S.M. Maffioletti

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.

Stem Cell Transplantation for Muscular Dystrophy: The Challenge of Immune Response

Treating muscle disorders poses several challenges to the rapidly evolving field of regenerative medicine. Considerable progress has been made in isolating, characterizing, and expanding myogenic stem cells and, although we are now envisaging strategies to generate very large numbers of transplantable cells (e.g., by differentiating induced pluripotent stem cells), limitations directly linked to the interaction between transplanted cells and the host will continue to hamper a successful outcome. Among these limitations, host inflammatory and immune responses challenge the critical phases after cell delivery, including engraftment, migration, and differentiation. Therefore, it is key to study the mechanisms and dynamics that impair the efficacy of cell transplants in order to develop strategies that can ultimately improve the outcome of allogeneic and autologous stem cell therapies, in particular for severe disease such as muscular dystrophies. In this review we provide an overview of the main players and issues involved in this process and discuss potential approaches that might be beneficial for future regenerative therapies of skeletal muscle.

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.

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering

Summary Generating 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.

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.

Reversible immortalisation, human artificial chromosomes, and induced pluripotency: new gene and cell therapy technologies for Duchenne muscular dystrophy

Abstract Background Duchenne muscular dystrophy is caused by mutations in the gene that encodes dystrophin, a major component of muscle fibres. The combination of gene therapy with cell therapy to treat the disorder is encouraging, but it is challenging because dystrophin is the largest human gene, and skeletal muscle the most abundant human tissue. We aimed to assess use of autologous stem cells with human artificial chromosomes (HACs) containing the entire dystrophin locus (DYS-HACs) as a solution to these challenges. Methods We describe two complementary strategies for the generation of genetically corrected myogenic stem cells from patients with Duchenne muscular dystrophy, one from muscle biopsy samples and the other from induced pluripotent stem (iPS) cells. We also describe the generation of a multifunctional DYS-HAC applicable in both strategies. Notably, these technologies are also being developed in genome-integration-free platforms. Findings Reversible immortalisation with lentivirally delivered excisable telomerase and BMI1 complementary DNAs allowed bypassing of replicative senescence and DYS-HAC transfer in muscle-derived cells. When isolation of tissue-derived progenitors proved challenging, we successfully derived skeletal myogenic cells from Duchenne muscular dystrophy iPS cells reprogrammed with non-integrating technologies and corrected them with the DYS-HAC. Finally we describe the engineering of a next-generation, multifunctional DYS-HAC that can provide complete genetic correction, enhanced proliferation, controllable cell death (as a safety system), and inducible myogenesis in both tissue-derived or iPS cell-derived stem cells. Interpretation This project provides the foundation for preclinical and clinical development of an autologous ex-vivo gene therapy protocol for Duchenne muscular dystrophy based on HACs and myogenic cells. Funding National Institute for Health Research, Medical Research Council, Takeda New Frontier Science, Muscular Dystrophy UK, Duchenne Childrens Trust, Duchenne Research Fund, Duchenne Parent Project Onlus.