Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne's muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan-null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan-null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.

SummaryGenerating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development.

Musculoskeletal modelling has become a valuable tool with which to understand how neural, muscular, skeletal and other tissues are integrated to produce movement. Most musculoskeletal modelling work has to date focused on humans or their close relatives, with few examples of quadrupedal animal limb models. A musculoskeletal model of the mouse hindlimb could have broad utility for questions in medicine, genetics, locomotion and neuroscience. This is due to this species’ position as a premier model of human disease, having an array of genetic tools for manipulation of the animal in vivo, and being a small quadruped, a category for which few models exist. Here, the methods used to develop the first three‐dimensional (3D) model of a mouse hindlimb and pelvis are described. The model, which represents bones, joints and 39 musculotendon units, was created through a combination of previously gathered muscle architecture data from microdissections, contrast‐enhanced micro‐computed tomography (CT) scanning and digital segmentation. The model allowed muscle moment arms as well as muscle forces to be estimated for each musculotendon unit throughout a range of joint rotations. Moment arm analysis supported the reliability of musculotendon unit placement within the model, and comparison to a previously published rat hindlimb model further supported the model's reliability. A sensitivity analysis performed on both the force‐generating parameters and muscle's attachment points of the model indicated that the maximal isometric muscle moment is generally most sensitive to changes in either tendon slack length or the coordinates of insertion, although the degree to which the moment is affected depends on several factors. This model represents the first step in the creation of a fully dynamic 3D computer model of the mouse hindlimb and pelvis that has application to neuromuscular disease, comparative biomechanics and the neuromechanical basis of movement. Capturing the morphology and dynamics of the limb, it enables future dissection of the complex interactions between the nervous and musculoskeletal systems as well as the environment.

Mice are one of the most commonly used laboratory animals, with an extensive array of disease models in existence, including for many neuromuscular diseases. The hindlimb is of particular interest due to several close muscle analogues/homologues to humans and other species. A detailed anatomical study describing the adult morphology is lacking, however. This study describes in detail the musculoskeletal geometry and skeletal muscle architecture of the mouse hindlimb and pelvis, determining the extent to which the muscles are adapted for their function, as inferred from their architecture. Using I2KI enhanced microCT scanning and digital segmentation, it was possible to identify 39 distinct muscles of the hindlimb and pelvis belonging to nine functional groups. The architecture of each of these muscles was determined through microdissections, revealing strong architectural specialisations between the functional groups. The hip extensors and hip adductors showed significantly stronger adaptations towards high contraction velocities and joint control relative to the distal functional groups, which exhibited larger physiological cross sectional areas and longer tendons, adaptations for high force output and elastic energy savings. These results suggest that a proximo-distal gradient in muscle architecture exists in the mouse hindlimb. Such a gradient has been purported to function in aiding locomotor stability and efficiency. The data presented here will be especially valuable to any research with a focus on the architecture or gross anatomy of the mouse hindlimb and pelvis musculature, but also of use to anyone interested in the functional significance of muscle design in relation to quadrupedal locomotion.