Skeletal muscle stem cells

In this review we shall discuss recent publications on the heterogeneity of muscle stem cells, signaling pathways that affect their behaviour and regulatory mechanisms that underlie their myogenic fate, with reference to insights provided by work on skeletal muscle formation in the embryo as well as the adult, with the mouse as a model of reference.     

Skeletal muscle stem and progenitor cells: reconciling genetics and lineage

Skeletal muscle provides a unique paradigm for studying stem to differentiated cell transitions, as well as the acquisition of cellular identity. Embryological and genetic studies over the last decades have unveiled key signaling pathways and regulatory genes which are involved in this process. In the adult, regeneration from fiber-associated satellite cells as well as non-muscle cells have opened the perspective for cell therapy studies. Paradoxically, however, the lineage has remained largely elusive. Recent studies have provided clues regarding the cellular organization in this lineage. Furthermore, the complexity of the genetic networks regulating global and local myogenic programs can be correlated with location and lineage. Finally, prenatal and postnatal developmental strategies have similarities and differences which will also be highlighted.     

Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane

We have demonstrated previously that adult human synovial membrane-derived mesenchymal stem cells (hSM-MSCs) have myogenic potential in vitro (De Bari, C., F. Dell'Accio, P. Tylzanowski, and F.P. Luyten. 2001. Arthritis Rheum. 44:1928-1942). In the present study, we have characterized their myogenic differentiation in a nude mouse model of skeletal muscle regeneration and provide proof of principle of their potential use for muscle repair in the mdx mouse model of Duchenne muscular dystrophy. When implanted into regenerating nude mouse muscle, hSM-MSCs contributed to myofibers and to long term persisting functional satellite cells. No nuclear fusion hybrids were observed between donor human cells and host mouse muscle cells. Myogenic differentiation proceeded through a molecular cascade resembling embryonic muscle development. Differentiation was sensitive to environmental cues, since hSM-MSCs injected into the bloodstream engrafted in several tissues, but acquired the muscle phenotype only within skeletal muscle. When administered into dystrophic muscles of immunosuppressed mdx mice, hSM-MSCs restored sarcolemmal expression of dystrophin, reduced central nucleation, and rescued the expression of mouse mechano growth factor.     

Skeletal muscle stem cells express anti-apoptotic ErbB receptors during activation from quiescence

To be effective for tissue repair, satellite cells (the stem cells of adult muscle) must survive the initial activation from quiescence. Using an in vitro model of satellite cell activation, we show that erbB1, erbB2 and erbB3, members of the EGF receptor tyrosine kinase family, appear on satellite cells within 6 h of activation. We show that signalling via erbB2 provides an anti-apoptotic survival mechanism for satellite cells during the first 24 h, as they progress to a proliferative state. Inhibition of erbB2 signalling with AG825 reduced satellite cell numbers, concomitant with elevated caspase-8 activation and TUNEL labelling of apoptotic satellite cells. In serum-free conditions, satellite cell apoptosis could be largely prevented by a mixture of erbB1, erbB3 and erbB4 ligand growth factors, but not by neuregulin alone (erbB3/erbB4 ligand). Furthermore, using inhibitors specific to discrete intracellular signalling pathways, we identify MEK as a pro-apoptotic mediator, and the erbB-regulated factor STAT3 as an anti-apoptotic mediator during satellite cell activation. These results implicate erbB2 signalling in the preservation of a full compliment of satellite cells as they activate in the context of a damaged muscle.     

Skeletal muscle stem cell birth and properties

Development and maintenance of an abundant tissue such as skeletal muscle poses several challenges. Curiously, not all skeletal muscle stem cells are born alike, since diverse genetic pathways can specify their birth. Stem and progenitor cells that establish the tissue during development, those that maintain its homeostasis, as well as participate in its regeneration have generated considerable interest. The ability to distinguish stem cells from more committed progenitors throughout prenatal and postnatal life has guided researchers to identify stem cell properties and characterise their niche. These properties include markers that influence cell behaviour and mode of division during normal development, after trauma and cell transplantations. This review addresses these issues from a developmental perspective.     

Skeletal myogenesis by human embryonic stem cells

We have examined the myogenic potential of human embryonic stem （hES） cells in a xeno-transplantation animal model. Here we show that precursors differentiated from hES cells can undergo myogenesis in an adult environment and give rise to a range of cell types in the myogenic lineage. This study provides direct evidences that hES cells can regenerate both muscle and satellite cells in vivo and are another promising cell type for treating muscle degenerative disorders in addition to other myogenic cell types.     

