Thomas Payne

Effect of VEGF on the Regenerative Capacity of Muscle Stem Cells in Dystrophic Skeletal Muscle

We have isolated a population of muscle-derived stem cells (MDSCs) that, when compared with myoblasts, display an improved regeneration capacity, exhibit better cell survival, and improve myogenesis and angiogenesis. In addition, we and others have observed that the origin of the MDSCs may reside within the blood vessel walls (endothelial cells and pericytes). Here, we investigated the role of vascular endothelial growth factor (VEGF)-mediated angiogenesis in MDSC transplantation-based skeletal muscle regeneration in mdx mice (an animal model of muscular dystrophy). We studied MDSC and MDSC transduced to overexpress VEGF; no differences were observed in vitro in terms of phenotype or myogenic differentiation. However, after in vivo transplantation, we observe an increase in angiogenesis and endogenous muscle regeneration as well as a reduction in muscle fibrosis in muscles transplanted with VEGF-expressing cells when compared to control cells. In contrast, we observe a significant decrease in vascularization and an increase in fibrosis in the muscles transplanted with MDSCs expressing soluble forms-like tyrosine kinase 1 (sFlt1) (VEGF-specific antagonist) when compared to control MDSCs. Our results indicate that VEGF-expressing cells do not increase the number of dystrophin-positive fibers in the injected mdx muscle, when compared to the control MDSCs. Together the results suggest that the transplantation of VEGF-expressing MDSCs improved skeletal muscle repair through modulation of angiogenesis, regeneration and fibrosis in the injected mdx skeletal muscle.

Regeneration of dystrophin-expressing myocytes in the mdx heart by skeletal muscle stem cells.

Cell transplantation holds promise as a potential treatment for cardiac dysfunction. Our group has isolated populations of murine skeletal muscle-derived stem cells (MDSCs) that exhibit stem cell-like properties. Here, we investigated the fate of MDSCs after transplantation into the hearts of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy (DMD). Transplanted MDSCs generated large grafts consisting primarily of numerous dystrophin-positive myocytes and, to a lesser degree, dystrophin-negative nonmyocytes that expressed an endothelial phenotype. Most of the dystrophin-positive myocytes expressed a skeletal muscle phenotype and did not express a cardiac phenotype. However, some donor myocytes, located at the graft–host myocardium border, were observed to express cardiac-specific markers. More than half of these donor cells that exhibited a cardiac phenotype still maintained a skeletal muscle phenotype, demonstrating a hybrid state. Sex-mismatched donors and hosts revealed that many donor-derived cells that acquired a cardiac phenotype did so through fusion with host cardiomyocytes. Connexin43 gap junctions were not expressed by donor-derived myocytes in the graft. Scar tissue formation in the border region may inhibit the fusion and gap junction connections between donor and host cells. This study demonstrates that MDSC transplantation warrants further investigation as a potential therapy for cardiac dysfunction in DMD.

Modeling Stem Cell Population Growth: Incorporating Terms for Proliferative Heterogeneity

Expansion of the undifferentiated stem cell phenotype is one of the most challenging aspects in stem cell research. Clinical protocols for stem cell therapeutics will require standardization of defined culture conditions. A first step in the development of predictable and reproducible, scalable bioreactor processes is the development of mathematical growth models. This paper provides practical models for describing cell growth in general, which are particularly well suited for examining stem cell populations. The nonexponential kinetics of stem cells derive from proliferative heterogeneity, which is biologically recognized as mitosis, quiescence, senescence, differentiation, or death. Here, we examined the assumptions of the Sherley model, which describes heterogeneous expansion in the absence of cell loss. We next incorporated terms into the model to account for A) cell loss or apoptosis and B) cell differentiation. We conclude that the basic assumptions of the model are valid and a high correlation between the modified equations and experimental data obtained using muscle‐derived stem cells was observed. Finally, we demonstrate an improved estimation of the kinetic parameters. This study contributes to both the biological and mathematical understanding of stem cell dynamics. Further, it is expected that the models will prove useful in establishing standardization of cell culture conditions and scalable systems and will be required to develop clinical protocols for stem cell therapeutics.

Myogenic endothelial cells purified from human skeletal muscle improve cardiac function after transplantation into infarcted myocardium.

