Collapse
Affiliation(s)
- Aurora Fusto
- Department of Neuroscience, University of Padua, Padua 35128, Italy
| | - Louise A Moyle
- Donnelly Centre, University of Toronto, Toronto, ON M5S3E1, Canada
- Institute of Biomaterials and Biochemical Engineering, University of Toronto, Toronto, ON M5S3G9, Canada
| | - Penney M Gilbert
- Donnelly Centre, University of Toronto, Toronto, ON M5S3E1, Canada
- Institute of Biomaterials and Biochemical Engineering, University of Toronto, Toronto, ON M5S3G9, Canada
- Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S3G5, Canada
- Department of Biochemistry, University of Toronto, Toronto, ON M5S1A8, Canada
| | - Elena Pegoraro
- Department of Neuroscience, University of Padua, Padua 35128, Italy
| |
Collapse
|
|
43
|
Tey SR, Robertson S, Lynch E, Suzuki M. Coding Cell Identity of Human Skeletal Muscle Progenitor Cells Using Cell Surface Markers: Current Status and Remaining Challenges for Characterization and Isolation. Front Cell Dev Biol 2019; 7:284. [PMID: 31828070 PMCID: PMC6890603 DOI: 10.3389/fcell.2019.00284] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2019] [Accepted: 11/01/2019] [Indexed: 12/12/2022] Open
Abstract
Skeletal muscle progenitor cells (SMPCs), also called myogenic progenitors, have been studied extensively in recent years because of their promising therapeutic potential to preserve and recover skeletal muscle mass and function in patients with cachexia, sarcopenia, and neuromuscular diseases. SMPCs can be utilized to investigate the mechanisms of natural and pathological myogenesis via in vitro modeling and in vivo experimentation. While various types of SMPCs are currently available from several sources, human pluripotent stem cells (PSCs) offer an efficient and cost-effective method to derive SMPCs. As human PSC-derived cells often display varying heterogeneity in cell types, cell enrichment using cell surface markers remains a critical step in current procedures to establish a pure population of SMPCs. Here we summarize the cell surface markers currently being used to detect human SMPCs, describing their potential application for characterizing, identifying and isolating human PSC-derived SMPCs. To date, several positive and negative markers have been used to enrich human SMPCs from differentiated PSCs by cell sorting. A careful analysis of current findings can broaden our understanding and reveal potential uses for these surface markers with SMPCs.
Collapse
Affiliation(s)
- Sin-Ruow Tey
- Department of Comparative Biosciences, University of Wisconsin, Madison, WI, United States
| | - Samantha Robertson
- Department of Comparative Biosciences, University of Wisconsin, Madison, WI, United States
| | - Eileen Lynch
- Department of Comparative Biosciences, University of Wisconsin, Madison, WI, United States
| | - Masatoshi Suzuki
- Department of Comparative Biosciences, University of Wisconsin, Madison, WI, United States.,The Stem Cell and Regenerative Medicine Center, University of Wisconsin, Madison, WI, United States
| |
Collapse
|
|
44
|
A Three-Dimensional Culture Model of Reversibly Quiescent Myogenic Cells. Stem Cells Int 2019; 2019:7548160. [PMID: 31827532 PMCID: PMC6885280 DOI: 10.1155/2019/7548160] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2019] [Revised: 09/24/2019] [Accepted: 10/03/2019] [Indexed: 12/13/2022] Open
Abstract
Satellite cells (SC) are the stem cells of skeletal muscles. They are quiescent in adult animals but resume proliferation to allow muscle hypertrophy or regeneration after injury. The mechanisms balancing quiescence, self-renewal, and differentiation of SC are difficult to analyze in vivo owing to their complexity and in vitro because the staminal character of SC is lost when they are removed from the niche and is not adequately reproduced in the culture models currently available. To overcome these difficulties, we set up a culture model of the myogenic C2C12 cell line in suspension. When C2C12 cells are cultured in suspension, they enter a state of quiescence and form three-dimensional aggregates (myospheres) that produce the extracellular matrix and express markers of quiescent SC. In the initial phase of culture, a portion of the cells fuses in syncytia and abandons the myospheres. The remaining cells are mononucleated and quiescent but resume proliferation and differentiation when plated in a monolayer. The notch pathway controls the quiescent state of the cells as shown by the fact that its inhibition leads to the resumption of differentiation. Within this context, notch3 appears to play a central role in the activity of this pathway since the expression of notch1 declines soon after aggregation. In summary, the culture model of C2C12 in suspension may be used to study the cellular interactions of muscle stem cells and the pathways controlling SC quiescence entrance and maintenance.
Collapse
|
|
45
|
Picchiarelli G, Demestre M, Zuko A, Been M, Higelin J, Dieterlé S, Goy MA, Mallik M, Sellier C, Scekic-Zahirovic J, Zhang L, Rosenbohm A, Sijlmans C, Aly A, Mersmann S, Sanjuan-Ruiz I, Hübers A, Messaddeq N, Wagner M, van Bakel N, Boutillier AL, Ludolph A, Lagier-Tourenne C, Boeckers TM, Dupuis L, Storkebaum E. FUS-mediated regulation of acetylcholine receptor transcription at neuromuscular junctions is compromised in amyotrophic lateral sclerosis. Nat Neurosci 2019; 22:1793-1805. [PMID: 31591561 PMCID: PMC6858880 DOI: 10.1038/s41593-019-0498-9] [Citation(s) in RCA: 79] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2019] [Accepted: 08/15/2019] [Indexed: 12/13/2022]
Abstract
Neuromuscular junction (NMJ) disruption is an early pathogenic event in amyotrophic lateral sclerosis (ALS). Yet, direct links between NMJ pathways and ALS-associated genes such as FUS, whose heterozygous mutations cause aggressive forms of ALS, remain elusive. In a knock-in Fus-ALS mouse model, we identified postsynaptic NMJ defects in newborn homozygous mutants, attributable to mutant FUS toxicity in skeletal muscle. Adult heterozygous knock-in mice displayed smaller neuromuscular endplates that denervated before motor neuron loss, consistent with ‘dying-back’ neuronopathy. FUS was enriched in subsynaptic myonuclei, and this innervation-dependent enrichment was distorted in FUS-ALS. Mechanistically, FUS collaborates with the ETS-transcription factor ERM to stimulate transcription of acetylcholine receptor (AchR) genes. FUS-ALS patient iPSC-derived motor neuron-myotube co-cultures revealed endplate maturation defects due to intrinsic FUS toxicity in both motor neurons and myotubes. Thus, FUS regulates AChR gene expression in subsynaptic myonuclei and muscle-intrinsic toxicity of ALS-mutant FUS may contribute to dying-back motor neuronopathy.
Collapse
Affiliation(s)
| | - Maria Demestre
- Institute of Anatomy and Cell Biology, Ulm University, Ulm, Germany
| | - Amila Zuko
- Department of Molecular Neurobiology, Donders Institute for Brain, Cognition and Behaviour and Faculty of Science, Radboud University, Nijmegen, The Netherlands
| | - Marije Been
- Department of Molecular Neurobiology, Donders Institute for Brain, Cognition and Behaviour and Faculty of Science, Radboud University, Nijmegen, The Netherlands
| | - Julia Higelin
- Institute of Anatomy and Cell Biology, Ulm University, Ulm, Germany
| | | | | | - Moushami Mallik
- Department of Molecular Neurobiology, Donders Institute for Brain, Cognition and Behaviour and Faculty of Science, Radboud University, Nijmegen, The Netherlands.,Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Muenster, Germany.,Faculty of Medicine, University of Muenster, Muenster, Germany
| | - Chantal Sellier
- IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France
| | | | - Li Zhang
- Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Muenster, Germany.,Faculty of Medicine, University of Muenster, Muenster, Germany
| | | | - Céline Sijlmans
- Department of Molecular Neurobiology, Donders Institute for Brain, Cognition and Behaviour and Faculty of Science, Radboud University, Nijmegen, The Netherlands
| | - Amr Aly
- Institute of Anatomy and Cell Biology, Ulm University, Ulm, Germany
| | - Sina Mersmann
- Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Muenster, Germany.,Faculty of Medicine, University of Muenster, Muenster, Germany
| | | | | | - Nadia Messaddeq
- IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France
| | - Marina Wagner
- Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Muenster, Germany.,Faculty of Medicine, University of Muenster, Muenster, Germany
| | - Nick van Bakel
- Department of Molecular Neurobiology, Donders Institute for Brain, Cognition and Behaviour and Faculty of Science, Radboud University, Nijmegen, The Netherlands
| | - Anne-Laurence Boutillier
- Laboratoire de Neurosciences Cognitives et Adaptatives, Université de Strasbourg, Centre National de la Recherche Scientifique, UMR 7364, Strasbourg, France
| | - Albert Ludolph
- Department of Neurology, Oberer Eselsberg 45, Ulm, Germany
| | - Clotilde Lagier-Tourenne
- Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA.,Broad Institute of Harvard University and MIT, Cambridge, MA, USA
| | - Tobias M Boeckers
- Institute of Anatomy and Cell Biology, Ulm University, Ulm, Germany. .,DZNE, Ulm site, Ulm, Germany.
| | - Luc Dupuis
- Université de Strasbourg, INSERM, UMR-S1118, Strasbourg, France.
| | - Erik Storkebaum
- Department of Molecular Neurobiology, Donders Institute for Brain, Cognition and Behaviour and Faculty of Science, Radboud University, Nijmegen, The Netherlands. .,Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Muenster, Germany. .,Faculty of Medicine, University of Muenster, Muenster, Germany.
| |
Collapse
|
|
46
|
Gene expression profiling of skeletal myogenesis in human embryonic stem cells reveals a potential cascade of transcription factors regulating stages of myogenesis, including quiescent/activated satellite cell-like gene expression. PLoS One 2019; 14:e0222946. [PMID: 31560727 PMCID: PMC6764674 DOI: 10.1371/journal.pone.0222946] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2019] [Accepted: 09/10/2019] [Indexed: 01/05/2023] Open
Abstract
Human embryonic stem cell (hESC)-derived skeletal muscle progenitors (SMP)—defined as PAX7-expressing cells with myogenic potential—can provide an abundant source of donor material for muscle stem cell therapy. As in vitro myogenesis is decoupled from in vivo timing and 3D-embryo structure, it is important to characterize what stage or type of muscle is modeled in culture. Here, gene expression profiling is analyzed in hESCs over a 50 day skeletal myogenesis protocol and compared to datasets of other hESC-derived skeletal muscle and adult murine satellite cells. Furthermore, day 2 cultures differentiated with high or lower concentrations of CHIR99021, a GSK3A/GSK3B inhibitor, were contrasted. Expression profiling of the 50 day time course identified successively expressed gene subsets involved in mesoderm/paraxial mesoderm induction, somitogenesis, and skeletal muscle commitment/formation which could be regulated by a putative cascade of transcription factors. Initiating differentiation with higher CHIR99021 concentrations significantly increased expression of MSGN1 and TGFB-superfamily genes, notably NODAL, resulting in enhanced paraxial mesoderm and reduced ectoderm/neuronal gene expression. Comparison to adult satellite cells revealed that genes expressed in 50-day cultures correlated better with those expressed by quiescent or early activated satellite cells, which have the greatest therapeutic potential. Day 50 cultures were similar to other hESC-derived skeletal muscle and both expressed known and novel SMP surface proteins. Overall, a putative cascade of transcription factors has been identified which regulates four stages of myogenesis. Subsets of these factors were upregulated by high CHIR99021 or their binding sites were significantly over-represented during SMP activation, ranging from quiescent to late-activated stages. This analysis serves as a resource to further study the progression of in vitro skeletal myogenesis and could be mined to identify novel markers of pluripotent-derived SMPs or regulatory transcription/growth factors. Finally, 50-day hESC-derived SMPs appear similar to quiescent/early activated satellite cells, suggesting they possess therapeutic potential.
Collapse
|
|
47
|
Cho KA, Lee HJ, Jeong H, Kim M, Jung SY, Park HS, Ryu KH, Lee SJ, Jeong B, Lee H, Kim HS. Tonsil-derived stem cells as a new source of adult stem cells. World J Stem Cells 2019; 11:506-518. [PMID: 31523370 PMCID: PMC6716082 DOI: 10.4252/wjsc.v11.i8.506] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/13/2019] [Revised: 07/25/2019] [Accepted: 07/30/2019] [Indexed: 02/06/2023] Open
Abstract
Located near the oropharynx, the tonsils are the primary mucosal immune organ. Tonsil tissue is a promising alternative source for the high-yield isolation of adult stem cells, and recent studies have reported the identification and isolation of tonsil-derived stem cells (T-SCs) from waste surgical tissue following tonsillectomies in relatively young donors (i.e., under 10 years old). As such, T-SCs offer several advantages, including superior proliferation and a shorter doubling time compared to bone marrow-derived mesenchymal stem cells (MSCs). T-SCs also exhibit multi-lineage differentiation, including mesodermal, endodermal (e.g., hepatocytes and parathyroid-like cells), and even ectodermal cells (e.g., Schwann cells). To this end, numbers of researchers have evaluated the practical use of T-SCs as an alternative source of autologous or allogenic MSCs. In this review, we summarize the details of T-SC isolation and identification and provide an overview of their application in cell therapy and regenerative medicine.
