Cardiomyocytes derived from human embryonic stem (hES) cells potentially offer large numbers of cells to facilitate repair of the infarcted heart. However, this approach has been limited by inefficient differentiation of hES cells into cardiomyocytes, insufficient purity of cardiomyocyte preparations and poor survival of hES cell-derived myocytes after transplantation. Seeking to overcome these challenges, we generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation that relies on activin A and BMP4. We then identified a cocktail of pro-survival factors that limits cardiomyocyte death after transplantation. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls receiving noncardiac hES cell derivatives or vehicle. The ability of hES cell-derived cardiomyocytes to partially remuscularize myocardial infarcts and attenuate heart failure encourages their study under conditions that closely match human disease.

https://courses.washington.edu/conj504/readings/murry_reading2.pdf

Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts

Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.

Three populations of myogenic cells were isolated from normal mouse skeletal muscle based on their adhesion characteristics and proliferation behaviors. Although two of these populations displayed satellite cell characteristics, a third population of long-time proliferating cells expressing hematopoietic stem cell markers was also identified. This third population comprises cells that retain their phenotype for more than 30 passages with normal karyotype and can differentiate into muscle, neural, and endothelial lineages both in vitro and in vivo. In contrast to the other two populations of myogenic cells, the transplantation of the long-time proliferating cells improved the efficiency of muscle regeneration and dystrophin delivery to dystrophic muscle. The long-time proliferating cells' ability to proliferate in vivo for an extended period of time, combined with their strong capacity for self-renewal, their multipotent differentiation, and their immune-privileged behavior, reveals, at least in part, the basis for the improvement of cell transplantation. Our results suggest that this novel population of muscle-derived stem cells will significantly improve muscle cell–mediated therapies.

/pdf/identification-of-a-novel-population-of-muscle-stem-cells-in-5ecsw5351v.pdf

Identification of a novel population of muscle stem cells in mice potential for muscle regeneration

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin+ cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34+ cells and Bcl-2+ cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34+Bcl-2+ enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.

https://rupress.org/jcb/article-pdf/150/5/1085/1294415/9912110.pdf

Clonal Isolation of Muscle-Derived Cells Capable of Enhancing Muscle Regeneration and Bone Healing

Skeletal muscle damaged by injury or by degenerative diseases such as muscular dystrophy is able to regenerate new muscle fibers. Regeneration mainly depends upon satellite cells, myogenic progenitors localized between the basal lamina and the muscle fiber membrane. However, other cell types outside the basal lamina, such as pericytes, also have myogenic potency. Here, we discuss the main properties of satellite cells and other myogenic progenitors as well as recent efforts to obtain myogenic cells from pluripotent stem cells for patient-tailored cell therapy. Clinical trials utilizing these cells to treat muscular dystrophies, heart failure, and stress urinary incontinence are also briefly outlined.

/pdf/repairing-skeletal-muscle-regenerative-potential-of-skeletal-3wighi892t.pdf

Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells

Transgenic CD1 mice expressing beta-galactosidase were used as myoblast donors. The myoblasts were injected in normal or mdx muscles previously irradiated and injected with notexin. Twenty-eight days after myoblast transplantation, the percentage of muscle fibers beta-glactosidase-positive was low in mice not immunosuppressed but was high (80%) in those treated with FK506. In mdx mice, muscle fibers expressing beta-galactosidase were also dystrophin positive. Most of the mice not treated with FK506 produced antibodies against the donor myoblasts. These results indicate that FK506 is a very useful immunosuppressive drug for myoblast transplantation in mice. Irradiation and notexin injection used in our experiments are, however, not feasible in humans. Other manipulations capable of increasing the participation of donor myoblasts to regeneration will therefore have to be identified before new clinical trials are attempted.

