Jinhong Meng

Contribution of Human Muscle-Derived Cells to Skeletal Muscle Regeneration in Dystrophic Host Mice

Background Stem cell transplantation is a promising potential therapy for muscular dystrophies, but for this purpose, the cells need to be systemically-deliverable, give rise to many muscle fibres and functionally reconstitute the satellite cell niche in the majority of the patients skeletal muscles. Human skeletal muscle-derived pericytes have been shown to form muscle fibres after intra-arterial transplantation in dystrophin-deficient host mice. Our aim was to replicate and extend these promising findings. Methodology/Principal Findings Isolation and maintenance of human muscle derived cells (mdcs) was performed as published for human pericytes. Mdscs were characterized by immunostaining, flow cytometry and RT-PCR; also, their ability to differentiate into myotubes in vitro and into muscle fibres in vivo was assayed. Despite minor differences between human mdcs and pericytes, mdscs contributed to muscle regeneration after intra-muscular injection in mdx nu/nu mice, the CD56+ sub-population being especially myogenic. However, in contrast to human pericytes delivered intra-arterially in mdx SCID hosts, mdscs did not contribute to muscle regeneration after systemic delivery in mdx nu/nu hosts. Conclusions/Significance Our data complement and extend previous findings on human skeletal muscle-derived stem cells, and clearly indicate that further work is necessary to prepare pure cell populations from skeletal muscle that maintain their phenotype in culture and make a robust contribution to skeletal muscle regeneration after systemic delivery in dystrophic mouse models. Small differences in protocols, animal models or outcome measurements may be the reason for differences between our findings and previous data, but nonetheless underline the need for more detailed studies on muscle-derived stem cells and independent replication of results before use of such cells in clinical trials.

Stem cells to treat muscular dystrophies – Where are we?

The muscular dystrophies are inherited disorders characterised by progressive muscle wasting and weakness. Stem cell therapy is considered to be one of the most promising strategies for treating muscular dystrophies. In this review, we first examine the evidence that a stem cell could be used to treat muscular dystrophies, and then discuss the criteria that an ideal stem cell should meet. We also highlight the importance of standard operation procedures to be followed for ensuring the consistent and reproducible efficacy of a particular stem cell. While at the moment the scientific community is looking for an ideal stem cell to treat muscular dystrophies, it is clear that in order for this field to benefit from therapeutic stem cell applications, additional careful investigations are required.

The contribution of human synovial stem cells to skeletal muscle regeneration

Stem cell therapy holds promise for treating muscle diseases. Although satellite cells regenerate skeletal muscle, they only have a local effect after intra-muscular transplantation. Alternative cell types, more easily obtainable and systemically-deliverable, were therefore sought. Human synovial stem cells (hSSCs) have been reported to regenerate muscle fibres and reconstitute the satellite cell pool. We therefore determined if these cells are able to regenerate skeletal muscle after intra-muscular injection into cryodamaged muscles of Rag2-/gamma chain-/C5-mice. We found that hSSCs possess only limited capacity to undergo myogenic differentiation in vitro or to contribute to muscle regeneration in vivo. However, this is enhanced by over-expression of human MyoD1. Interestingly, hSSCs express extracellular matrix components laminin alpha2 and collagen VI within grafted muscles. Therefore, despite their limited capacity to regenerate skeletal muscle, hSSCs could play a role in treating muscular dystrophies secondary to defects in extracellular matrix proteins.

Human skeletal muscle-derived CD133(+) cells form functional satellite cells after intramuscular transplantation in immunodeficient host mice.

