骨髓间充质干细胞成肌分化在骨骼肌再生中的应用

骨骼肌良好的再生能力是由于肌卫星细胞的存在,然而肌卫星细胞的数量仅占骨骼肌细胞数量的1%~ 5%,当肌肉损伤时,仅依靠这些卫星细胞还不足以促进骨骼肌修复与再生,并且这种再生能力会随着年龄的增大而衰减,并不能修复损伤严重的骨骼肌。骨髓间充质干细胞（BMSC）因其多向分化潜能,旁分泌潜能,免疫调节能力及容易获取等特点广泛用于损伤骨骼肌的修复与再生。但在某种程度上,仅仅采用BMSC治疗损伤的骨骼肌仍不能达到满意的效果。因此,大量研究采用药物、生物材料、细胞及细胞因子对BMSC进行预处理不仅可改善它的移植率,还可显著促进其向骨骼肌分化,从而最大限度的发掘骨骼肌间充质干细胞的成肌分化潜能以促进骨骼肌的修复。因此,本篇综述旨在概括BMSC成肌分化在骨骼肌再生中的应用。     

Myogenic differentiation of mesenchymal stem cells co‐cultured with primary myoblasts

TE (tissue engineering) of skeletal muscle is a promising method to reconstruct loss of muscle tissue. This study evaluates MSCs (mesenchymal stem cells) as new cell source for this application. As a new approach to differentiate the MSCs towards the myogenic lineage, co‐cultivation with primary myoblasts has been developed and the myogenic potential of GFP (green fluorescent protein)‐transduced rat MSC co‐cultured with primary rat myoblasts was assessed by ICC (immunocytochemistry). Myogenic potential of MSC was analysed by ICC, FACS and qPCR (quantitative PCR). MSC—myoblast fusion phenomena leading to hybrid myotubes were evaluated using a novel method to evaluate myotube fusion ratios based on phase contrast and fluorescence microscopy. Furthermore, MSC constitutively expressed the myogenic markers MEF2 (myogenic enhancer factor 2) and α‐sarcomeric actin, and MEF2 expression was up‐regulated upon co‐cultivation with primary myoblasts and the addition of myogenic medium supplements. Significantly higher numbers of MSC nuclei were involved in myotube formations when bFGF (basic fibroblast growth factor) and dexamethasone were added to co‐cultures. In summary, we have determined optimal co‐culture conditions for MSC myogenic differentiation up to myotube formations as a promising step towards applicability of MSC as a cell source for skeletal muscle TE as well as other muscle cell‐based therapies.     

Engraftment of mesenchymal stem cells into dystrophin-deficient mice is not accompanied by functional recovery

Mesenchymal stem cell preparations have been proposed for muscle regeneration in musculoskeletal disorders. Although MSCs have great in vitro expansion potential and possess the ability to differentiate into several mesenchymal lineages, myogenesis has proven to be much more difficult to induce. We have recently demonstrated that Pax3, the master regulator of the embryonic myogenic program, enables the in vitro differentiation of a murine mesenchymal stem cell line (MSCB9-Pax3) into myogenic progenitors. Here we show that injection of these cells into cardiotoxin-injured muscles of immunodeficient mice leads to the development of muscle tumors, resembling rhabdomyosarcomas. We then extended these studies to primary human mesenchymal stem cells (hMSCs) isolated from bone marrow. Upon genetic modification with a lentiviral vector encoding PAX3, hMSCs activated the myogenic program as demonstrated by expression of myogenic regulatory factors. Upon transplantation, the PAX3-modified MSCs did not generate rhabdomyosarcomas but rather, resulted in donor-derived myofibers. These were found at higher frequency in PAX3-transduced hMSCs than in mock-transduced MSCs. Nonetheless, neither engraftment of PAX3-modified or unmodified MSCs resulted in improved contractility. Thus these findings suggest that limitations remain to be overcome before MSC preparations result in effective treatment for muscular dystrophies.     

Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources for the cell therapy of muscle diseases and can serve as powerful experimental tools for skeletal muscle research, provided an effective method to induce skeletal muscle cells is established. However, the current methods for myogenic differentiation from human ES cells are still inefficient for clinical use, while myogenic differentiation from human iPS cells remains to be accomplished. Here, we aimed to establish a practical differentiation method to induce skeletal myogenesis from both human ES and iPS cells. To accomplish this goal, we developed a novel stepwise culture method for the selective expansion of mesenchymal cells from cell aggregations called embryoid bodies. These mesenchymal cells, which were obtained by dissociation and re-cultivation of embryoid bodies, uniformly expressed CD56 and the mesenchymal markers CD73, CD105, CD166, and CD29, and finally differentiated into mature myotubes in vitro. Furthermore, these myogenic mesenchymal cells exhibited stable long-term engraftment in injured muscles of immunodeficient mice in vivo and were reactivated upon subsequent muscle damage, increasing in number to reconstruct damaged muscles. Our simple differentiation system facilitates further utilization of ES and iPS cells in both developmental and pathological muscle research and in serving as a practical donor source for cell therapy of muscle diseases.     

