Radbod Darabi

Functional skeletal muscle regeneration from differentiating embryonic stem cells

Little progress has been made toward the use of embryonic stem (ES) cells to study and isolate skeletal muscle progenitors. This is due to the paucity of paraxial mesoderm formation during embryoid body (EB) in vitro differentiation and to the lack of reliable identification and isolation criteria for skeletal muscle precursors. Here we show that expression of the transcription factor Pax3 during embryoid body differentiation enhances both paraxial mesoderm formation and the myogenic potential of the cells within this population. Transplantation of Pax3-induced cells results in teratomas, however, indicating the presence of residual undifferentiated cells. By sorting for the PDGF-α receptor, a marker of paraxial mesoderm, and for the absence of Flk-1, a marker of lateral plate mesoderm, we derive a cell population from differentiating ES cell cultures that has substantial muscle regeneration potential. Intramuscular and systemic transplantation of these cells into dystrophic mice results in extensive engraftment of adult myofibers with enhanced contractile function without the formation of teratomas. These data demonstrate the therapeutic potential of ES cells in muscular dystrophy.

Human ES- and iPS-derived myogenic progenitors restore DYSTROPHIN and improve contractility upon transplantation in dystrophic mice.

A major obstacle in the application of cell-based therapies for the treatment of neuromuscular disorders is obtaining the appropriate number of stem/progenitor cells to produce effective engraftment. The use of embryonic stem (ES) or induced pluripotent stem (iPS) cells could overcome this hurdle. However, to date, derivation of engraftable skeletal muscle precursors that can restore muscle function from human pluripotent cells has not been achieved. Here we applied conditional expression of PAX7 in human ES/iPS cells to successfully derive large quantities of myogenic precursors, which, upon transplantation into dystrophic muscle, are able to engraft efficiently, producing abundant human-derived DYSTROPHIN-positive myofibers that exhibit superior strength. Importantly, transplanted cells also seed the muscle satellite cell compartment, and engraftment is present over 11 months posttransplant. This study provides the proof of principle for the derivation of functional skeletal myogenic progenitors from human ES/iPS cells and highlights their potential for future therapeutic application in muscular dystrophies.

An ex vivo gene therapy approach to treat muscular dystrophy using inducible pluripotent stem cells

Duchenne muscular dystrophy is a progressive and incurable neuromuscular disease caused by genetic and biochemical defects of the dystrophin-glycoprotein complex. Here we show the regenerative potential of myogenic progenitors derived from corrected dystrophic induced pluripotent stem (iPS) cells generated from fibroblasts of mice lacking both dystrophin and utrophin. We correct the phenotype of dystrophic iPS cells using a Sleeping Beauty transposon carrying the micro-utrophin (μUTRN) gene, differentiate these cells into skeletal muscle progenitors, and transplant them back into dystrophic mice. Engrafted muscles displayed large numbers of micro-utrophin-positive myofibers, with biochemically restored dystrophin-glycoprotein complex and improved contractile strength. The transplanted cells seed the satellite cell compartment, responded properly to injury and exhibit neuromuscular synapses. We also detect muscle engraftment after systemic delivery of these corrected progenitors. These results represent an important advance toward the future treatment of muscular dystrophies using genetically corrected autologous iPS cells.

Assessment of the Myogenic Stem Cell Compartment Following Transplantation of Pax3/Pax7-Induced Embryonic Stem Cell-Derived Progenitors

An effective long‐term cell therapy for skeletal muscle regeneration requires donor contribution to both muscle fibers and the muscle stem cell pool. Although satellite cells have these abilities, their therapeutic potential so far has been limited due to their scarcity in adult muscle. Myogenic progenitors obtained from Pax3‐engineered mouse embryonic stem (ES) cells have the ability to generate myofibers and to improve the contractility of transplanted muscles in vivo, however, whether these cells contribute to the muscle stem cell pool and are able to self‐renew in vivo are still unknown. Here, we addressed this question by investigating the ability of Pax3, which plays a critical role in embryonic muscle formation, and Pax7, which is important for maintenance of the muscle satellite cell pool, to promote the derivation of self‐renewing functional myogenic progenitors from ES cells. We show that Pax7, like Pax3, can drive the expansion of an ES‐derived myogenic progenitor with significant muscle regenerative potential. We further demonstrate that a fraction of transplanted cells remains mononuclear, and displays key features of skeletal muscle stem cells, including satellite cell localization, response to reinjury, and contribution to muscle regeneration in secondary transplantation assays. The ability to engraft, self‐renew, and respond to injury provide foundation for the future therapeutic application of ES‐derived myogenic progenitors in muscle disorders. STEM CELLS 2011;29:777–790