Collapse
Affiliation(s)
- Kyung-Ah Cho
- Department of Microbiology, College of Medicine, Ewha Womans University, Seoul 07985, South Korea
| | - Hyun Jung Lee
- Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, South Korea
| | - Hansaem Jeong
- College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, South Korea
| | - Miri Kim
- College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, South Korea
| | - Soo Yeon Jung
- Department of Otorhinolaryngology, College of Medicine, Ewha Womans University, Seoul 07985, South Korea
| | - Hae Sang Park
- Department of Otorhinolaryngology, College of Medicine, Hallym University, Chuncheon 24252, South Korea
| | - Kyung-Ha Ryu
- Department of Pediatrics, College of Medicine, Ewha Womans University, Seoul 07985, South Korea
| | - Seung Jin Lee
- College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, South Korea
| | - Byeongmoon Jeong
- Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, South Korea
| | - Hyukjin Lee
- College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, South Korea
| | - Han Su Kim
- Department of Otorhinolaryngology, College of Medicine, Ewha Womans University, Seoul 07985, South Korea
| |
Collapse
|
|
48
|
Lynch E, Semrad T, Belsito VS, FitzGibbons C, Reilly M, Hayakawa K, Suzuki M. C9ORF72-related cellular pathology in skeletal myocytes derived from ALS-patient induced pluripotent stem cells. Dis Model Mech 2019; 12:12/8/dmm039552. [PMID: 31439573 PMCID: PMC6737948 DOI: 10.1242/dmm.039552] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Accepted: 07/10/2019] [Indexed: 12/25/2022] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a late-onset neuromuscular disease with no cure and limited treatment options. Patients experience a gradual paralysis leading to death from respiratory complications on average only 2-5 years after diagnosis. There is increasing evidence that skeletal muscle is affected early in the disease process, yet the pathological processes occurring in the skeletal muscle of ALS patients are still mostly unknown. Specifically, the most common genetic cause of ALS, a hexanucleotide repeat expansion in the C9ORF72 gene, has yet to be fully characterized in the context of skeletal muscle. In this study, we used the protocol previously developed in our lab to differentiate skeletal myocytes from induced pluripotent stem cells (iPSCs) of C9ORF72 ALS (C9-ALS) patients in order to create an in vitro disease model of C9-ALS skeletal muscle pathology. Of the three C9ORF72 mutation hallmarks, we did not see any evidence of haploinsufficiency, but we did detect RNA foci and dipeptide repeat (DPR) proteins. Additional abnormalities included changes in the expression of mitochondrial genes and a susceptibility to oxidative stress, indicating that mitochondrial dysfunction may be a critical feature of C9-ALS skeletal muscle pathology. Finally, the C9-ALS myocytes had increased expression and aggregation of TDP-43. Together, these data show that skeletal muscle cells experience pathological changes due to the C9ORF72 mutation. Our in vitro model could facilitate further study of cellular and molecular pathology in ALS skeletal muscle in order to discover new therapeutic targets against this devastating disease. This article has an associated First Person interview with the first author of the paper. Summary: Evidence of protein aggregation and mitochondrial dysfunction were found in skeletal myocytes differentiated from ALS-patient induced pluripotent stem cells with the C9ORF72 mutation.