OBJECTIVES The aim of this study was to evaluate the therapeutic potential of human skeletal muscle-derived myoendothelial cells for myocardial infarct repair. BACKGROUND We have recently identified and purified a novel population of myoendothelial cells from human skeletal muscle. These cells coexpress myogenic and endothelial cell markers and produce robust muscle regeneration when injected into cardiotoxin-injured skeletal muscle. METHODS Myoendothelial cells were isolated from biopsies of human skeletal muscle using a fluorescence-activated cell sorter along with populations of regular myoblasts and endothelial cells. Acute myocardial infarction was induced in male immune-deficient mice, and cells were directly injected into the ischemic area. Cardiac function was assessed by echocardiography, and donor cell engraftment, angiogenesis, scar tissue, endogenous cardiomyocyte proliferation, and apoptosis were all evaluated by immunohistochemistry. RESULTS A greater improvement in left ventricular function was observed after intramyocardial injection of myoendothelial cells when compared with that seen in hearts injected with myoblast or endothelial cells. Transplanted myoendothelial cells generated robust engraftments within the infarcted myocardium, and also stimulated angiogenesis, attenuation of scar tissue, and proliferation and survival of endogenous cardiomyocytes more effectively than transplanted myoblasts or endothelial cells. CONCLUSIONS Our findings suggest that myoendothelial cells represent a novel cell population from human skeletal muscle that may hold promise for cardiac repair.

Differential efficacy of gels derived from small intestinal submucosa as an injectable biomaterial for myocardial infarct repair

Injectable biomaterials have been recently investigated as a therapeutic approach for cardiac repair. Porcine-derived small intestinal submucosa (SIS) material is currently used in the clinic to promote accelerated wound healing for a variety of disorders. In this study, we hypothesized that gels derived from SIS extracellular matrix would be advantageous as an injectable material for cardiac repair. We evaluated 2 forms of SIS gel, types B (SIS-B) and C (SIS-C), for their ability to provide a therapeutic effect when injected directly into ischemic myocardium using a murine model of an acute myocardial infarction. Echocardiography analysis at both 2 and 6 weeks after infarction demonstrated preservation of end-systolic left ventricular geometry and improvement of cardiac contractility in the hearts injected with SIS-B when compared with control hearts injected with saline. However, the SIS-C gel provided no functional efficacy in comparison with control. Histological analysis revealed that SIS-B reduced infarct size and induced angiogenesis relative to control, whereas injection of SIS-C had minimal effect on these histological parameters. Characterization of both gels revealed differential growth factor content with SIS-B exhibiting higher levels of basic fibroblast growth factor than SIS-C, which may explain, at least in part, the differential histological and functional results. This study suggests that SIS gel offers therapeutic potential as an injectable material for the repair of ischemic myocardium. Further understanding of SIS gel characteristics, such as biological and physical properties, that are critical determinants of efficacy would be important for optimization of this biomaterial for cardiac repair.

The Use of Ex Vivo Gene Transfer Based on Muscle-Derived Stem Cells for Cardiovascular Medicine

Cell transplantation is a potential therapy for patients suffering from congestive heart failure. Many cell types have been experimentally tested for their ability to improve cardiac function. In this review, we discuss the potential of cell transplantation into the heart using various cell sources and introduce an attractive new cell source: Muscle-derived stem cells (MDSCs) are capable of delivering therapeutic genes and potentially differentiating toward a cardiomyocyte lineage within an injected heart. MDSCs are an attractive, alternate cell source because in addition to being multipotent (i.e., capable of differentiating into various lineages), they are easily accessible via simple biopsy of the patients own muscle. This review will describe the isolation and unique characteristics of MDSCs and outline their potential use in regenerative medicine.