Skeletal muscle generated from induced pluripotent stem cells-induction and application

Human induced pluripotent stem cells(hiPS cells or hiPSCs) can be derived from cells of patients with severe muscle disease. If skeletal muscle induced from patientiPSCs shows disease-specific phenotypes, it can be useful for studying the disease pathogenesis and for drug development. On the other hand, human iPSCs from healthy donors or hereditary muscle diseaseiPSCs whose genomes are edited to express normal protein are expected to be a cell source for cell therapy. Several protocols for the derivation of skeletal muscle from human iPSCs have been reported to allow the development of efficient treatments for devastating muscle diseases. In 2017, the focus of research is shifting to another stage:(1) the establishment of mature myofibers that are suitable for study of the pathogenesis of muscle disease;(2) setting up a highthroughput drug screening system; and(3) the preparation of highly regenerative, non-oncogenic cells in large quantities for cell transplantation, etc.     

Skeletal muscle satellite cells cultured in simulated microgravity

Summary Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and, therefore, provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (∼ 200 gm) were used for all studies and were composed of greater than 75% satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D culture and 3-D HARV culture. Plating efficiency (cells attached ÷ cells plated ×100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and nonsatellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability because glucose levels in medium from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARV were joined together by cells into 3-D aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a 3-D level of organization that could provide a more suitable model to study postnatal muscle development than is currently available with standard culture methods.     

Skeletal muscle atrophy leads to loss and dysfunction of muscle precursor cells

Atrophy of skeletal muscle leads to decreases in myofiber size and nuclear number; however, the effects of atrophic conditions on muscle precursor cells (MPC) are largely unknown. MPC lie outside myofibers and represent the main source of additional myonuclei necessary for muscle growth and repair. In the present study, we examined the properties of MPC after hindlimb suspension (HS)-induced atrophy and subsequent recovery of the mouse hindlimb muscles. We demonstrated that the number of MPC in atrophied muscles was decreased. RT-PCR analysis of cells isolated from atrophied muscles indicated that several mRNA characteristic of the myogenic program in MPC were absent. Cells isolated from atrophied muscles failed to properly proliferate and undergo differentiation into multinucleated myotubes. Thus atrophy led to a decrease in MPC and caused dysfunction in those MPC that remained. Upon regrowth of the atrophied muscles, these deleterious effects were reversed. Our data suggest that preventing loss or dysfunction of MPC may be a new pharmacological target during muscle atrophy. satellite cells; hindlimb suspension; proliferation; differentiation; myotubes     

Skeletal muscle differentiation potential of human adult bone marrow cells

Murine bone marrow (BM) cells have been shown to undergo myogenic differentiation and participate in muscle repair in different muscle regeneration models. In the present paper, we report on a subset of cells (CD45+/desmin+) with myogenic potential being present at very low frequencies in human adult BM. By a simple culture method, we were able to obtain in vitro multinucleated myotubes in up to 20% of the cultures. Myotubes were generated using both BM flushed from rib fragments obtained during thoracotomy and BM derived from iliac crest aspirates. Cells of the different adherent and non-adherent fractions expressed numerous muscle specific markers by immunocytochemistry, real-time RT-PCR, flow cytometry, and Western blot analyses. Moreover, direct injection of whole BM into the right tibialis anterior muscle of immunodeficient mice (NOD/RAG) that had previously been treated with cardiotoxin to induce muscle degeneration, showed a variable but significant level of human cell engraftment (from 0.06 to 0.26% Dys+/FISH+ fibers). These data suggest that cells with skeletal muscle differentiation potential are present in adult human BM can differentiate in vitro and give rise to myogenic cells in vivo in immunodeficient mice after muscle damage. Further improvements might allow new approaches to cell-mediated therapies for muscular diseases.     

Skeletal muscle satellite cells appear during late chicken embryogenesis.

The emergence of avian satellite cells during development has been studied using markers that distinguish adult from fetal cells. Previous studies by us have shown that myogenic cultures from fetal (Embryonic Day 10) and adult 12-16 weeks) chicken pectoralis muscle (PM) each regulate expression of the embryonic isoform of fast myosin heavy chain (MHC) differently. In fetal cultures, embryonic MHC is coexpressed with a ventricular MHC in both myocytes (differentiated myoblasts) and myotubes. In contrast, myocytes and newly formed myotubes in adult cultures express ventricular but not embryonic MHC. In the current study, the appearance of myocytes and myotubes which express ventricular but not embryonic MHC was used to determine when adult myoblasts first emerge during avian development. By examining patterns of MHC expression in mass and clonal cultures prepared from embryonic and posthatch chicken skeletal muscle using double-label immunofluorescence with isoform-specific monoclonal antibodies, we show that a significant number of myocytes and myotubes which stain for ventricular but not embryonic MHC are first seen in cultures derived from PM during fetal development (Embryonic Day 18) and comprise the majority, if not all, of the myoblasts present at hatching and beyond. These results suggest that adult type myoblasts become dominant in late embryogenesis. We also show that satellite cell cultures derived from adult slow muscle give results similar to those of cultures derived from adult fast muscle. Cultures derived from Embryonic Day 10 hindlimb form myocytes and myotubes that coexpress ventricular and embryonic MHCs in a manner similar to cells of the Embryonic Day 10 PM. Thus, adult and fetal expression patterns of ventricular and embryonic MHCs are correlated with developmental age but not muscle fiber type.     