Functional Myogenic Engraftment from Mouse iPS Cells

Direct reprogramming of adult fibroblasts to a pluripotent state has opened new possibilities for the generation of patient- and disease-specific stem cells. However the ability of induced pluripotent stem (iPS) cells to generate tissue that mediates functional repair has been demonstrated in very few animal models of disease to date. Here we present the proof of principle that iPS cells may be used effectively for the treatment of muscle disorders. We combine the generation of iPS cells with conditional expression of Pax7, a robust approach to derive myogenic progenitors. Transplantation of Pax7-induced iPS-derived myogenic progenitors into dystrophic mice results in extensive engraftment, which is accompanied by improved contractility of treated muscles. These findings demonstrate the myogenic regenerative potential of iPS cells and provide rationale for their future therapeutic application for muscular dystrophies.

Engraftment of mesenchymal stem cells into dystrophin-deficient mice is not accompanied by functional recovery

Mesenchymal stem cell preparations have been proposed for muscle regeneration in musculoskeletal disorders. Although MSCs have great in vitro expansion potential and possess the ability to differentiate into several mesenchymal lineages, myogenesis has proven to be much more difficult to induce. We have recently demonstrated that Pax3, the master regulator of the embryonic myogenic program, enables the in vitro differentiation of a murine mesenchymal stem cell line (MSCB9-Pax3) into myogenic progenitors. Here we show that injection of these cells into cardiotoxin-injured muscles of immunodeficient mice leads to the development of muscle tumors, resembling rhabdomyosarcomas. We then extended these studies to primary human mesenchymal stem cells (hMSCs) isolated from bone marrow. Upon genetic modification with a lentiviral vector encoding PAX3, hMSCs activated the myogenic program as demonstrated by expression of myogenic regulatory factors. Upon transplantation, the PAX3-modified MSCs did not generate rhabdomyosarcomas but rather, resulted in donor-derived myofibers. These were found at higher frequency in PAX3-transduced hMSCs than in mock-transduced MSCs. Nonetheless, neither engraftment of PAX3-modified or unmodified MSCs resulted in improved contractility. Thus these findings suggest that limitations remain to be overcome before MSC preparations result in effective treatment for muscular dystrophies.

Apoptosis signaling pathways in osteoarthritis and possible protective role of melatonin

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive erosion of articular cartilage. As chondrocytes are the only cell type forming the articular cartilage, their gradual loss is the main cause of OA. There is a substantial body of published research that suggests reactive oxygen species (ROS) are major causative factors for chondrocyte damage and OA development. Oxidative stress elicited by ROS is capable of oxidizing and subsequently disrupting cartilage homeostasis, promoting catabolism via induction of cell death and damaging numerous components of the joint. IL‐1β and TNF‐α are crucial inflammatory factors that play pivotal roles in the pathogenesis of OA. In this process, the mitochondria are the major source of ROS production in cells, suggesting a role of mitochondrial dysfunction in this type of arthritis. This may also be promoted by inflammatory cytokines such as IL‐1β and TNF‐α which contribute to chondrocyte death. In patients with OA, the expression of endoplasmic reticulum (ER) stress‐associated molecules is positively correlated with cartilage degeneration. Melatonin and its metabolites are broad‐spectrum antioxidants and free radical scavengers which regulate a variety of molecular pathways such as inflammation, proliferation, apoptosis, and metastasis in different pathophysiological situations. Herein, we review the effects of melatonin on OA, focusing on its ability to regulate apoptotic processes and ER and mitochondrial activity. We also evaluate likely protective effects of melatonin on OA pathogenesis.

