The development of cell or gene therapies for diseases involving cells that are widely distributed throughout the body has been severely hampered by the inability to achieve the disseminated delivery of cells or genes to the affected tissues or organ. Here we report the results of bone marrow transplantation studies in the mdx mouse, an animal model of Duchenne's muscular dystrophy, which indicate that the intravenous injection of either normal haematopoietic stem cells or a novel population of muscle-derived stem cells into irradiated animals results in the reconstitution of the haematopoietic compartment of the transplanted recipients, the incorporation of donor-derived nuclei into muscle, and the partial restoration of dystrophin expression in the affected muscle. These results suggest that the transplantation of different stem cell populations, using the procedures of bone marrow transplantation, might provide an unanticipated avenue for treating muscular dystrophy as well as other diseases where the systemic delivery of therapeutic cells to sites throughout the body is critical. Our studies also suggest that the inherent developmental potential of stem cells isolated from diverse tissues or organs may be more similar than previously anticipated.

During embryonic development, skeletal muscles arise from somites, which derive from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation of monolayer cultures of mouse embryonic stem (ES) cells into PSM-like cells without the introduction of transgenes or cell sorting. We show that primary and secondary skeletal myogenesis can be recapitulated in vitro from the PSM-like cells, providing an efficient, serum-free protocol for the generation of striated, contractile fibers from mouse and human pluripotent cells. The mouse ES cells also differentiate into Pax7(+) cells with satellite cell characteristics, including the ability to form dystrophin(+) fibers when grafted into muscles of dystrophin-deficient mdx mice, a model of Duchenne muscular dystrophy (DMD). Fibers derived from ES cells of mdx mice exhibit an abnormal branched phenotype resembling that described in vivo, thus providing an attractive model to study the origin of the pathological defects associated with DMD.

Satellite cells are dormant progenitors located at the periphery of skeletal myofibers that can be triggered to proliferate for both self-renewal and differentiation into myogenic cells. In addition to anatomic location, satellite cells are typified by markers such as M-cadherin, Pax7, Myf5, and neural cell adhesion molecule-1. The Pax3 and Pax7 transcription factors play essential roles in the early specification, migration, and myogenic differentiation of satellite cells. In addition to muscle-committed satellite cells, multi-lineage stem cells encountered in embryonic, as well as adult, tissues exhibit myogenic potential in experimental conditions. These multi-lineage stem cells include side-population cells, muscle-derived stem cells (MDSCs), and mesoangioblasts. Although the ontogenic derivation, identity, and localization of these non-conventional myogenic cells remain elusive, recent results suggest their ultimate origin in blood vessel walls. Indeed, purified pericytes and endothelium-related cells demonstrate high myogenic potential in culture and in vivo. Allogeneic myoblasts transplanted into Duchenne muscular dystrophy (DMD) patients have been, in early trials, largely inefficient owing to immune rejection, rapid death, and limited intramuscular migration--all obstacles that are now being alleviated, at least in part, by more efficient immunosuppression and escalated cell doses. As an alternative to myoblast transplantation, stem cells such as mesoangioblasts and CD133+ progenitors administered through blood circulation have recently shown great potential to regenerate dystrophic muscle.

Summary Maintenance of body temperature is essential for survival of homeotherms. Brown adipose tissue (BAT) is a specialized fat tissue that is dedicated to thermoregulation1. Due to its remarkable capacity to dissipate stored energy and its demonstrated presence in adult humans2-5, BAT holds great promise for the treatment of obesity and metabolic syndrome1. Rodent data suggest the existence of two types of brown fat cells: the constitutive BAT (cBAT), which is of embryonic origin and anatomically located in the interscapular region of mice, and the recruitable BAT (rBAT) that resides within white adipose tissue (WAT)6 and skeletal muscle7, that has alternatively been called beige8, brite9, or inducible BAT10. Bone morphogenetic proteins (BMPs) regulate the formation and thermogenic activity of BAT10-12. We here provide evidence for a systemically active regulatory mechanism that serves to control whole body BAT-activity for thermoregulation and energy homeostasis. Genetic ablation of type 1A BMP-receptor (Bmpr1A) in brown adipogenic progenitor cells leads to a severe paucity of cBAT. This in turn increases sympathetic input to WAT, thereby promoting the formation of rBAT within white fat depots. This previously unknown compensatory mechanism, aimed at restoring total brown fat-mediated thermogenic capacity in the body, is sufficient to maintain normal temperature homeostasis and resistance to diet-induced obesity. These data suggest an important physiological cross-talk between the constitutive and recruitable brown fat cells. This sophisticated regulatory mechanism of body temperature may participate in the control of energy balance and metabolic disease.

Severe childhood autosomal recessive muscular dystrophy (SCARMD) is a progressive muscle-wasting disorder common in North Africa that segregates with microsatellite markers at chromosome 13q12. Here, it is shown that a mutation in the gene encoding the 35-kilodalton dystrophin-associated glycoprotein, gamma-sarcoglycan, is likely to be the primary genetic defect in this disorder. The human gamma-sarcoglycan gene was mapped to chromosome 13q12, and deletions that alter its reading frame were identified in three families and one of four sporadic cases of SCARMD. These mutations not only affect gamma-sarcoglycan but also disrupt the integrity of the entire sarcoglycan complex.

The dystrophin associated proteins (DAPs) are good candidates for harboring primary mutations in the genetically heterogeneous autosomal recessive muscular dystrophies (ARMD). The transmembrane components of the DAPs can be separated into the dystroglycan and the sarcoglycan complexes. Here we report the isolation of cDNAs encoding the 43 kD sarcoglycan protein beta-sarcoglycan (A3b) and the localization of the human gene to chromosome 4q12. We describe a young girl with ARMD with truncating mutations on both alleles. Immunostaining of her muscle biopsy shows specific loss of the components of the sarcoglycan complex (beta-sarcoglycan, alpha-sarcoglycan (adhalin), and 35 kD sarcoglycan). Thus secondary destabilization of the sarcoglycan complex may be an important pathophysiological event in ARMD.

Muscle biopsies from six patients with Duchenne muscular dystrophy (DMD) participating in a myoblast transplantation clinical trial were reexamined using a fluorescence in situ hybridization (FISH)-based method. Donor nuclei were detected in all biopsies analyzed, including nine where no donor myoblasts were previously thought to be present. In three patients, more than 10% of the original number of donor cells were calculated as present 6 months after implantation. Half of the detected donor nuclei were fused into host myofibers, and of these, nearly 50% produced dystrophin. These findings demonstrate that although donor myoblasts have persisted after injection, their microenvironment influences whether they fuse and express dystrophin. Our methodology could be used for developing new approaches to improve myoblast transfer efficacy and for the analysis of future gene- and/or cell-based therapies of numerous genetic disorders.

Gene delivery by transplantation of normal myoblasts has been proposed as a treatment of the primary defect, lack of the muscle protein dystrophin, that causes Duchenne muscular dystrophy (DMD), a lethal human muscle degenerative disorder. To test this possibility, we transplanted normal myoblasts from a father or an unaffected sibling into the muscle of eight boys with DMD, and assessed their production of dystrophin. Three patients with deletions in the dystrophin gene expressed normal dystrophin transcripts in muscle biopsy specimens taken from the transplant site one month after myoblast injection. Using the polymerase chain reaction we established that the dystrophin in these biopsies derived from donor myoblast DNA. These results show that transplanted myoblasts persist and produce dystrophin in muscle fibres of DMD patients.