Skeletal muscle precursors do not arise from bone marrow cells

Cell and Tissue Research - This paper tests the hypothesis that bone marrow stem cells can give rise to circulating muscle precursor cells. Irradiated host mice were reconstituted with bone marrow...     

Skeletal muscle progenitor cells and the role of Pax genes

Satellite cells, which lie under the basal lamina of muscle fibres, are marked by the expression of Pax7, and in many muscles of Pax3 also. A pure population of satellite cells, isolated from a Pax3(GFP/+) mouse line by flow cytometry, contribute very efficiently to skeletal muscle regeneration and also self-renew, thus demonstrating their role as muscle stem cells. Pax3/7 regulates the entry of these cells into the myogenic programme via the activation of the myogenic determination gene, MyoD. Pax7 is also essential for the survival of satellite cells. This dual role underlines the importance of ensuring that a tissue stem cell that has lost its myogenic instruction should not be left to run amok, with the potential risk of tissue deregulation and cancer. A somite-derived population of Pax3/Pax7 positive cells is responsible for muscle growth during development and gives rise to the satellite cells of postnatal muscles. In the absence of both Pax3 and Pax7, these cells die or assume other cell fates. Pax3/7 lies genetically upstream of both MyoD and Myf5, which determine the skeletal muscle fate of these cells. To cite this article: M. Buckingham, C. R. Biologies 330 (2007).     

Skeletal muscle tissue engineering

The reconstruction of skeletal muscle tissue either lost by traumatic injury or tumor ablation or functional damage due to myopathies is hampered by the lack of availability of functional substitution of this native tissue. Until now, only few alternatives exist to provide functional restoration of damaged muscle tissues. Loss of muscle mass and their function can surgically managed in part using a variety of muscle transplantation or transposition techniques. These techniques represent a limited degree of success in attempts to restore the normal functioning, however they are not perfect solutions. A new alternative approach to addressing difficult tissue reconstruction is to engineer new tissues. Although those tissue engineering techniques attempting regeneration of human tissues and organs have recently entered into clinical practice, the engineering of skeletal muscle tissue ist still a scientific challenge. This article reviews some of the recent findings resulting from tissue engineering science related to the attempt of creation and regeneration of functional skeletal muscle tissue.     

Skeletal muscle cell populations

Summary Cell suspensions from the breast muscles of 10-day old chicken embryos were separated into non-myogenic, fibroblast-like cell fractions and a mononucleated, myogenic cell fraction by Percoll^TM density centrifugation. lsolated populations were characterized by their morphology in both mass cultures and individual macroscopic clones and by the immunocytochemical detection of skeletal muscle-and smooth muscle-specific proteins in individual cells. Cell populations were also characterized by their protein patterns using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The less dense, non-myogenic cells comprised 16% of the cells. In culture they were predominantly flatiened, stellate cells and gave rise to clones lacking myotubes. These fibroblast-like cells were negative for skeletal muscle myosin or muscle type creatine phosphokinase. Less than 0.1% of these cells demonstrated strong fluorescence when stained with anti-desmin or anti-smooth muscle specific actin. This observation suggested that the vast majority of these cells were not related to vascular smooth muscle cells. Also, over 99% of the non-myogenic cells did not display characteristic properties of endothelial cells. The denser myogenic cell fraction comprised over 80% of the cells and in clonal cultures gave rise to about 70% myogenic clones. An additional 30% of clones from this fraction were non-myogenic indicating heterogeneity in this population. We conclude that Percoll centrifugation can be employed for the isolation of myogenic and non-myogenic cell populations directly from the embryonic muscle. Moreover, this procedure allows the direct analysis of cell-specific proteins (e.g., by gel electrophoresis) without the need for cell culturing. The results thus obtained closely reflect the status of the cells in the intact muscle.     

The potential of muscle stem cells.

Skeletal muscle contains two types of stem cells: satellite cells, which function as myogenic precursors, and a population of multipotent adult stem cells. Satellite cells are believed to form a stable, self-renewing pool of stem cells in adult muscle where they function in tissue growth and repair. An additional stem cell population in adult muscle displays a remarkable capacity to differentiate into hematopoietic cells as well as muscle following transplantation. This article discusses the characteristics and properties of these cell populations, the relationship between them, and the potential for stem cell-based muscle therapeutics.     

Efficient generation of iPS cells from skeletal muscle stem cells

Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that "epigenetic memory" significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle.