A subpopulation of murine bone marrow cells fully differentiates along the myogenic pathway and participates in muscle repair in the mdx dystrophic mouse

Bone marrow (BM) transplantation in mice suggests the existence of pluripotent cells able to differentiate into skeletal muscle tissue, although sustained myofiber reconstitution has not yet been achieved. We investigated the myogenic potential of mouse BM cells and evaluated whether a BM fraction enriched for cells expressing skeletal muscle markers would ameliorate muscle repair, when compared to whole BM, into the dystrophic mdx mouse. We demonstrate that cells expressing striated-muscle-specific proteins are already present in the BM independently from experimentally forced myogenic conversion. We observed the presence of both markers of early myogenic program such as Pax3, Myf5, MyoD, desmin, and late myogenesis such as myosin heavy chain and alpha-sarcomeric actin. These myogenic cells are more represented in the early nonadherent BM fraction, which generates clones able to fully differentiate into myotubes. Transplantation in mdx mice by intravenous injection of whole BM and a tenfold BM myogenic enriched fraction resulted in BM reconstitution and limited dystrophin restoration. Taken together, these data show that a fraction of BM cells have a definite potential for differentiation along the skeletal muscle pathway and can be recruited by muscle repair mechanisms. They also indicate that factors limiting the degree of muscle recruitment and the host stem cell competition should be assessed in order to evaluate the usefulness of BM-derived myogenic cells into the context of cell-mediated gene therapy of inherited muscle diseases.     

Generation of skeletal muscle from transplanted embryonic stem cells in dystrophic mice

Embryonic stem (ES) cells have great therapeutic potential because of their capacity to proliferate extensively and to form any fully differentiated cell of the body, including skeletal muscle cells. Successful generation of skeletal muscle in vivo, however, requires selective induction of the skeletal muscle lineage in cultures of ES cells and following transplantation, integration of appropriately differentiated skeletal muscle cells with recipient muscle. Duchenne muscular dystrophy (DMD), a severe progressive muscle wasting disease due to a mutation in the dystrophin gene and the mdx mouse, an animal model for DMD, are characterized by the absence of the muscle membrane associated protein, dystrophin. Here, we show that co-culturing mouse ES cells with a preparation from mouse muscle enriched for myogenic stem and precursor cells, followed by injection into mdx mice, results occasionally in the formation of normal, vascularized skeletal muscle derived from the transplanted ES cells. Study of this phenomenon should provide valuable insights into skeletal muscle development in vivo from transplanted ES cells.     

The expression of myogenic regulatory factors and muscle growth factors in the masticatory muscles of dystrophin-deficient (MDX) mice

The activities of myogenic regulatory factors (MRF) and muscle growth factors increase in muscle that is undergoing regeneration, and may correspond to some specific changes. Little is known about the role of MRFs in masticatory muscles in mdx mice (the model of Duchenne muscular dystrophy) and particularly about their mRNA expression during the process of muscle regeneration. Using Taqman RT-PCR, we examined the mRNA expression of the MRFs myogenin and MyoD1 (myogenic differentiation 1), and of the muscle growth factors myostatin, IGF1 (insulin-like growth factor) and MGF (mechanogrowth factor) in the masseter, temporal and tongue masticatory muscles of mdx mice (n = 6 to 10 per group). The myogenin mRNA expression in the mdx masseter and temporal muscle was found to have increased (P < 0.05), whereas the myostatin mRNA expressions in the mdx masseter (P < 0.005) and tongue (P < 0.05) were found to have diminished compared to those for the controls. The IGF and MGF mRNA amounts in the mdx mice remained unchanged. Inside the mdx animal group, gender-related differences in the mRNA expressions were also found. A higher mRNA expression of myogenin and MyoD1 in the mdx massterer and temporal muscles was found in females in comparison to males, and the level of myostatin was higher in the masseter and tongue muscle (P < 0.001 for all comparisons). Similar gender-related differences were also found within the control groups. This study reveals the intermuscular differences in the mRNA expression pattern of myogenin and myostatin in mdx mice. The existence of these differences implies that dystrophinopathy affects the skeletal muscles differentially. The finding of gender-related differences in the mRNA expression of the examined factors may indicate the importance of hormonal influences on muscle regeneration.     

Endothelial cells within embryonic skeletal muscles: a potential source of myogenic progenitors

We investigated whether the vessel-associated or endothelial cells within mouse embryo muscles can be a source of myogenic progenitors. Immunodetection of the stem cell surface markers, CD34 and Flk1, which are known to characterize the endothelial lineage, was done throughout the course of embryo muscle development. Both markers appeared to be restricted to the vessel-associated cells. On the basis of CD34 labeling, the reactive cells were purified by magnetic-bead selection from the limb muscles of 17-dpc desmin+/-LacZ mouse embryos and characterized by fluorescence-activated cell sorting. The cells in the selected CD34(+) population appeared to be approximately 95% positive for Flk1, but usually negative for CD45. We demonstrated that in vitro the CD34(+)/Flk1(+) population differentiated into endothelial cells and skeletal myofibers. When transplanted into mdx mouse muscle, this population displayed a high propensity to disperse within the recipient muscle, fuse with the host myofibers, and restore dystrophin expression. The marked ability of the embryonic muscle endothelial cells to activate myogenic program could be related to their somitic origin.     

Contribution of human muscle-derived cells to skeletal muscle regeneration in dystrophic host mice

Background

Stem cell transplantation is a promising potential therapy for muscular dystrophies, but for this purpose, the cells need to be systemically-deliverable, give rise to many muscle fibres and functionally reconstitute the satellite cell niche in the majority of the patient''s skeletal muscles. Human skeletal muscle-derived pericytes have been shown to form muscle fibres after intra-arterial transplantation in dystrophin-deficient host mice. Our aim was to replicate and extend these promising findings.

Methodology/Principal Findings

Isolation and maintenance of human muscle derived cells (mdcs) was performed as published for human pericytes. Mdscs were characterized by immunostaining, flow cytometry and RT-PCR; also, their ability to differentiate into myotubes in vitro and into muscle fibres in vivo was assayed. Despite minor differences between human mdcs and pericytes, mdscs contributed to muscle regeneration after intra-muscular injection in mdx nu/nu mice, the CD56+ sub-population being especially myogenic. However, in contrast to human pericytes delivered intra-arterially in mdx SCID hosts, mdscs did not contribute to muscle regeneration after systemic delivery in mdx nu/nu hosts.

Conclusions/Significance

Our data complement and extend previous findings on human skeletal muscle-derived stem cells, and clearly indicate that further work is necessary to prepare pure cell populations from skeletal muscle that maintain their phenotype in culture and make a robust contribution to skeletal muscle regeneration after systemic delivery in dystrophic mouse models. Small differences in protocols, animal models or outcome measurements may be the reason for differences between our findings and previous data, but nonetheless underline the need for more detailed studies on muscle-derived stem cells and independent replication of results before use of such cells in clinical trials.     

Stem cells from umbilical cord blood differentiate into myotubes and express dystrophin in vitro only after exposure to in vivo muscle environment

BACKGROUND INFORMATION: Duchenne muscular dystrophy is a disease characterized by progressive and irreversible muscle degeneration for which there is no therapy. HUCB (human umbilical cord blood) has been considered as an important source of haematopoietic and mesenchymal stem cells, each having been shown to differentiate into distinct cell types. However, it remains unclear if these cells are able to differentiate into muscle cells. RESULTS: We have showed that stem cells from HUCB did not differentiate into myotubes or express dystrophin when cultured in muscle-conditioned medium or with human muscle cells. However, delivery of GFP (green fluorescent protein)-transduced mononucleated cells from HUCB, which comprises both haematopoietic and mesenchymal populations, into quadriceps muscle of mdx (mouse dystrophy X-chromosome linked) mice resulted in the expression of human myogenic markers. After recovery of these cells from mdx muscle and in vitro cultivation, they were able to fuse and form GFP-positive myotubes that expressed dystrophin. CONCLUSIONS: These results indicate that chemical factors and cell-to-cell contact provided by in vitro conditions were not enough to trigger the differentiation of stem cells into muscle cells. Nevertheless, we showed that the HUCB-derived stem cells were capable of acquiring a muscle phenotype after exposure to an in vivo muscle environment, which was required to activate the differentiation programme.     

Differences in the Expression and Distribution of Flotillin-2 in Chick,Mice and Human Muscle Cells

Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.     