Stem cell therapy is a promising strategy for treatment of muscular dystrophies. In addition to muscle fiber formation, reconstitution of functional stem cell pool by donor cells is vital for long-term treatment. We show here that some CD133(+) cells within human muscle are located underneath the basal lamina of muscle fibers, in the position of the muscle satellite cell. Cultured hCD133(+) cells are heterogeneous and multipotent, capable of forming myotubes and reserve satellite cells in vitro. They contribute to extensive muscle regeneration and satellite cell formation following intramuscular transplantation into irradiated and cryodamaged tibialis anterior muscles of immunodeficient Rag2-/γ chain-/C5-mice. Some donor-derived satellite cells expressed the myogenic regulatory factor MyoD, indicating that they were activated. In addition, when transplanted host muscles were reinjured, there was significantly more newly-regenerated muscle fibers of donor origin in treated than in control, nonreinjured muscles, indicating that hCD133(+) cells had given rise to functional muscle stem cells, which were able to activate in response to injury and contribute to a further round of muscle regeneration. Our findings provide new evidence for the location and characterization of hCD133(+) cells, and highlight that these cells are highly suitable for future clinical application.Stem cell therapy is a promising strategy for treatment of muscular dystrophies. In addition to muscle fiber formation, reconstitution of functional stem cell pool by donor cells is vital for long-term treatment. We show here that some CD133+ cells within human muscle are located underneath the basal lamina of muscle fibers, in the position of the muscle satellite cell. Cultured hCD133+ cells are heterogeneous and multipotent, capable of forming myotubes and reserve satellite cells in vitro. They contribute to extensive muscle regeneration and satellite cell formation following intramuscular transplantation into irradiated and cryodamaged tibialis anterior muscles of immunodeficient Rag2-/γ chain-/C5-mice. Some donor-derived satellite cells expressed the myogenic regulatory factor MyoD, indicating that they were activated. In addition, when transplanted host muscles were reinjured, there was significantly more newly-regenerated muscle fibers of donor origin in treated than in control, nonreinjured muscles, indicating that hCD133+ cells had given rise to functional muscle stem cells, which were able to activate in response to injury and contribute to a further round of muscle regeneration. Our findings provide new evidence for the location and characterization of hCD133+ cells, and highlight that these cells are highly suitable for future clinical application.

Repeated low doses of morpholino antisense oligomer: an intermediate mouse model of spinal muscular atrophy to explore the window of therapeutic response

The human SMN2 transgenic mice are well-established models of spinal muscular atrophy (SMA). While the severe type I mouse model has a rapidly progressive condition mimicking type I SMA in humans, the mild type III mice do not faithfully recapitulate chronic SMA variants affecting children. A SMA mouse model that clinically mimics the features of type II and III SMA in human is therefore needed. In this study, we generated intermediately affected SMA mice by delivering low-dose morpholino oligomer (PMO25) into the existing severe SMA mice. We show that a single low-dose administration of PMO25 moderately extended the survival of severe type I SMA mice. The neuromuscular pathology is also modestly but significantly improved in these mice. A second administration of PMO25 at postnatal day 5 (PND5) demonstrated an additive effect on survival. Additional systemic administration of low-dose PMO25 at 2-week intervals suppressed the occurrence of distal necrosis beyond postnatal day 100, and induced more complete phenotypic rescue than a single bolus high-dose injection at PND0. Our study demonstrates that survival of motor neuron (SMN) is required early at a critical threshold to prevent symptoms and suggests that subsequent systemic administration of low-dose PMO25 in SMA mice can provide therapeutic benefit and phenotypic rescue, presumably via peripheral SMN restoration. Our work also provides additional insight into the time window of response to administration of antisense oligonucleotides to SMA mice with an intermediate phenotype. This information is crucial at a time when a number of therapeutic interventions are in clinical trials in SMA patients.

The effect of the muscle environment on the regenerative capacity of human skeletal muscle stem cells