Very efficient myoblast allotransplantation in mice under FK506 immunosuppression

Myoblast transplantation is a potential treatment for Duchenne muscular dystrophy. One of the problems possibly responsible for the limited success of clinical trials is the rapid death of the myoblasts after transplantation. To investigate this problem, myoblasts expressing beta-galactosidase were injected in the tibialis anterior muscles of mice. Beta-galactosidase activity was reduced by 74.7% after 3 days. Myoblast death observed at 3 days was reduced to 57.2% when the hosts were irradiated. This result suggested that host cells were contributing to this phenomenon. Transplantation in SCID and FK506-treated mice did not reduce cell death, indicating that mortality was not due to an acute specific reaction. In contrast, administration of the anti-LFA-1 (TIB-213) mAb markedly reduced myoblast death at 3 days without altering leukocyte tissue infiltration. We postulated that neutrophils were mediating myoblast mortality by an LFA-1-dependent mechanism. To test this hypothesis, IL-1beta-activated myoblasts were loaded with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethylester) (DCFH), a marker for oxidative stress. Addition of neutrophils and zymosan-activated serum resulted in a time-dependent DCFH fluorescence; this neutrophil-induced oxidation was considerably inhibited by TIB-213. These results indicate that an effective control of the inflammatory reaction will be necessary for any new clinical trials of myoblast transplantation and suggest that neutrophil-mediated myoblast injury occurs by an LFA-1-dependent pathway.

Prevention by anti-LFA-1 of acute myoblast death following transplantation.

Human and mouse (C57BL/10SnJ+/+) myoblasts were injected separately in the muscles of C57BL/10SnJ mdx/mdx mice. Mouse myoblasts (C57BL/10SnJ+/+) were also injected in normal mice (C57BL/10SnJ+/+ and BALB/c +/+). Some muscles that received a xenotransplantation (i.e., human myoblasts) were previously injected with a myotoxin, i.e., notexin. This treatment was not used for the allografts (i.e., mouse myoblasts). Human myoblast injections did not increase the number of dystrophin-positive cells above the background level due to backmutation. Moreover, the human myoblasts detected with an anti-HLA antibody decreased rapidly during the 6-week followup. The injection of normal mouse myoblasts in mdx mice did, however, increase the number of dystrophin-positive fibers. Moreover, numerous cells expressing mouse MHC class II, macrophages, granulocytes, neutrophils, natural killer cells, and a subset of T lymphocytes were detected by immunohistochemistry in cryostat sections of myoblast injected muscles. These cells were present within 1 week of the myoblast injection in the muscle regions containing injected human or mouse myoblasts, and progressively decreased during the 6-week follow-up in the human myoblast transplantation. Lymphocyte infiltration reached a significant level following xeno- and alloincompatible transplantations. Antibodies against the human myoblasts and against alloincompatible myoblasts were also detected in the serum of the recipients. These results suggest that humoral and cellular immune reactions are responsible for the poor outcome of myoblast transplantation in mice and could be involved in failure of transplantation in Duchenne muscular dystrophy patients. These results indicate that adequate immunousuppression must be used in these patients.© 1995 John Wiley &Sons, Inc.

Lymphocyte infiltration following allo-and xenomyoblast transplantation in mdx mice

The therapeutic potential of adenovirus-mediated gene transfer using first-generation vectors is severely limited by the fact that only transient expression is achievable in immunocompetent animals. The loss in transgene expression can be attributed at least in part to the appearance of detrimental immune responses directed toward vector and/or transgene-encoded determinants. FK506 and cyclosporin A both reduced these immune responses. These immunosuppressants, however, may induce many severe side effects during prolonged use. An alternative strategy has been developed to overcome these problems following in vivo transfection of muscles of adult immunocompetent mice with a delta E1/E3a adenoviral vector encoding a beta-galactosidase (beta-Gal) expression cassette. YTS 177 (an anti-CD4 monoclonal antibody) as well as CTLA4Ig, a recombinant protein, partially controlled the immune responses. They were indeed able to reduce the muscle infiltration by CD4+ and CD8+ cells but they failed to repress the humoral response. Co-administration of YTS 191 (an anti-CD4), YTS 169 (an anti-CD8), and TIB 213 (an anti-CD11a) was, however, very efficient in blocking both cellular and humoral immune reactions. This combination of monoclonal antibodies allowed strong and stable transgene expression over 1 month. These data underline the potential of monoclonal antibodies as immunosuppressive adjunct treatment for adenovirus-mediated gene transfer.

B. Guerette

Papers

Very efficient myoblast allotransplantation in mice under FK506 immunosuppression

Prevention by anti-LFA-1 of acute myoblast death following transplantation.

Lymphocyte infiltration following allo-and xenomyoblast transplantation in mdx mice

Prevention of Immune Reactions Triggered by First-Generation Adenoviral Vectors by Monoclonal Antibodies and CTLA4Ig

Myoblast allotransplantation in mice: degree of success varies depending on the efficacy of various immunosuppressive treatments.