The therapeutic applications of multipotential mesenchymal/stromal stem cells in skeletal tissue repair

Four decades after the first isolation and characterization of clonogenic bone marrow stromal cells or mesenchymal stem cells (MSC) in the laboratory of Dr. Alexander Friedenstien, the therapeutic application of their progeny following ex vivo expansion are only now starting to be realized in the clinic. The multipotency, paracrine effects, and immune-modulatory properties of MSC present them as an ideal stem cell candidate for tissue engineering and regenerative medicine. In recent years it has come to light that MSC encompass plasticity that extends beyond the conventional bone, adipose, cartilage, and other skeletal structures, and has expanded to the differentiation of liver, kidney, muscle, skin, neural, and cardiac cell lineages. This review will specifically focus on the skeletal regenerative capacity of bone marrow derived MSC alone or in combination with growth factors, biocompatible scaffolds, and following genetic modification.     

Myogenic potential of human alveolar mucosa derived cells

Difficulties related to the obtainment of stem/progenitor cells from skeletal muscle tissue make the search for new sources of myogenic cells highly relevant. Alveolar mucosa might be considered as a perspective candidate due to availability and high proliferative capacity of its cells. Human alveolar mucosa cells (AMC) were obtained from gingival biopsy samples collected from 10 healthy donors and cultured up to 10 passages. AMC matched the generally accepted multipotent mesenchymal stromal cells criteria and possess population doubling time, caryotype and immunophenotype stability during long-term cultivation. The single myogenic induction of primary cell cultures resulted in differentiation of AMC into multinucleated myotubes. The myogenic differentiation was associated with expression of skeletal muscle markers: skeletal myosin, skeletal actin, myogenin and MyoD1. Efficiency of myogenic differentiation in AMC cultures was similar to that in skeletal muscle cells. Furthermore, some of differentiated myotubes exhibited contractions in vitro. Our data confirms the sufficiently high myogenic potential and proliferative capacity of AMC and their ability to maintain in vitro proliferation-competent myogenic precursor cells regardless of the passage number.     

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.     

Murine and human myogenic cells identified by elevated aldehyde dehydrogenase activity: implications for muscle regeneration and repair

Background

Despite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity.

Methodology/Principal Findings

Skeletal muscle progenitors were isolated from murine and human skeletal muscle by a modified preplate technique and unfractionated enzymatic digestion, respectively. ALDH^hi subpopulations isolated by fluorescence activate cell sorting demonstrated increased proliferation and myogenic differentiation capacities compared to their ALDH^lo counterparts when cultivated in oxidative and inflammatory stress media conditions. This behavior correlated with increased intracellular levels of reduced glutathione and superoxide dismutase. ALDH^hi murine myoblasts were observed to exhibit an increased muscle regenerative potential compared to ALDH^lo myoblasts, undergo multipotent differentiation (osteogenic and chondrogenic), and were found predominately in the SAC fraction, characteristics that are also observed in murine MDSCs. Likewise, human ALDH^hi hMDCs demonstrated superior muscle regenerative capacity compared to ALDH^lo hMDCs.

Conclusions

The methodology of isolating myogenic cells on the basis of elevated ALDH activity yielded cells with increased stress resistance, a behavior that conferred increased regenerative capacity of dystrophic murine skeletal muscle. This result demonstrates the critical role of stress resistance in myogenic cell therapy as well as confirms the role of ALDH as a marker for rapid isolation of murine and human myogenic progenitors for cell therapy.     

The emerging biology of satellite cells and their therapeutic potential

Adult skeletal muscle contains an abundant and highly accessible population of muscle stem and progenitor cells called satellite cells. The primary function of satellite cells is to mediate postnatal muscle growth and repair. Owing to their availability and remarkable capacity to regenerate damaged muscle, satellite cells and their descendent myoblasts have been considered as powerful candidates for cell-based therapies to treat muscular dystrophies and other neuromuscular diseases. However, regenerative medicine in muscle repair requires a thorough understanding of, and the ability to manipulate, the molecular mechanisms that control the proliferation, self-renewal and myogenic differentiation of satellite cells. Here, we review the latest advances in our current understanding of the quiescence, activation, proliferation and self-renewal of satellite cells and the challenges in the development of satellite cell-based regenerative medicine.     

