Therapeutic potential of stem/progenitor cells in human skeletal muscle for cardiovascular regeneration

Although myoblast transplantation in patients with ischemic heart failure results in a significant improvement of cardiac function, subsequent studies have consistently shown the myotubes formation in the absence of electromechanical coupling with the neighboring host myocardium, accompanied with the short-term release of paracrine effectors from implanted cells. One major pitfall of using myoblasts is that transplanted cells do not differentiate into cardiomyocytes, which may cause the inherent proarrhythmogenic events. Therefore, whether a discrete subpopulation in heterogeneous muscle-cell cultures is responsible for substantial cardiovascular regeneration has yet to be investigated. We describe here the isolation of progenitor cells from human skeletal muscle. These cells proliferated as non-adherent myospheres in suspension and displayed early embryonic factors and mesenchymal cell-like characteristics. Flow cytometric analyses demonstrated that CD56/N-CAM/Leu-19, a neural cell adhesion molecule abundantly present in myoblasts, was absent in myospheres but was expressed in an adherent cell population containing myogenic precursors. Myosphere-derived progenitor cells (MDPCs) differentiated in culture to produce cardiac, smooth muscle, and endothelial cells. Transplantation of MDPCs into ischemic hearts in NOD/scid mice promoted angiogenesis with substantial cardiovascular regeneration. Our results provide a foundation to further study the cell and biological function of human MDPCs which may have potential therapeutic implications.     

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles⁷.     

骨髓间质干细胞修复受损心肌研究进展

骨髓间充质干细胞是一种多潜能干细胞。在体外培养时,多种诱导因素可使其分化为心肌细胞等。目前进行的动物实验和临床研究表明骨髓间充质干细胞具有促进血管增生以及改善心肌梗死后心脏功能的作用,为受损心肌的治疗提供了广阔前景。但是其修复受损心肌的机制仍具有很大争议。本文就以上内容进行综述。     

The role of stem cells in skeletal and cardiac muscle repair.

In postnatal muscle, skeletal muscle precursors (myoblasts) can be derived from satellite cells (reserve cells located on the surface of mature myofibers) or from cells lying beyond the myofiber, e.g., interstitial connective tissue or bone marrow. Both of these classes of cells may have stem cell properties. In addition, the heretical idea that post-mitotic myonuclei lying within mature myofibers might be able to re-form myoblasts or stem cells is examined and related to recent observations for similar post-mitotic cardiomyocytes. In adult hearts (which previously were not considered capable of repair), the role of replicating endogenous cardiomyocytes and the recruitment of other (stem) cells into cardiomyocytes for new cardiac muscle formation has recently attracted much attention. The relative contribution of these various sources of precursor cells in postnatal muscles and the factors that may enhance stem cell participation in the formation of new skeletal and cardiac muscle in vivo are the focus of this review. We concluded that, although many endogenous cell types can be converted to skeletal muscle, the contribution of non-myogenic cells to the formation of new postnatal skeletal muscle in vivo appears to be negligible. Whether the recruitment of such cells to the myogenic lineage can be significantly enhanced by specific inducers and the appropriate microenvironment is a current topic of intense interest. However, dermal fibroblasts appear promising as a realistic alternative source of exogenous myoblasts for transplantation purposes. For heart muscle, experiments showing the participation of bone marrow-derived stem cells and endothelial cells in the repair of damaged cardiac muscle are encouraging.     

Directed In Vitro Myogenesis of Human Embryonic Stem Cells and Their In Vivo Engraftment

Development of human embryonic stem cell (hESC)-based therapy requires derivation of in vitro expandable cell populations that can readily differentiate to specified cell types and engraft upon transplantation. Here, we report that hESCs can differentiate into skeletal muscle cells without genetic manipulation. This is achieved through the isolation of cells expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), following embryoid body (EB) formation. The ESC-derived cells differentiated into myoblasts in vitro as evident by upregulation of various myogenic genes, irrespective of the presence of serum in the medium. This result is further corroborated by the presence of sarcomeric myosin and desmin, markers for terminally differentiated cells. When transplanted in vivo, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. These hESC-derived cells, which readily undergo myogenic differentiation in culture medium containing serum, could be a viable cell source for skeletal muscle repair and tissue engineering to ameliorate various muscle wasting diseases.     