Collapse
Affiliation(s)
- Eileen Lynch
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Theran Semrad
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Vincent S Belsito
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Claire FitzGibbons
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Megan Reilly
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Koji Hayakawa
- Department of Toxicology, Faculty of Veterinary Medicine, Okayama University of Science, Imabari, Ehime 794-8555, Japan
| | - Masatoshi Suzuki
- Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA .,Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison, Madison, WI 53706, USA
| |
Collapse
|
|
49
|
Jiwlawat N, Lynch EM, Napiwocki BN, Stempien A, Ashton RS, Kamp TJ, Crone WC, Suzuki M. Micropatterned substrates with physiological stiffness promote cell maturation and Pompe disease phenotype in human induced pluripotent stem cell-derived skeletal myocytes. Biotechnol Bioeng 2019; 116:2377-2392. [PMID: 31131875 DOI: 10.1002/bit.27075] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2018] [Revised: 04/19/2019] [Accepted: 05/21/2019] [Indexed: 12/23/2022]
Abstract
Recent advances in bioengineering have enabled cell culture systems that more closely mimic the native cellular environment. Here, we demonstrated that human induced pluripotent stem cell (iPSC)-derived myogenic progenitors formed highly-aligned myotubes and contracted when seeded on two-dimensional micropatterned platforms. The differentiated cells showed clear nuclear alignment and formed elongated myotubes dependent on the width of the micropatterned lanes. Topographical cues from micropatterning and physiological substrate stiffness improved the formation of well-aligned and multinucleated myotubes similar to myofibers. These aligned myotubes exhibited spontaneous contractions specifically along the long axis of the pattern. Notably, the micropatterned platforms developed bundle-like myotubes using patient-derived iPSCs with a background of Pompe disease (glycogen storage disease type II) and even enhanced the disease phenotype as shown through the specific pathology of abnormal lysosome accumulations. A highly-aligned formation of matured myotubes holds great potential in further understanding the process of human muscle development, as well as advancing in vitro pharmacological studies for skeletal muscle diseases.
Collapse
Affiliation(s)
- Nunnapas Jiwlawat
- Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin
| | - Eileen M Lynch
- Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin
| | - Brett N Napiwocki
- Wisconsin Institute for Discovery, University of Wisconsin, Madison, Wisconsin.,Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin
| | - Alana Stempien
- Wisconsin Institute for Discovery, University of Wisconsin, Madison, Wisconsin.,Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin
| | - Randolph S Ashton
- Wisconsin Institute for Discovery, University of Wisconsin, Madison, Wisconsin.,Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin.,The Stem Cell and Regenerative Medicine Center, University of Wisconsin, Madison, Wisconsin
| | - Timothy J Kamp
- Department of Medicine, University of Wisconsin, Madison, Wisconsin.,The Stem Cell and Regenerative Medicine Center, University of Wisconsin, Madison, Wisconsin.,Department of Cell and Regenerative Biology, University of Wisconsin, Madison, Wisconsin
| | - Wendy C Crone
- Wisconsin Institute for Discovery, University of Wisconsin, Madison, Wisconsin.,Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin.,The Stem Cell and Regenerative Medicine Center, University of Wisconsin, Madison, Wisconsin.,Department of Engineering Physics, University of Wisconsin, Madison, Wisconsin
| | - Masatoshi Suzuki
- Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin.,Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin.,The Stem Cell and Regenerative Medicine Center, University of Wisconsin, Madison, Wisconsin
| |
Collapse
|
|
50
|
Danisovic L, Culenova M, Csobonyeiova M. Induced Pluripotent Stem Cells for Duchenne Muscular Dystrophy Modeling and Therapy. Cells 2018; 7:253. [PMID: 30544588 PMCID: PMC6315586 DOI: 10.3390/cells7120253] [Citation(s) in RCA: 28] [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: 11/21/2018] [Revised: 11/30/2018] [Accepted: 12/05/2018] [Indexed: 02/07/2023] Open
Abstract
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, caused by mutation of the DMD gene which encodes the protein dystrophin. This dystrophin defect leads to the progressive degeneration of skeletal and cardiac muscles. Currently, there is no effective therapy for this disorder. However, the technology of cell reprogramming, with subsequent controlled differentiation to skeletal muscle cells or cardiomyocytes, may provide a unique tool for the study, modeling, and treatment of Duchenne muscular dystrophy. In the present review, we describe current methods of induced pluripotent stem cell generation and discuss their implications for the study, modeling, and development of cell-based therapies for Duchenne muscular dystrophy.
Collapse
Affiliation(s)
- Lubos Danisovic
- Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, Sasinkova 4, 811 08 Bratislava, Slovakia.
| | - Martina Culenova
- Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, Sasinkova 4, 811 08 Bratislava, Slovakia.
| | - Maria Csobonyeiova
- Institute of Histology and Embryology, Faculty of Medicine, Comenius University, Sasinkova 4, 811 08 Bratislava, Slovakia.
| |
Collapse
|