A role for cell sex in stem cell-mediated skeletal muscle regeneration: female cells have higher muscle regeneration efficiency.

We have shown that muscle-derived stem cells (MDSCs) transplanted into dystrophic (mdx) mice efficiently regenerate skeletal muscle. However, MDSC populations exhibit heterogeneity in marker profiles and variability in regeneration abilities. We show here that cell sex is a variable that considerably influences MDSCs' regeneration abilities. We found that the female MDSCs (F-MDSCs) regenerated skeletal muscle more efficiently. Despite using additional isolation techniques and cell cloning, we could not obtain a male subfraction with a regeneration capacity similar to that of their female counterparts. Rather than being directly hormonal or caused by host immune response, this difference in MDSCs' regeneration potential may arise from innate sex-related differences in the cells' stress responses. In comparison with F-MDSCs, male MDSCs have increased differentiation after exposure to oxidative stress induced by hydrogen peroxide, which may lead to in vivo donor cell depletion, and a proliferative advantage for F-MDSCs that eventually increases muscle regeneration. These findings should persuade researchers to report cell sex, which is a largely unexplored variable, and consider the implications of relying on cells of one sex.

m-Calpain activation is regulated by its membrane localization and by its binding to phosphatidylinositol 4,5-bisphosphate.

m-Calpain plays a critical role in cell migration enabling rear de-adhesion of adherent cells by cleaving structural components of the adhesion plaques. Growth factors and chemokines regulate keratinocyte, fibroblast, and endothelial cell migration by modulating m-calpain activity. Growth factor receptors activate m-calpain secondary to phosphorylation on serine 50 by ERK. Concurrently, activated m-calpain is localized to its inner membrane milieu by binding to phosphatidylinositol 4,5-bisphosphate (PIP(2)). Opposing this, CXCR3 ligands inhibit cell migration by blocking m-calpain activity secondary to a PKA-mediated phosphorylation in the C2-like domain. The failure of m-calpain activation in the absence of PIP(2) points to a key regulatory role, although whether this PIP(2)-mediated membrane localization is regulatory for m-calpain activity or merely serves as a docking site for ERK phosphorylation is uncertain. Herein, we report the effects of two CXCR3 ligands, CXCL11/IP-9/I-TAC and CXCL10/IP-10, on the EGF- and VEGF-induced redistribution of m-calpain in human fibroblasts and endothelial cells. The two chemokines block the tail retraction and, thus, the migration within minutes, preventing and reverting growth factor-induced relocalization of m-calpain to the plasma membrane of the cells. PKA phosphorylation of m-calpain blocks the binding of the protease to PIP(2). Unexpectedly, we found that this was due to membrane anchorage itself and not merely serine 50 phosphorylation, as the farnesylation-induced anchorage of m-calpain triggers a strong activation of this protease, leading notably to an increased cell death. Moreover, the ERK and PKA phosphorylations have no effect on this membrane-anchored m-calpain. However, the presence of PIP(2) is still required for the activation of the anchored m-calpain. In conclusion, we describe a novel mechanism of m-calpain activation by interaction with the plasma membrane and PIP(2) specifically, this phosphoinositide acting as a cofactor for the enzyme. The phosphorylation of m-calpain by ERK and PKA by growth factors and chemokines, respectively, act in cells to regulate the enzyme only indirectly by controlling its redistribution.

Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential.

Muscle-derived stem cells (MDSCs) can differentiate into multiple lineages, including haematopoietic lineages. However, it is unknown whether MDSCs preserve their myogenic potential after differentiation into other lineages. To address this issue, we isolated from dystrophic muscle a population of MDSCs that express stem-cell markers and can differentiate into various lineages. After systemic delivery of three MDSC clones into lethally irradiated mice, we found that differentiation of the donor cells into various lineages of the haematopoietic system resulted in repopulation of the recipients' bone marrow. Donor-derived bone-marrow cells, isolated from these recipients by fluorescence-activated cell sorting (FACS), also repopulated the bone marrow of secondary, lethally irradiated, recipients and differentiated into myogenic cells both in vitro and in vivo in normal mdx mice. These findings demonstrate that MDSC clones retain their myogenic potential after haematopoietic differentiation.

Both actin and polyproline interactions of profilin-1 are required for migration, invasion and capillary morphogenesis of vascular endothelial cells.

