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Skeletal muscle is a complex tissue made up of multinucleated muscle fibers that exert force and are supported by several types of cells. Many serious and fatal diseases affect skeletal muscle, so the construction of bioengineered models of this complex cellular composition and function is useful for studying muscle pathophysiology and developing treatments. Recently, the team of Professor Francesco Saverio Tedesco from University College London conducted research on three-dimensional hydrogels combined with human induced pluripotent stem cells to build bioengineered skeletal muscle for tissue, disease and treatment modeling. The research results were published in Nature protocols on February 15, 2023 under the title "3D human induced pluripotent stem cell–derived bioengineered skeletal muscles for tissue, disease and therapy modeling".
1. Bioengineering skeletal muscle construction ideas and processes
Figure 1 Pilot testing and quality control of biomaterials and cells
This section briefly describes the steps required to generate monophyletic or multilineage 3D muscle types. The ability to support the formation of neatly aligned multinucleated skeletal muscle fibers, such as the use of primary or immortal human myocytes, is first confirmed to be more readily available than hiPSC-derived myoblasts. Next, quality control is performed on hiPSC initiation population type, genome stability, pluripotency, and ensuring free of mycoplasma contamination before differentiation into specific derivatives. Media containing serum, such as FBS, may produce different results, so batch testing and stocking from batches that produce the best results is required.
Figure 2 Workflow for generating and analyzing three-dimensional skeletal muscle structure using hiPSC-derived primary cells
The overall process consists of three main phases as follows. The first step is primary cell monolayer expansion for fabricating engineered muscle (step 1). Up to four different cell lines (including two different method options derived from progenitor cells) are used here: myofibers, motor neurons, endothelial cells, and pericytes. The second step is to generate and cultivate a three-dimensional artificial muscle structure (step 2). The choice of which progenitor cells to expand is based on the type of construct required (with or without vascular cells or innervation). The third step is to use engineered muscles in downstream applications. Some alternative or novel methods for deriving myoblasts and non-myoblastic cell lines can also be adapted to our method and generate bioengineered skeletal muscle tissue.
2. Bioengineered skeletal muscle characterization and application
Figure 3 Derived 3D bioengineered muscle characterization
Fig. 4 HiPSC-derived three-dimensional muscle was used to model muscular dystrophy caused by nuclear envelope abnormalities
Figure 5 Examples of in vitro and in vivo application of hiPSC-derived 3D artificial muscles: testing gene therapy vectors, assessing cell migration capacity and studying biocompatibility
The 3D artificial muscle platform can be used to simulate the properties of normal or patient human skeletal muscle, such as muscle fiber formation, arrangement, and growth, interaction with supporting cell types, and the establishment of muscle stem cell banks. Through this platform, this paper pioneered a hiPSC-based 3D model of muscular dystrophy, revealing key cellular phenotypic features of severe muscle disease caused by nuclear envelope protein defects. Three-dimensional muscle is superior to traditional two-dimensional cell monolayer when used to simulate skeletal muscle lamellar lesions, and the abnormal shape associated with potential mutations in the nuclear envelope gene LMNA nucleus is better reproduced in three-dimensional state. Using this strategy, the kernel spindle length is defined in three dimensions as an objective phenotypic reading for the screening item. Nuclear malformation features are mutation-specific in relation to the severity of clinical presentation, with the most common and severe LMNA R249W mutation having the greatest degree of nuclear deformation and elongation.
The researchers then measured key downstream skeletal muscle functions, such as contractility and calcium dynamics. This data can be used to develop therapeutic pipelines, based on small molecules or advanced products such as gene therapy vectors. The multilinear nature of the 3D construct enables testing of the cell specificity, toxicity, and efficacy of drugs and advanced therapies, including viral vectors and cell therapies, on in vitro humanized homologous platforms. Open up new avenues beyond disease modeling for personalized medicine, including drug and therapy testing, toxicity studies, and in vivo implanted tissue replacement. hiPSC-derived three-dimensional muscles can be used to monitor expression in living tissues and monitor in real time. (1) non-integrated, non-viral vectors for preclinical gene and cell therapy studies of DMD and (2) adeno-associated viral vector (AAV) serotypes (AAV9) for neuromuscular gene therapy clinical trials, emphasizing dose-response correlation of different transgenic doses and laying the groundwork for further work to investigate the cellular and lineage specificity of different AAV serotypes for precision medicine. The use of this 3D platform to evaluate advanced therapies other than viral vectors and demonstrated that it can be used to evaluate myogenic cell transplantation and migration within homologous 3D intramuscular, provides a unique strategy for evaluating myogenic cell transplantation in the quasi-in in vivo environment.
The three-dimensional organoid platform greatly refines, reduces, and replaces animal use in preclinical research. Three-dimensional artificial muscle, patient specificity, multicellularity, and homology reduce the number of animals needed to work in vivo, validate a limited number of disease pathogenesis or therapeutic/toxicity test experiments. Biocompatibility studies were conducted showing strong biocompatibility, and hiPSC-derived three-dimensional muscles (both monophyletic and multilineage) were subjected to acute volumetric muscle injury or loss, vascularization and integration with host tissues in immunodeficient mice, providing the basis for in vivo testing of alternative strategies using this approach to organize alternative strategies as well as select therapies from in vitro work.
The platform will innovate and accelerate the development of science and technology by: (1) studying human somatogenesis to understand early disease phenotypes that cannot be captured by derived skeletal muscle cells; (2) Includes further analysis of cellular components from less studied but critical cell niches, as well as supercell types such as fiber-adipogenic progenitor cells and tendon junctions; (3) Master emerging microfabrication and microfluidic technologies to generate advanced muscle-on-chip models for precision medicine.
In summary, this paper uses human induced pluripotent stem cells for modular three-dimensional bioengineering of multilineage skeletal muscle, in which cells first differentiate into myoblasts, nerves, and vascular progenitor cells, and then bind under three-dimensional hydrogel tension to produce a well-ordered myofiber scaffold containing a vascular network and motor neurons. Three-dimensional bioengineered muscle reproduces morphological and functional characteristics of human skeletal muscle, including the establishment of a cell bank of labeled muscle stem cells. Bioengineered muscles provide a high-fidelity platform to study muscle pathologies, such as the malformed nuclei that occur in muscular dystrophy caused by mutated lamin proteins. This method is easy for experienced operators in cell culture and can be set up in as little as 9 to 30 days, depending on the number of cell lines in the construct. This paper also provides examples of the application of this advanced platform in vitro testing of gene and cell therapies and in vivo research, providing proof of principle for its potential as a tool for developing the next generation of neuromuscular or musculoskeletal therapies.
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