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.
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•Human iPSC-derived 3D artificial muscles show features of normal skeletal muscle•Multiple muscular dystrophy iPSC lines can be differentiated in 3D artificial muscles•Artificial muscle constructs model severe, incurable forms of muscular dystrophy•Isogenic vascular-like networks and motor neurons develop within artificial muscles
Maffioletti et al. generate human 3D artificial skeletal muscles from healthy donors and patient-specific pluripotent stem cells. These human artificial muscles accurately model severe genetic muscle diseases. They can be engineered to include other cell types present in skeletal muscle, such as vascular cells and motor neurons.
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.
Satellite cells are responsible for skeletal muscle regeneration. Upon activation, they proliferate as transient amplifying myoblasts, most of which fuse into regenerating myofibers. Despite their ...remarkable differentiation potential, these cells have limited migration capacity, which curtails clinical use for widespread forms of muscular dystrophy. Conversely, skeletal muscle perivascular cells have less myogenic potential but better migration capacity than satellite cells. Here we show that modulation of Notch and PDGF pathways, involved in developmental specification of pericytes, induces perivascular cell features in adult mouse and human satellite cell-derived myoblasts. DLL4 and PDGF-BB-treated cells express markers of perivascular cells and associate with endothelial networks while also upregulating markers of satellite cell self-renewal. Moreover, treated cells acquire trans-endothelial migration ability while remaining capable of engrafting skeletal muscle upon intramuscular transplantation. These results extend our understanding of muscle stem cell fate plasticity and provide a druggable pathway with clinical relevance for muscle cell therapy.
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•DLL4 and PDGF-BB change satellite cell morphology, proliferation, and differentiation•DLL4 and PDGF-BB induce both perivascular and satellite cell gene expression•Treated satellite cells acquire perivascular cell properties and improved migration•Human satellite cell-derived myoblasts respond to DLL4 and PDGF-BB treatment
Gerli and Moyle and colleagues show that treatment with molecules involved in developmental specification of pericytes (DLL4 and PDGF-BB) alters satellite cell fate and provides them with features potentially relevant for novel cell therapy protocols.
Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is ...Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large‐capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS‐HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS‐HAC into DMD satellite cell‐derived myoblasts and perivascular cell‐derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic in vitro (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next‐generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy.
Synopsis
Reversible immortalisation of human dystrophic muscle progenitors with a novel human artificial chromosome (HAC) containing the entire dystrophin locus (DYS‐HAC2) enables genetic correction & provide evidence of translation of HAC technology into DMD muscle progenitors for ex vivo gene therapy.
Lentivirally delivered hTERT and Bmi1 cDNAs extend proliferation of human muscle progenitors (both myoblasts and pericyte‐derived mesoangioblasts) and can be reverted by administering Cre recombinase and ganciclovir.
Extension of the proliferative ability of muscle progenitors derived from Duchenne muscular dystrophy patients enables their genetic correction with a novel DYS‐HAC.
A next‐generation DYS‐HAC containing multiple gene functions was engineered to enable simultaneous delivery of: (i) genomic integration‐free reversible immortalisation, (ii) genetic correction, (iii) inducible differentiation, (iv) controllable cell death.
Reversible immortalisation of human dystrophic muscle progenitors with a novel human artificial chromosome (HAC) containing the entire dystrophin locus (DYS‐HAC2) enables genetic correction & provide evidence of translation of HAC technology into DMD muscle progenitors for ex vivo gene therapy.
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 Children's Trust, Duchenne Research Fund, Duchenne Parent Project Onlus.
The Polycomb group gene BMI1 is essential for efficient muscle regeneration in a mouse model of Duchenne muscular dystrophy, and its enhanced expression in adult skeletal muscle satellite cells ...ameliorates the muscle strength in this model. Here, we show that the impact of mild BMI1 overexpression observed in mouse models is translatable to human cells. In human myoblasts, BMI1 overexpression increases mitochondrial activity, leading to an enhanced energetic state with increased ATP production and concomitant protection against DNA damage both in vitro and upon xenografting in a severe dystrophic mouse model. These preclinical data in mouse models and human cells provide a strong rationale for the development of pharmacological approaches to target BMI1-mediated mitochondrial regulation and protection from DNA damage to sustain the regenerative potential of the skeletal muscle in conditions of chronic muscle wasting.
•BMI1 overexpression enhances energetic state in human dystrophic myoblasts•It protects the cells from DNA damage both in vitro and in vivo in a dystrophic model•BMI1 expression is reduced in chronic muscle wasting independently of its etiology
In this article, Marino and colleagues show that BMI1 overexpression in human myoblasts enhanced their energetic state while conferring protection from DNA damage both in vitro and in vivo upon xenografting in a dystrophic mouse model. These preclinical data provide a strong rationale for the development of pharmacological approaches to target BMI1-mediated mitochondrial regulation and protection from DNA damage to sustain the regenerative potential of the skeletal muscle.
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.
Intra‐arterial transplantation of mesoangioblasts proved safe and partially efficacious in preclinical models of muscular dystrophy. We now report the first‐in‐human, exploratory, non‐randomized ...open‐label phase I–IIa clinical trial of intra‐arterial HLA‐matched donor cell transplantation in 5 Duchenne patients. We administered escalating doses of donor‐derived mesoangioblasts in limb arteries under immunosuppressive therapy (tacrolimus). Four consecutive infusions were performed at 2‐month intervals, preceded and followed by clinical, laboratory, and muscular MRI analyses. Two months after the last infusion, a muscle biopsy was performed. Safety was the primary endpoint. The study was relatively safe: One patient developed a thalamic stroke with no clinical consequences and whose correlation with mesoangioblast infusion remained unclear. MRI documented the progression of the disease in 4/5 patients. Functional measures were transiently stabilized in 2/3 ambulant patients, but no functional improvements were observed. Low level of donor DNA was detected in muscle biopsies of 4/5 patients and donor‐derived dystrophin in 1. Intra‐arterial transplantation of donor mesoangioblasts in human proved to be feasible and relatively safe. Future implementation of the protocol, together with a younger age of patients, will be needed to approach efficacy.
Synopsis
This study reports a safe, first‐in‐human mesoangioblast cell therapy to treat Duchenne muscular dystrophy (DMD) in 5 young patients, using a successful preclinical strategy, as an exploratory non‐randomized open‐label phase I–IIa clinical trial of intra‐arterial HLA‐matched donor cell transplantation.
Five patients affected by DMD were treated by intra‐arterial infusions of escalating doses of HLA‐matched donor mesoangioblasts.
The trial was overall safe but showed minimal, if any, efficacy.
Data analysis suggested to treat patients at an earlier stage of the disease, optimize in in vitro models each single step of the transplantation protocol, and use genetically corrected, autologous mesoangioblasts in a future trial.
This study reports a safe, first‐in‐human mesoangioblast cell therapy to treat Duchenne muscular dystrophy (DMD) in 5 young patients, using a successful preclinical strategy, as an exploratory non‐randomized open‐label phase I‐IIa clinical trial of intra‐arterial HLA‐matched donor cell transplantation.
In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle ...development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded
(also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.
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