The objective of the present study was to evaluate how different ligand interactions of profilin-1 (Pfn1), an actin-binding protein that is upregulated during capillary morphogenesis of vascular endothelial cells (VEC), contribute to migration and capillary forming ability of VEC. We adopted a knockdown-knockin experimental system to stably express either fully functional form or mutants of Pfn1 that are impaired in binding to two of its major ligands, actin (H119E mutant) and proteins containing polyproline domains (H133S mutant), in a human dermal microvascular cell line (HmVEC) against near-null endogenous Pfn1 background. We found that silencing endogenous Pfn1 expression in HmVEC leads to slower random migration, reduced velocity of membrane protrusion and a significant impairment in matrigel-induced cord formation. Only re-expression of fully functional but not any of the two ligand-binding deficient mutants of Pfn1 rescues the above defects. We further show that loss of Pfn1 expression in VEC inhibits three-dimensional capillary morphogenesis, MMP2 secretion and ECM invasion. VEC invasion through ECM is also inhibited when actin and polyproline interactions of Pfn1 are disrupted. Together, these experimental data demonstrate that Pfn1 regulates VEC migration, invasion and capillary morphogenesis through its interaction with both actin and proline-rich ligands.

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.

Prospective identification of myogenic endothelial cells in human skeletal muscle.

We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.

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

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.

High harvest yield, high expansion, and phenotype stability of CD146 mesenchymal stromal cells from whole primitive human umbilical cord tissue.

Human umbilical cord blood is an excellent primitive source of noncontroversial stem cells for treatment of hematologic disorders; meanwhile, new stem cell candidates in the umbilical cord (UC) tissue could provide therapeutic cells for nonhematologic disorders. We show novel in situ characterization to identify and localize a panel of some markers expressed by mesenchymal stromal cells (MSCs; CD44, CD105, CD73, CD90) and CD146 in the UC. We describe enzymatic isolation and purification methods of different UC cell populations that do not require manual separation of the vessels and stroma of the coiled, helical-like UC tissue. Unique quantitation of in situ cell frequency and stromal cell counts upon harvest illustrate the potential to obtain high numerical yields with these methods. UC stromal cells can differentiate to the osteogenic and chondrogenic lineages and, under specific culturing conditions, they exhibit high expandability with unique long-term stability of their phenotype. The remarkable stability of the phenotype represents a novel finding for human MSCs, from any source, and supports the use of these cells as highly accessible stromal cells for both basic studies and potentially therapeutic applications such as allogeneic clinical use for musculoskeletal disorders.

Sex differences in muscle-derived stem cells and skeletal muscle.

Sex is well known to influence life expectancy and disposition to disease. Stem and progenitor cells are believed to persist throughout life, and they contribute to the repair and healthy maintenance of tissue; consequently, sex-related differences demonstrated by stem cells may provide insight to sex-related differences in aging, disease, and healing. However, cell sex is an often overlooked variable in stem cell biology.

The role of the biochemical and biophysical environment in chondrogenic stem cell differentiation assays and cartilage tissue engineering.

The field of regenerative medicine offers hope for the development of a cell-based therapy for the repair of articular cartilage (AC). Yet, the greatest challenge in the use of stem cells for tissue repair, is understanding how the cells respond to stimuli and using that knowledge to direct cell fate. Novel methods that utilize stem cells in cartilage regeneration will require specific spatio-temporal controls of the biochemical and biophysical signaling environments. Current chondrogenic differentiation research focuses on the roles of biochemical stimuli like growth factors, hormones, and small molecules, and the role of the physical environment and mechanical stimuli, such as compression and shear stress, which likely act through mechanical receptors. Numerous signals are associated with chondrogenic-like activity of cells in different systems, however many variables for a controlled method still need to be optimized; e.g., spatial and temporal application of the stimuli, and time of transplantation of an engineered construct. Understanding the necessary microenvironmental signals for cell differentiation will advance cell therapy for cartilage repair.

Development of a tissue-engineered vascular graft combining a biodegradable scaffold, muscle-derived stem cells and a rotational vacuum seeding technique.

There is a clinical need for a tissue-engineered vascular graft (TEVG), and combining stem cells with biodegradable tubular scaffolds appears to be a promising approach. The goal of this study was to characterize the incorporation of muscle-derived stem cells (MDSCs) within tubular poly(ester urethane) urea (PEUU) scaffolds in vitro to understand their interaction, and to evaluate the mechanical properties of the constructs for vascular applications. Porous PEUU scaffolds were seeded with MDSCs using our recently described rotational vacuum seeding device, and cultured inside a spinner flask for 3 or 7 days. Cell viability, number, distribution and phenotype were assessed along with the suture retention strength and uniaxial mechanical behavior of the TEVGs. The seeding device allowed rapid even distribution of cells within the scaffolds. After 3 days, the constructs appeared completely populated with cells that were spread within the polymer. Cells underwent a population doubling of 2.1-fold, with a population doubling time of 35 h. Stem cell antigen-1 (Sca-1) expression by the cells remained high after 7 days in culture (77+/-20% vs. 66+/-6% at day 0) while CD34 expression was reduced (19+/-12% vs. 61+/-10% at day 0) and myosin heavy chain expression was scarce (not quantified). The estimated burst strength of the TEVG constructs was 2127+/-900 mm Hg and suture retention strength was 1.3+/-0.3N. We conclude from this study that MDSCs can be rapidly seeded within porous biodegradable tubular scaffolds while maintaining cell viability and high proliferation rates and without losing stem cell phenotype for up to 7 days of in-vitro culture. The successful integration of these steps is thought necessary to provide rapid availability of TEVGs, which is essential for clinical translation.