BackgroundMuscle stem cell transplantation is a possible treatment for muscular dystrophy. In addition to the intrinsic properties of the stem cells, the local and systemic environment plays an important role in determining the fate of the grafted cells. We therefore investigated the effect of modulating the host muscle environment in different ways (irradiation or cryoinjury or a combination of irradiation and cryoinjury) in two immunodeficient mouse strains (mdx nude and recombinase-activating gene (Rag)2-/γ chain-/C5-) on the regenerative capacity of two types of human skeletal muscle-derived stem cell (pericytes and CD133+ cells).MethodsHuman skeletal muscle-derived pericytes or CD133+ cells were transplanted into muscles of either mdx nude or recombinase-activating gene (Rag)2-/γ chain-/C5- host mice. Host muscles were modulated prior to donor cell transplantation by either irradiation, or cryoinjury, or a combination of irradiation and cryoinjury. Muscles were analysed four weeks after transplantation, by staining transverse cryostat sections of grafted muscles with antibodies to human lamin A/C, human spectrin, laminin and Pax 7. The number of nuclei and muscle fibres of donor origin and the number of satellite cells of both host and donor origin were quantified.ResultsWithin both host strains transplanted intra-muscularly with both donor cell types, there were significantly more nuclei and muscle fibres of donor origin in host muscles that had been modulated by cryoinjury, or irradiation+cryoinjury, than by irradiation alone. Irradiation has no additive effects in further enhancing the transplantation efficiency than cryodamage. Donor pericytes did not give rise to satellite cells. However, using CD133+ cells as donor cells, there were significantly more nuclei, muscle fibres, as well as satellite cells of donor origin in Rag2-/γ chain-/C5- mice than mdx nude mice, when the muscles were injured by either cryodamage or irradiation+cryodamage.ConclusionsRag2-/γ chain-/C5- mice are a better recipient mouse strain than mdx nude mice for human muscle stem cell transplantation. Cryodamage of host muscle is the most effective method to enhance the transplantation efficiency of human skeletal muscle stem cells. This study highlights the importance of modulating the muscle environment in preclinical studies to optimise the efficacy of transplanted stem cells.

Lentiviral vectors can be used for full-length dystrophin gene therapy

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a ‘template-switching’ lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne Muscular Dystrophy

Autologous stem cells that have been genetically modified to express dystrophin are a possible means of treating Duchenne Muscular Dystrophy (DMD). To maximize the therapeutic effect, dystrophin construct needs to contain as many functional motifs as possible, within the packaging capacity of the viral vector. Existing dystrophin constructs used for transduction of muscle stem cells do not contain the nNOS binding site, an important functional motif within the dystrophin gene. In this proof-of-concept study, using stem cells derived from skeletal muscle of a DMD patient (mdcs) transplanted into an immunodeficient mouse model of DMD, we report that two novel dystrophin constructs, C1 (ΔR3-R13) and C2 (ΔH2-R23), can be lentivirally transduced into mdcs and produce dystrophin. These dystrophin proteins were functional in vivo, as members of the dystrophin glycoprotein complex were restored in muscle fibres containing donor-derived dystrophin. In muscle fibres derived from cells that had been transduced with construct C1, the largest dystrophin construct packaged into a lentiviral system, nNOS was restored. The combination of autologous stem cells and a lentivirus expressing a novel dystrophin construct which optimally restores proteins of the dystrophin glycoprotein complex may have therapeutic application for all DMD patients, regardless of their dystrophin mutation.

Publisher Correction: Necroptosis mediates myofibre death in dystrophin-deficient mice

The original version of this article contained an error in Fig. 3. In panel c, the labels ‘mdx’ and ‘mdx Ripk3-/-‘ were inadvertently inverted. This has now been corrected in the PDF and HTML versions of the Article.

Necroptosis mediates myofibre death in dystrophin-deficient mice

Duchenne muscular dystrophy (DMD) is a severe degenerative disorder caused by mutations in the dystrophin gene. Dystrophin-deficient muscles are characterised by progressive myofibre necrosis in which inflammation plays a deleterious role. However, the molecular mechanisms underlying inflammation-induced necrosis in muscle cells are unknown. Here we show that necroptosis is a mechanism underlying myofibre death in dystrophin-deficient muscle. RIPK1, RIPK3 and MLKL are upregulated in dystrophic mouse myofibres. In human DMD samples, there is strong immunoreactivity to RIPK3 and phospho-MLKL in myofibres. In vitro, TNFα can elicit necroptosis in C2C12 myoblasts, and RIPK3 overexpression sensitises myoblasts to undergo TNF-induced death. Furthermore, genetic ablation of Ripk3 in mdx mice reduces myofibre degeneration, inflammatory infiltrate, and muscle fibrosis, and eventually improves muscle function. These findings provide the first evidence of necroptotic cell death in a disease affecting skeletal muscle and identify RIPK3 as a key player in the degenerative process in dystrophin-deficient muscles.Muscular dystrophies are characterised by extensive myofibre cell death. Here Morgan et al. show that RIPK3-mediated necroptosis contributes to myofibre cell death in Duchenne muscular dystrophy, and that RIPK3 deletion protects dystrophic mice against myofibre degeneration.