Engraftment of embryonic stem cell-derived myogenic progenitors in a dominant model of muscular dystrophy

Muscular dystrophies (MDs) consist of a genetically heterogeneous group of disorders, recessive or dominant, characterized by progressive skeletal muscle weakening. To date, no effective treatment is available. Experimental strategies pursuing muscle regeneration through the transplantation of stem cell preparations have brought hope to patients affected by this disorder. Efficacy has been demonstrated in recessive MD models through contribution of wild-type nuclei to the muscle fiber heterokaryon; however, to date, there has been no study investigating the efficacy of a cell therapy in a dominant model of MD. We have recently demonstrated that Pax3-induced embryonic stem (ES) cell-derived myogenic progenitors are able to engraft and improve muscle function in mdx mice, a recessive mouse model for Duchenne MD. To assess whether this therapeutic effect can be extended to a dominant type of muscle disorder, here we transplanted these cells into FRG1 transgenic mice, a dominant model that has been associated with facioscapulohumeral muscular dystrophy. Our results show that Pax3-induced ES-derived myogenic progenitors are capable of significant engraftment after intramuscular or systemic transplantation into Frg1 mice. Analyses of contractile parameters revealed functional improvement in treated muscles of male mice, but not females, which are less severely affected. This study is the first to use Frg1 transgenic mice to assess muscle regeneration as well as to support the use of a cell-based therapy for autosomal dominant types of MD.

A New Immuno‐, Dystrophin‐Deficient Model, the NSG‐mdx4Cv Mouse, Provides Evidence for Functional Improvement Following Allogeneic Satellite Cell Transplantation

Transplantation of a myogenic cell population into an immunodeficient recipient is an excellent way of assessing the in vivo muscle‐generating capacity of that cell population. To facilitate both allogeneic and xenogeneic transplantations of muscle‐forming cells in mice, we have developed a novel immunodeficient muscular dystrophy model, the NSG‐mdx4Cv mouse. The IL2Rg mutation, which is linked to the Dmd gene on the X chromosome, simultaneously depletes NK cells and suppresses thymic lymphomas, issues that limit the utility of the SCID/mdx model. The NSG‐mdx4Cv mouse presents a muscular dystrophy of similar severity to the conventional mdx mouse. We show that this animal supports robust engraftment of both pig and dog muscle mononuclear cells. The question of whether satellite cells prospectively isolated by flow cytometry can confer a functional benefit upon transplantation has been controversial. Using allogeneic Pax7‐ZsGreen donors and NSG‐mdx4Cv recipients, we demonstrate definitively that as few as 900 FACS‐isolated satellite cells can provide functional regeneration in vivo, in the form of an increased mean maximal force‐generation capacity in cell‐transplanted muscles, compared to a sham‐injected control group. These studies highlight the potency of satellite cells to improve muscle function and the utility of the NSG‐mdx4Cv model for studies on muscle regeneration and Duchenne muscular dystrophy therapy. STEM Cells 2013;31:1611–1620

Engraftment of ES-Derived Myogenic Progenitors in a Severe Mouse Model of Muscular Dystrophy.

Controlled myogenic differentiation of mouse embryonic stem cells by Pax3 combined with purification of PDGFαR+Flk-1- paraxial mesoderm results in the efficient in vitro generation of early skeletal myogenic progenitors. Upon transplantation into dystrophin-deficient mdx mice, these progenitors promote significant regeneration that is accompanied by improvement in muscle contractility. In this study, we aimed to raise the bar and assess the therapeutic potential of these cells in a more clinically relevant model of muscular dystrophy: the dystrophin-utrophin double-knockout (dKO) mouse. Unlike mdx mice, which display a mild phenotype, dKO mice are severely ill, displaying progressive muscle wasting, impaired mobility, and premature death. Here we show that in this very severe model of DMD, transplantation of Pax3-induced ES-derived skeletal myogenic progenitors results in significant engraftment as evidenced by the presence of Dystrophin+ myofibers with restoration of β-dystroglycan and eNOS within the sarcolemma, and enhanced strengthen of treated muscles. These findings demonstrate that ES-derived myogenic cell preparations are capable of engrafting in severely dystrophic muscle, and promote significant regeneration, providing a rationale for further studies on the potential therapeutic application of these cells in muscular dystrophies.