Tissue engineering with muscle-derived stem cells.

Tissue engineering and cell therapy approaches aim to take advantage of the repopulating ability and plasticity of multipotent stem cells to regenerate lost or diseased tissue. Researchers continue to investigate stem cells in mature tissues and demonstrate the potential ability of organ-specific cells to differentiate into multiple lineages. One stem cell that displays such promise is the muscle-derived stem cell (MDSC). Data supporting the existence of MDSCs have emerged as part of investigations to improve myoblast cell transplantation for the treatment of muscular dystrophies. As these efforts continue, the potential for MDSC-based therapy for other musculoskeletal injuries, as well as for cardiac and smooth muscle injuries, is currently being explored.

Gene therapy and tissue engineering based on muscle-derived stem cells.

Skeletal muscle represents a convenient source of stem cells for cell-based tissue and genetic engineering. Muscle-derived stem cells (MDSCs) exhibit both multipotentiality and self-renewal capabilities, and are considered to be distinct from the well-studied satellite cell, another type of muscle stem cell that is capable of self-renewal and myogenic lineage differentiation. The MDSC appears to have less restricted differentiation capabilities as compared with the satellite cell, and may be a precursor of the satellite cell. This review considers the evidence for the existence of MDSCs as well as their origin. We will discuss recent investigations highlighting the potential of stem cell transplantation for the treatment of skeletal, cardiac and smooth muscle injuries and disease. We will highlight challenges in bridging the gap between understanding basic stem cell biology and clinical utilization for cell therapy.

Heterogeneous structure of stem cells dynamics: statistical models and quantitative predictions.

Understanding stem cell (SC) population dynamics is essential for developing models that can be used in basic science and medicine, to aid in predicting cells fate. These models can be used as tools e.g. in studying patho-physiological events at the cellular and tissue level, predicting (mal)functions along the developmental course, and personalized regenerative medicine. Using time-lapsed imaging and statistical tools, we show that the dynamics of SC populations involve a heterogeneous structure consisting of multiple sub-population behaviors. Using non-Gaussian statistical approaches, we identify the co-existence of fast and slow dividing subpopulations, and quiescent cells, in stem cells from three species. The mathematical analysis also shows that, instead of developing independently, SCs exhibit a time-dependent fractal behavior as they interact with each other through molecular and tactile signals. These findings suggest that more sophisticated models of SC dynamics should view SC populations as a collective and avoid the simplifying homogeneity assumption by accounting for the presence of more than one dividing sub-population, and their multi-fractal characteristics.

Arsenic induces sustained impairment of skeletal muscle and muscle progenitor cell ultrastructure and bioenergetics.

Over 4 million individuals in the United States, and over 140 million individuals worldwide, are exposed daily to arsenic-contaminated drinking water. Human exposures can range from below the current limit of 10 µg/L to over 1mg/L, with 100 µg/L promoting disease in a large portion of those exposed. Although increased attention has recently been paid to myopathy following arsenic exposure, the pathogenic mechanisms underlying clinical symptoms remain poorly understood. This study tested the hypothesis that arsenic induces lasting muscle mitochondrial dysfunction and impairs metabolism. Compared to nonexposed controls, mice exposed to drinking water containing 100 µg/L arsenite for 5 weeks demonstrated impaired muscle function, mitochondrial myopathy, and altered oxygen consumption that were concomitant with increased mitochondrial fusion gene transcription. There were no differences in the levels of inorganic arsenic or its monomethyl and dimethyl metabolites between controls and exposed muscles, confirming that arsenic does not accumulate in muscle. Nevertheless, muscle progenitor cells isolated from exposed mice recapitulated the aberrant myofiber phenotype and were more resistant to oxidative stress, generated more reactive oxygen species, and displayed autophagic mitochondrial morphology, compared to cells isolated from nonexposed mice. These pathological changes from a possible maladaptive oxidative stress response provide insight into declines in muscle functioning caused by exposure to this common environmental contaminant.

Neuromuscular electrical stimulation as a method to maximize the beneficial effects of muscle stem cells transplanted into dystrophic skeletal muscle.