Derivation of multipotent nestin+/CD271−/STRO-1− mesenchymal-like precursors from human embryonic stem cells in chemically defined conditions

The successful establishment of stem cell-based therapies requires multipotent, immunocompatible stem cells, highly efficient strategies for direct differentiation, and most importantly, optimal culture conditions for large-scale expansion of such cell populations. Other than adult tissues, human embryonic stem cells (hESCs) represent another infinitely expansible source for mesenchymal stem cell (MSC) derivation. Here, we reproducibly derived a population of Nestin⁺/CD271^?/STRO-1^? mesenchymal-like precursors from hESCs (hESC-MPs) in chemically defined conditions, without requiring any serum or serum replacement of animal origin, based on a Y-27632-assisted monolayer culture system. These cells showed slim fibroblastic morphology, and satisfied the criteria of MSCs including self-renewal, the expression of multiple MSC-specific markers and the ability to differentiate into osteoblasts, adipocytes and chondrocytes. Compared with previously reported hESC-derived MSCs, our hESC-MPs were more multipotent, and could differentiate into representative derivatives of all three embryonic germ layers including mature smooth muscle cells, cardiomyocytes, functional hepatocytes and neural cells expressing various neurotransmitter phenotypes, making them an attractive cell source for regenerative medicine.     

Small molecules that recapitulate the early steps of urodele amphibian limb regeneration and confer multipotency

In urodele amphibians, an early step in limb regeneration is skeletal muscle fiber dedifferentiation into a cellulate that proliferates to contribute new limb tissue. However, mammalian muscle cannot dedifferentiate after injury. We have developed a novel, small-molecule-based method to induce dedifferentiation in mammalian skeletal muscle. Muscle cellularization was induced by the small molecule myoseverin. Candidate small molecules were tested for the induction of proliferation in the cellulate. We observed that treatment with the small molecules BIO (glycogen synthase-3 kinase inhibitor), lysophosphatidic acid (pleiotropic activator of G-protein-coupled receptors), SB203580 (p38 MAP kinase inhibitor), or SQ22536 (adenylyl cyclase inhibitor) induced proliferation. Moreover, these proliferating cells were multipotent, as confirmed by the chemical induction of mesodermal-derived cell lineages. Microarray analysis showed that the multipotent, BIO-treated cellulate possessed a markedly different gene expression pattern than lineage-restricted C2C12 myoblasts, especially for genes related to signal transduction and differentiation. Sequential small molecule treatment of the muscle cellulate with BIO, SB203580, or SQ22536 and the aurora B kinase inhibitor, reversine, induced the formation of cells with neurogenic potential (ectodermal lineage), indicating the acquirement of pluripotency. This is the first demonstration of a small molecule method that induces mammalian muscle to undergo dedifferentiation and rededifferentiation into alternate cell lineages. This method induces dedifferentiation in a simple, stepwise approach and has therapeutic potential to enhance tissue regeneration in mammals.     

Human epicardium-derived cells fuse with high efficiency with skeletal myotubes and differentiate toward the skeletal muscle phenotype: a comparison study with stromal and endothelial cells

Recent studies have underscored a role for the epicardium as a source of multipotent cells. Here, we investigate the myogenic potential of adult human epicardium-derived cells (EPDCs) and analyze their ability to undergo skeletal myogenesis when cultured with differentiating primary myoblasts. Results are compared to those obtained with mesenchymal stromal cells (MSCs) and with endothelial cells, another mesodermal derivative. We demonstrate that EPDCs spontaneously fuse with pre-existing myotubes with an efficiency that is significantly higher than that of other cells. Although at a low frequency, endothelial cells may also contribute to myotube formation. In all cases analyzed, after entering the myotube, nonmuscle nuclei are reprogrammed to express muscle-specific genes. The fusion competence of nonmyogenic cells in vitro parallels their ability to reconstitute dystrophin expression in mdx mice. We additionally show that vascular cell adhesion molecule 1 (VCAM1) expression levels of nonmuscle cells are modulated by soluble factors secreted by skeletal myoblasts and that VCAM1 function is required for fusion to occur. Finally, treatment with interleukin (IL)-4 or IL-13, two cytokines released by differentiating myotubes, increases VCAM1 expression and enhances the rate of fusion of EPDCs and MSCs, but not that of endothelial cells.     

Isolation,Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.     

Conditional TGF‐β1 treatment increases stem cell‐like cell population in myoblasts

The limitation in successfully acquiring large populations of stem cell has impeded their application. A new method based on the dedifferentiation of adult somatic cells to generate induced multipotent stem cells would allow us to obtain a large amount of autologous stem cells for regenerative medicine. The current work was proposed to induce a sub‐population of cells with characteristics of muscle stem cells from myoblasts through conditional treatment of transforming growth factor (TGF)‐β₁. Our results show that a lower concentration of TGF‐β₁ is able to promote C2C12 myoblasts to express stem cell markers as well as to repress myogenic proteins, which involves a mechanism of dedifferentiation. Moreover, TGF‐β₁ treatment promoted the proliferation‐arrested C2C12 myoblasts to re‐enter the S‐phase. We also investigated the multi‐differentiation potentials of the dedifferentiated cells. TGF‐β₁ pre‐treated C2C12 myoblasts were implanted into mice to repair dystrophic skeletal muscle or injured bone. In addition to the C2C12 myoblasts, similar effects of TGF‐β₁ were also observed in the primary myoblasts of mice. Our results suggest that TGF‐β₁ is effective as a molecular trigger for the dedifferentiation of skeletal muscle myoblasts and could be used to generate a large pool of progenitor cells that collectively behave as multipotent stem cell‐like cells for regenerative medicine applications.     

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.     

Antioxidant Levels Represent a Major Determinant in the Regenerative Capacity of Muscle Stem Cells

Stem cells are classically defined by their multipotent, long-term proliferation, and self-renewal capabilities. Here, we show that increased antioxidant capacity represents an additional functional characteristic of muscle-derived stem cells (MDSCs). Seeking to understand the superior regenerative capacity of MDSCs compared with myoblasts in cardiac and skeletal muscle transplantation, our group hypothesized that survival of the oxidative and inflammatory stress inherent to transplantation may play an important role. Evidence of increased enzymatic and nonenzymatic antioxidant capacity of MDSCs were observed in terms of higher levels of superoxide dismutase and glutathione, which appears to confer a differentiation and survival advantage. Further when glutathione levels of the MDSCs are lowered to that of myoblasts, the transplantation advantage of MDSCs over myoblasts is lost when transplanted into both skeletal and cardiac muscles. These findings elucidate an important cause for the superior regenerative capacity of MDSCs, and provide functional evidence for the emerging role of antioxidant capacity as a critical property for MDSC survival post-transplantation.     

Harnessing the therapeutic potential of myogenic stem cells

The potential clinical use of stem cells for cell transplantation therapies to replace defective genes in myopathies is an area of intense investigation. Precursor cells derived from non-muscle tissue with myogenic potential have been identified in many tissues, including bone marrow and dermis, although the status of these putative stem cells requires clarification. The incorporation of circulating bone-marrow derived stem cells into regenerating adult skeletal muscle has been demonstrated in mice but the contribution of donor cells is so minimal that it would appear clinically irrelevant at this stage. The possibility of a true stem cell subpopulation within skeletal muscle that replenishes the satellite cells (conventional muscle precursors on the surface of myofibres) is also very attractive as a superior source of myoblasts for muscle construction. A full understanding of the intrinsic factors (i.e. gene expression within the stem cell) and extrinsic factors (i.e. signals from the external environment) which control the commitment of stem cells to the myogenic lineage, and the conditions which favour stem cell expansion in vivo is required before stem cells can be seriously considered for clinical cell therapy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Xenotransplantation of bovine bone marrow stromal cells into pig fetuses: incorporation into skeletal muscle.