Adult mesenchymal stem cells: a pluripotent population with multiple applications

Mesenchymal stem cells (MSCs) have been isolated not only from bone marrow, but also from many other tissues such as adipose tissue, skeletal muscle, liver, brain and pancreas. Because MSC were found to have the ability to differentiate into cells of multiple organs and systems such as bone, fat, cartilage, muscle, neurons, hepatocytes and insulin-producing cells, MSCs have generated a great deal of interest for their potential use in regenerative medicine and tissue engineering. Furthermore, given the ease of their isolation and their extensive expansion rate and differentiation potential, mesenchymal stem cells are among the first stem cell types that have a great potential to be introduced in the clinic. Finally, mesenchymal stem cells seem to be not only hypoimmunogenic and thus be suitable for allogeneic transplantation, but they are also able to produce immunosuppression upon transplantation. In this review we summarize the latest research in the use of mesenchymal stem cells in transplantation for generalized diseases, local implantation for local tissue defects, and as a vehicle for genes in gene therapy protocols.     

Allogeneic mesenchymal stem cells: agents of immune modulation

Adult mesenchymal stem cells possess a remarkably diverse array of immunosuppressive characteristics. The capacity to suppress the regular processes of allogeneic rejection, have allowed the use of tissue mismatched cells as therapeutic approaches in regenerative medicine and as agents of immune deviation. This review describes recent advances in understanding the mechanistic basis of mesenchymal stromal or stem cells (MSC) interaction with innate immunity. Particular emphasis is placed on the effect of Toll-like receptor signalling on MSC and a hypothesis that innate immune signals induce a 'licensing switch' in MSC is put forward. The mechanisms underlying MSC suppression of T cell responses and induction of regulatory populations are surveyed. Conflicting data regarding the influence of MSC on B cell function are outlined and discussed. Finally the limits to MSC mediated immune modulation are discussed with reference to the future clinical application of novel cell therapies.     

The Obestatin/GPR39 System Is Up-regulated by Muscle Injury and Functions as an Autocrine Regenerative System

The maintenance and repair of skeletal muscle are attributable to an elaborate interaction between extrinsic and intrinsic regulatory signals that regulate the myogenic process. In the present work, we showed that obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor are expressed in rat skeletal muscle and are up-regulated upon experimental injury. To define their roles in muscle regeneration, L6E9 cells were used to perform in vitro assays. For the in vivo assays, skeletal muscle tissue was obtained from male rats and maintained under continuous subcutaneous infusion of obestatin. In differentiating L6E9 cells, preproghrelin expression and correspondingly obestatin increased during myogenesis being sustained throughout terminal differentiation. Autocrine action was demonstrated by neutralization of the endogenous obestatin secreted by differentiating L6E9 cells using a specific anti-obestatin antibody. Knockdown experiments by preproghrelin siRNA confirmed the contribution of obestatin to the myogenic program. Furthermore, GPR39 siRNA reduced obestatin action and myogenic differentiation. Exogenous obestatin stimulation was also shown to regulate myoblast migration and proliferation. Furthermore, the addition of obestatin to the differentiation medium increased myogenic differentiation of L6E9 cells. The relevance of the actions of obestatin was confirmed in vivo by the up-regulation of Pax-7, MyoD, Myf5, Myf6, myogenin, and myosin heavy chain (MHC) in obestatin-infused rats when compared with saline-infused rats. These data elucidate a novel mechanism whereby the obestatin/GPR39 system is coordinately regulated as part of the myogenic program and operates as an autocrine signal regulating skeletal myogenesis.     

Bradykinin mediates myogenic differentiation in murine myoblasts through the involvement of SK1/Spns2/S1P2 axis

