3D human induced pluripotent stem cell-derived bioengineered skeletal muscles for tissue, disease and therapy modeling.

Skeletal muscle is a complex tissue composed of multinucleated myofibers responsible for force generation that are supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function are instrumental for investigating muscle pathophysiology and developing therapies. Here, we detail the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, which are first differentiated into myogenic, neural and vascular progenitor cells and then combined within 3D hydrogels under tension to generate an aligned myofiber scaffold containing vascular networks and motor neurons. 3D bioengineered muscles recapitulate morphological and functional features of human skeletal muscle, including establishment of a pool of cells expressing muscle stem cell markers. Importantly, bioengineered muscles provide a high-fidelity platform to study muscle pathology, such as emergence of dysmorphic nuclei in muscular dystrophies caused by mutant lamins. The protocol is easy to follow for operators with cell culture experience and takes between 9 and 30 d, depending on the number of cell lineages in the construct. We also provide examples of applications of this advanced platform for testing gene and cell therapies in vitro, as well as for in vivo studies, providing proof of principle of its potential as a tool to develop next-generation neuromuscular or musculoskeletal therapies.

Bioengineering human skeletal muscle models: Recent advances, current challenges and future perspectives.

Engineering models of human skeletal muscle tissue provides unique translational opportunities to investigate and develop therapeutic strategies for acute muscle injuries, and to establish personalised and precision medicine platforms for in vitro studies of severe neuromuscular and musculoskeletal disorders. Several myogenic and non-myogenic cell types can be isolated, generated, amplified and combined with scaffolds and biomaterials to achieve this aim. Novel bio-fabrication strategies, which include exogenous stimuli to enhance tissue maturation, promise to achieve an ever-increasing degree of tissue functionalisation both in vivo and in vitro. Here we review recent advances, current challenges and future perspectives to build human skeletal muscle tissue "in a dish", focusing on the cellular constituents and on applications for in vitro disease modelling. We also briefly discuss the impact that emerging technologies such as 3D bioprinting, organ-on-chip and organoids might have to circumvent technical hurdles in future studies.

Modeling Skeletal Muscle Laminopathies Using Human Induced Pluripotent Stem Cells Carrying Pathogenic LMNA Mutations.

Laminopathies are a clinically heterogeneous group of disorders caused by mutations in LMNA. The main proteins encoded by LMNA are Lamin A and C, which together with Lamin B1 and B2, form the nuclear lamina: a mesh-like structure located underneath the inner nuclear membrane. Laminopathies show striking tissue specificity, with subtypes affecting striated muscle, peripheral nerve, and adipose tissue, while others cause multisystem disease with accelerated aging. Although several pathogenic mechanisms have been proposed, the exact pathophysiology of laminopathies remains unclear, compounded by the rarity of these disorders and lack of easily accessible cell types to study. To overcome this limitation, we used induced pluripotent stem cells (iPSCs) from patients with skeletal muscle laminopathies such as LMNA-related congenital muscular dystrophy and limb-girdle muscular dystrophy 1B, to model disease phenotypes in vitro. iPSCs can be derived from readily accessible cell types, have unlimited proliferation potential and can be differentiated into cell types that would otherwise be difficult and invasive to obtain. iPSC lines from three skeletal muscle laminopathy patients were differentiated into inducible myogenic cells and myotubes. Disease-associated phenotypes were observed in these cells, including abnormal nuclear shape and mislocalization of nuclear lamina proteins. Nuclear abnormalities were less pronounced in monolayer cultures of terminally differentiated skeletal myotubes than in proliferating myogenic cells. Notably, skeletal myogenic differentiation of LMNA-mutant iPSCs in artificial muscle constructs improved detection of myonuclear abnormalities compared to conventional monolayer cultures across multiple pathogenic genotypes, providing a high-fidelity modeling platform for skeletal muscle laminopathies. Our results lay the foundation for future iPSC-based therapy development and screening platforms for skeletal muscle laminopathies.

Efficient derivation and inducible differentiation of expandable skeletal myogenic cells from human ES and patient-specific iPS cells.

Skeletal muscle is the most abundant human tissue; therefore, an unlimited availability of myogenic cells has applications in regenerative medicine and drug development. Here we detail a protocol to derive myogenic cells from human embryonic stem (ES) and induced pluripotent stem (iPS) cells, and we also provide evidence for its extension to human iPS cells cultured without feeder cells. The procedure, which does not require the generation of embryoid bodies or prospective cell isolation, entails four stages with different culture densities, media and surface coating. Pluripotent stem cells are disaggregated to single cells and then differentiated into expandable cells resembling human mesoangioblasts. Subsequently, transient Myod1 induction efficiently drives myogenic differentiation into multinucleated myotubes. Cells derived from patients with muscular dystrophy and differentiated using this protocol have been genetically corrected, and they were proven to have therapeutic potential in dystrophic mice. Thus, this platform has been demonstrated to be amenable to gene and cell therapy, and it could be extended to muscle tissue engineering and disease modeling.

Transplantation of induced pluripotent stem cell-derived mesoangioblast-like myogenic progenitors in mouse models of muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.