Bone marrow stromal cells are multipotent and have been shown to differentiate into a wide variety of mesenchymal cell types in vivo. In this study we tested the ability of bovine bone marrow stromal cells to contribute to the development of porcine skeletal muscle tissue. Fetal pigs were injected early in gestation with bone marrow stem cells originating from slaughtered steers. After approximately forty days of development the fetuses were harvested and sections of their skeletal muscle were analyzed for the presence of bovine cells. PCR was used to detect bovine DNA present in DNA extracted from the fetal pig skeletal muscle. We also used a PRINS (Oligonucleotide Primed In- Situ Synthesis) protocol to confirm the presence of bovine cells within the porcine skeletal muscle tissue sections. The results of both assays indicate that bovine bone marrow stromal cells can participate in the development of porcine skeletal muscle. This study helps to demonstrate the potential that bone marrow stromal cells have to contribute to advances in animal biotechnology and medicine.     

Isolation and enrichment of skeletal muscle progenitor cells from mouse bone marrow

There is great interest in the therapeutic potential of non-hematopoietic stem cells obtained from bone marrow called mesenchymal stem cells (MSCs). Rare myogenic progenitor cells in MSC cultures have been shown to convert into skeletal muscle cells in vitro and also in vivo after transplantation of bone marrow into mice. To be clinically useful, however, isolation and expansion of myogenic progenitor cells is important to improve the efficacy of cell transplantation in generating normal skeletal muscle cells. We introduced into MSCs obtained from mouse bone marrow, a plasmid vector in which an antibiotic (Zeocin) resistance gene is driven by MyoD and Myf5 enhancer elements, which are selectively active in skeletal muscle progenitor cells. Myogenic precursor cells were then isolated by antibiotic selection, expanded in culture, and shown to differentiate appropriately into multinucleate myotubes in vitro. Our results show that using a genetic selection strategy, an enriched population of myogenic progenitor cells, which will be useful for cell transplantation therapies, can be isolated from MSCs.     

Functional heterogeneity of side population cells in skeletal muscle

Skeletal muscle regeneration has been exclusively attributed to myogenic precursors, satellite cells. A stem cell-rich fraction referred to as side population (SP) cells also resides in skeletal muscle, but its roles in muscle regeneration remain unclear. We found that muscle SP cells could be subdivided into three sub-fractions using CD31 and CD45 markers. The majority of SP cells in normal non-regenerating muscle expressed CD31 and had endothelial characteristics. However, CD31(-)CD45(-) SP cells, which are a minor subpopulation in normal muscle, actively proliferated upon muscle injury and expressed not only several regulatory genes for muscle regeneration but also some mesenchymal lineage markers. CD31(-)CD45(-) SP cells showed the greatest myogenic potential among three SP sub-fractions, but indeed revealed mesenchymal potentials in vitro. These SP cells preferentially differentiated into myofibers after intramuscular transplantation in vivo. Our results revealed the heterogeneity of muscle SP cells and suggest that CD31(-)CD45(-) SP cells participate in muscle regeneration.     

Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source

Myoblasts, the precursors of skeletal muscle fibers, can be induced to withdraw from the cell cycle and differentiate in vitro. Recent studies have also identified undifferentiated subpopulations that can self-renew and generate myogenic cells (Baroffio, A., M. Hamann, L. Bernheim, M.-L. Bochaton-Pillat, G. Gabbiani, and C.R. Bader. 1996. Differentiation. 60:47-57; Yoshida, N., S. Yoshida, K. Koishi, K. Masuda, and Y. Nabeshima. 1998. J. Cell Sci. 111:769-779). Cultured myoblasts can also differentiate and contribute to repair and new muscle formation in vivo, a capacity exploited in attempts to develop myoblast transplantation (MT) for genetic modification of adult muscle. Our studies of the dynamics of MT demonstrate that cultures of myoblasts contain distinct subpopulations defined by their behavior in vitro and divergent responses to grafting. By comparing a genomic and a semiconserved marker, we have followed the fate of myoblasts transplanted into muscles of dystrophic mice, finding that the majority of the grafted cells quickly die and only a minority are responsible for new muscle formation. This minority is behaviorally distinct, slowly dividing in tissue culture, but rapidly proliferative after grafting, suggesting a subpopulation with stem cell-like characteristics.