Skeletal muscle tissue retains a remarkable regenerative capacity due to the activation of resident stem cells that in pathological conditions or after tissue damage proliferate and commit themselves into myoblasts. These immature myogenic cells undergo differentiation to generate new myofibers or repair the injured ones, giving a strong contribution to muscle regeneration. Cytokines and growth factors, potently released after tissue injury by leukocytes and macrophages, are not only responsible of the induction of the initial inflammatory response, but can also affect skeletal muscle regeneration. Growth factors exploit sphingosine kinase (SK), the enzyme that catalyzes the production of sphingosine 1-phosphate (S1P), to exert their biological effects in skeletal muscle. In this paper we show for the first time that bradykinin (BK), the leading member of kinin/kallikrein system, is able to induce myogenic differentiation in C2C12 myoblasts. Moreover, evidence is provided that SK1, the specific S1P-transporter spinster homolog 2 (Spns2) and S1P₂ receptor are involved in the action exerted by BK, since pharmacological inhibition/antagonism or specific down-regulation significantly alter BK-induced myogenic differentiation. Moreover, the molecular mechanism initiated by BK involves a rapid translocation of SK1 to plasma membrane, analyzed by time-lapse immunofluorescence analysis. The present study highlights the role of SK1/Spns2/S1P receptor 2 signaling axis in BK-induced myogenic differentiation, thus confirming the crucial involvement of this pathway in skeletal muscle cell biology.     

Differentiation of activated satellite cells in denervated muscle following single fusions in situ and in cell culture

Satellite cells represent a cellular source of regeneration in adult skeletal muscle. It remains unclear why a large pool of stem myoblasts in denervated muscle does not compensate for the loss of muscle mass during post-denervation atrophy. In this study, we present evidence that satellite cells in long-term denervated rat muscle are able to activate synthesis of contractile proteins after single fusions in situ. This process of early differentiation leads to formation of abnormally diminutive myotubes. The localization of such dwarf myotubes beneath the intact basal lamina on the surface of differentiated muscle fibers shows that they form by fusion of neighboring satellites or by the progeny of a single satellite cell following one or two mitotic divisions. We demonstrated single fusions of myoblasts using electron microscopy, immunocytochemical labeling and high resolution confocal digital imaging. Sequestration of nascent myotubes by the rapidly forming basal laminae creates a barrier that limits further fusions. The recruitment of satellite cells in the formation of new muscle fibers results in a progressive decrease in their local densities, spatial separation and ultimate exhaustion of the myogenic cell pool. To determine whether the accumulation of aberrant dwarf myotubes is explained by the intrinsic decline of myogenic properties of satellite cells, or depends on their spatial separation and the environment in the tissue, we studied the fusion of myoblasts isolated from normal and denervated muscle in cell culture. The experiments with a culture system demonstrated that the capacity of myoblasts to synthesize contractile proteins without serial fusions depended on cell density and the availability of partners for fusion. Satellite cells isolated from denervated muscle and plated at fusion-permissive densities progressed through the myogenic program and actively formed myotubes, which shows that their myogenic potential is not considerably impaired. The results of this study suggest that under conditions of denervation, progressive spatial separation and confinement of many satellite cells within the endomysial tubes of atrophic muscle fibers and progressive interstitial fibrosis are the important factors that prevent their normal differentiation. Our findings also provide an explanation of why denervated muscle partially and temporarily is able to restore its functional capacity following injury and regeneration: the release of satellite cells from their sublaminal location provides the necessary space for a more active regenerative process.     

Human Ng2+ adipose stem cells loaded in vivo on a new crosslinked hyaluronic acid‐lys scaffold fabricate a skeletal muscle tissue

Mesenchymal stem cell (MSC) therapy holds promise for treating diseases and tissue repair. Regeneration of skeletal muscle tissue that is lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. Human Adipose stem cells (ASCs) have been reported to regenerate muscle fibers and reconstitute the pericytic cell pool after myogenic differentiation in vitro. Our aim was to evaluate the differentiation potential of constructs made from a new cross‐linked hyaluronic acid (XHA) scaffold on which different sorted subpopulations of ASCs were loaded. Thirty days after engraftment in mice, we found that NG2⁺ ASCs underwent a complete myogenic differentiation, fabricating a human skeletal muscle tissue, while NG2^? ASCs merely formed a human adipose tissue. Myogenic differentiation was confirmed by the expression of MyoD, MF20, laminin, and lamin A/C by immunofluorescence and/or RT‐PCR. In contrast, adipose differentiation was confirmed by the expression of adiponectin, Glut‐4, and PPAR‐γ. Both tissues formed expressed Class I HLA, confirming their human origin and excluding any contamination by murine cells. In conclusion, our study provides novel evidence that NG2⁺ ASCs loaded on XHA scaffolds are able to fabricate a human skeletal muscle tissue in vivo without the need of a myogenic pre‐differentiation step in vitro. We emphasize the translational significance of our findings for human skeletal muscle regeneration. J. Cell. Physiol. 228: 1762–1773, 2013. © 2013 Wiley Periodicals, Inc.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.