Cellular therapy is a potential approach to improve the regenerative capacity of damaged or diseased skeletal muscle. However, its clinical use has often been limited by impaired donor cell survival, proliferation and differentiation following transplantation. Additionally, functional improvements after transplantation are all-too-often negligible. Because the host microenvironment plays an important role in the fate of transplanted cells, methods to modulate the microenvironment and guide donor cell behavior are warranted. The purpose of this study was to investigate whether the use of neuromuscular electrical stimulation (NMES) for 1 or 4 weeks following muscle-derived stem cell (MDSC) transplantation into dystrophic skeletal muscle can modulate the fate of donor cells and enhance their contribution to muscle regeneration and functional improvements. Animals submitted to 4 weeks of NMES after transplantation demonstrated a 2-fold increase in the number of dystrophin+ myofibers as compared to control transplanted muscles. These findings were concomitant with an increased vascularity in the MDSC+NMES group when compared to non-stimulated counterparts. Additionally, animals subjected to NMES (with or without MDSC transplantation) presented an increased maximal specific tetanic force when compared to controls. Although cell transplantation and/or the use of NMES resulted in no changes in fatigue resistance, the combination of both MDSC transplantation and NMES resulted in a faster recovery from fatigue, when compared to non-injected and non-stimulated counterparts. We conclude that NMES is a viable method to improve MDSC engraftment, enhance dystrophic muscle strength, and, in combination with MDSC transplantation, improve recovery from fatigue. These findings suggest that NMES may be a clinically-relevant adjunct approach for cell transplantation into skeletal muscle.

Human myogenic endothelial cells exhibit chondrogenic and osteogenic potentials at the clonal level.

We have previously reported the high regenerative potential of murine muscle-derived stem cells (mMDSCs) that are capable of differentiating into multiple mesodermal cell lineages, including myogenic, endothelial, chondrocytic, and osteoblastic cells. Recently, we described a putative human counterpart of mMDSCs, the myogenic endothelial cells (MECs), in adult human skeletal muscle, which efficiently repair/regenerate the injured and dystrophic skeletal muscle as well as the ischemic heart in animal disease models. Nevertheless it remained unclear whether human MECs, at the clonal level, preserve mMDSC-like chondrogenic and osteogenic potentials and classic stem cell characteristics including high proliferation and resistance to stress. Herein, we demonstrated that MECs, sorted from fresh postnatal human skeletal muscle biopsies, can be grown clonally and exhibit robust resistance to oxidative stress with no tumorigeneity. MEC clones were capable of differentiating into chondrocytes and osteoblasts under inductive conditions in vitro and participated in cartilage and bone formation in vivo. Additionally, adipogenic and angiogenic potentials of clonal MECs (cMECs) were observed. Overall, our study showed that cMECs not only display typical properties of adult stem cells but also exhibit chondrogenic and osteogenic capacities in vitro and in vivo, suggesting their potential applications in articular cartilage and bone repair/regeneration.

Human muscle-derived cell populations isolated by differential adhesion rates: phenotype and contribution to skeletal muscle regeneration in Mdx/SCID mice.

Muscle-derived stem cells (MDSCs) isolated from murine skeletal tissue by the preplate method have displayed the capability to commit to the myogenic lineage and regenerate more efficiently than myoblasts in skeletal and cardiac muscle in murine Duchenne Muscular Dystrophy mice (mdx). However, until now, these studies have not been translated to human muscle cells. Here, we describe the isolation, by a preplate technique, of candidate human MDSCs, which exhibit myogenic and regenerative characteristics similar to their murine counterparts. Using the preplate isolation method, we compared cells that adhere faster to the flasks, preplate 2 (PP2), and cells that adhere slower, preplate 6 (PP6). The human PP6 cells express several markers of mesenchymal stem cells and are distinct from human PP2 (a myoblast-like population) based on their expression of CD146 and myogenic markers desmin and CD56. After transplantation to the gastrocnemius muscle of mdx/SCID mice, we observe significantly higher levels of PP6 cells participating in muscle regeneration as compared with the transplantation of PP2 cells. This study supports some previous findings related to mouse preplate cells, and also identifies some differences between mouse and human muscle preplate cells.

Automated live cell imaging systems reveal dynamic cell behavior.

Automated time-lapsed microscopy provides unique research opportunities to visualize cells and subcellular components in experiments with time-dependent parameters. As accessibility to these systems is increasing, we review here their use in cell science with a focus on stem cell research. Although the use of time-lapsed imaging to answer biological questions dates back nearly 150 years, only recently have the use of an environmentally controlled chamber and robotic stage controllers allowed for high-throughput continuous imaging over long periods at the cell and subcellular levels. Numerous automated imaging systems are now available from both companies that specialize in live cell imaging and from major microscope manufacturers. We discuss the key components of robots used for time-lapsed live microscopic imaging, and the unique data that can be obtained from image analysis. We show how automated features enhance experimentation by providing examples of uniquely quantified proliferation and migration live cell imaging data. In addition to providing an efficient system that drastically reduces man-hours and consumes fewer laboratory resources, this technology greatly enhances cell science by providing a unique dataset of temporal changes in cell activity.