Human Skeletal Muscle Cells With a Slow Adhesion Rate After Isolation and an Enhanced Stress Resistance Improve Function of Ischemic Hearts

Identification of cells that are endowed with maximum potency could be critical for the clinical success of cell-based therapies. We investigated whether cells with an enhanced efficacy for cardiac cell therapy could be enriched from adult human skeletal muscle on the basis of their adhesion properties to tissue culture flasks following tissue dissociation. Cells that adhered slowly displayed greater myogenic purity and more readily differentiated into myotubes in vitro than rapidly adhering cells (RACs). The slowly adhering cell (SAC) population also survived better than the RAC population in kinetic in vitro assays that simulate conditions of oxidative and inflammatory stress. When evaluated for the treatment of a myocardial infarction (MI), intramyocardial injection of the SACs more effectively improved echocardiographic indexes of left ventricular (LV) remodeling and contractility than the transplantation of the RACs. Immunohistological analysis revealed that hearts injected with SACs displayed a reduction in myocardial fibrosis and an increase in infarct vascularization, donor cell proliferation, and endogenous cardiomyocyte survival and proliferation in comparison with the RAC-treated hearts. In conclusion, these results suggest that adult human skeletal muscle-derived cells are inherently heterogeneous with regard to their efficacy for enhancing cardiac function after cardiac implantation, with SACs outperforming RACs.

Sex of muscle stem cells does not influence potency for cardiac cell therapy

We have previously shown that populations of skeletal muscle-derived stem cells (MDSCs) exhibit sex-based differences for skeletal muscle and bone repair, with female cells demonstrating superior engrafting abilities to males in skeletal muscle while male cells differentiating more robustly toward the osteogenic and chondrogenic lineages. In this study, we tested the hypothesis that the therapeutic capacity of MDSCs transplanted into myocardium is influenced by sex of donor MDSCs or recipient. Male and female MDSCs isolated from the skeletal muscle of 3-week-old mice were transplanted into recipient male or female dystrophin-deficient (mdx) hearts or into the hearts of male SCID mice following acute myocardial infarction. In the mdx model, no difference was seen in engraftment or blood vessel formation based on donor cell or recipient sex. In the infarction model, MDSC-transplanted hearts showed higher postinfarction angiogenesis, less myocardial scar formation, and improved cardiac function compared to vehicle controls. However, sex of donor MDSCs had no significant effects on engraftment, angiogenesis, and cardiac function. VEGF expression, a potent angiogenic factor, was similar between male and female MDSCs. Our results suggest that donor MDSC or recipient sex has no significant effect on the efficiency of MDSC-triggered myocardial engraftment or regeneration following cardiac injury. The ability of the MDSCs to improve cardiac regeneration and repair through promotion of angiogenesis without differentiation into the cardiac lineage may have contributed to the lack of sex difference observed in these models.

923. Muscle Stem Cells Genetically Modified to Express a VEGF Antagonist Display an Impaired Ability for Cardiac Repair

Background: The exact mechanism by which cardiac cell transplantation exerts therapeutic improvements in cardiac function is unclear. One possible mechanism is that the transplanted cells act by promoting neoangiogenesis within the infarct through their release of angiogenic factors. Our previous research has demonstrated that skeletal muscle-derived stem cells (MDSCs) expressed vascular endothelial growth factor (VEGF), a potent angiogenic factor, after transplantation in infarcted hearts. These results led us to hypothesize that VEGF expression by the transplanted MDSCs mediates functional improvements. To test this hypothesis, we performed a set of experiments in which we genetically modified MDSCs to overexpress VEGF (gain-of-function) or overexpress soluble FLT-1, which is a soluble VEGF receptor and antagonist of VEGF (loss-of-function).

Modeling stem cell population growth: incorporating parameters for quiescence, differentiation and apoptosis

The use of stem cells in cell-mediated therapies or cell transplantation applications will require a controlled, scalable system for expansion of the cells and for control of cellular differentiation. Modeling stem cell population growth is one step towards developing such a system. Stem cell populations are heterogeneous and include cells which are non-mitotic. In particular, stem cells may be quiescent or mitotically active. The mitotically-active cells may 1) self-renew to give rise to similar cells, or 2) divide asymmetrically to give rise to precursor cells (which become may terminally differentiated). Cell populations also may include non-mitotic cells which are apoptotic. Non-exponential growth of the stem cell population is the result of the non-mitotic fraction. In this study, we extend a non-exponential model to incorporate parameters describing the non-dividing population. In particular, the models incorporate terms to account for 1) cell loss due to apoptosis or necrosis and 2) the non-dividing differentiated state of a cell. Murine muscle-derived stem cells (MDSC) will be used to experimentally test the model assumptions and determine the goodness of fit of the models using nonlinear regression. This analysis illustrates the dynamics of proliferation in stem cell populations where cells are undergoing both self-renewal and differentiation.