Expression of mdr1 is required for efficient long term regeneration of dystrophic muscle

The mouse mdr1a and mdr1b genes are expressed in skeletal muscle, though their precise role in muscle is unknown. Dystrophic muscle is characterized by repeated cycles of degeneration and regeneration. To explore the role of the mdr1 genes during muscle regeneration, we have created a triple knockout mouse lacking the mdr1a, mdr1b, and the dystrophin genes. The resulting ReX mice developed normally and were fertile. However, as adults, ReX had a higher proportion of degenerating muscle fibers and greater long-term loss of muscle mass than mdx. ReX muscles were also characterized by a reduced proportion of muscle side population (mSP) cells, of myogenic cells, and a reduced capacity for muscle regeneration. We found too that mSP cells derived from dystrophic muscle are more myogenic than those from normal muscle. Thus, in dystrophic muscle, the mdr1 gene plays an important role in the preservation of the mSP and of the myogenic regenerative potential. Moreover, our results suggest a hitherto unappreciated role of mdr1 in precursor cells of regenerating tissue; they therefore provide an important clue to the physiological significance of mdr1 expression in stem cells.     

Muscle reconstitution by muscle satellite cell descendants with stem cell-like properties

Recent studies have demonstrated that a distinct subpopulation with stem cell-like characteristics in myoblast culture is responsible for new muscle fiber formation after intramuscular transplantation. The identification and isolation of stem-like cells would have significant implications for successful myogenic cell transfer therapy in human muscle disorders. Using a clonal culture system for mouse muscle satellite cells, we have identified two cell types, designated 'round cells' and 'thick cells', in clones derived from single muscle satellite cells that have been taken from either slow or fast muscle. Clonal analysis of satellite cells revealed that the round cells are immediate descendants of quiescent satellite cells in adult muscle. In single-myofiber culture, round cells first formed colonies and then generated progeny, thick cells, that underwent both myogenic and osteogenic terminal differentiation under the appropriate culture conditions. Thick cells, but not round cells, responded to terminal differentiation-inducing signals. Round cells express Pax7, a specific marker of satellite cells, at high levels. Myogenic cell transfer experiments showed that round cells reconstitute myofibers more efficiently than thick cells. Furthermore, round cells restored dystrophin in myofibers of mdx nude mice, even when as few as 5000 cells were transferred into the gastrocnemius muscle. These results suggest that round cells are satellite-cell descendants with stem cell-like characteristics and represent a useful source of donor cells to improve muscle regeneration.     

Developmental behavior of embryonic myogenic progenitors transplanted into adult muscle as revealed by desmin LacZ recombinant gene.

We studied the behavior of myogenic progenitors from donor desmin(+/-) LacZ embryos after implantation into tibialis anterior muscle of 2-month-old mouse hosts. Myogenic progenitors were collected from 10-day post-coital mouse embryo somite dermomyotomes (DMs), forelimb buds (LBs), and trunks. The replacement of desmin by the LacZ coding sequence allowed specific monitoring of beta-galactosidase expression in donor myogenic cells. Immunostaining for myosin heavy chain and laminin expression was performed together with acetylcholine receptor histochemistry on sections of implanted muscle. Myogenic progenitors generated from DM, LB, and trunk were able to proliferate and adopt a myogenic pathway after transplantation into adult mouse muscle. Although their development appeared to be limited for DM and LB cell transplantation, the differentiation of myogenic progenitors occurred readily with trunk cell injection, suggesting that cell types associated with DM cells were involved in long-term myofiber differentiation (21 day). When neural tube/notochord (NTN) or sclerotomal (S) cells were co-transplanted with DM cells, myogenic nuclei were produced, indicating that both NTN and S are required for the differentiation of DMs grafted into adult muscle. These data are consistent with the differentiation of neural tissues and bone from NTN and S, respectively, and with the development of anatomic relations among all in vivo-differentiated tissues. These results suggest that embryonic trunk cells can be used to repair different types of injured tissues (especially skeletal muscle) under appropriate environmental conditions.     

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。     

成肌调节因子MyoD和myogenin在不同月龄DMD模型鼠mdx小鼠的表达

目的探讨成肌调节因子MyoD和myogenin在不同月龄DMD模型鼠mdx鼠的表达情况。方法取不同月龄DMD模型鼠mdx鼠以及相应的同龄正常C57鼠的腓肠肌,冰冻切片后用HE染色显示肌肉病理,SABC-DAB染色检测成肌调节因子MyoD和myogenin的表达。结果不同月龄mdx鼠肌肉坏死和再生程度不同,MyoD和myogenin在1月龄mdx鼠表达最强,在13月龄mdx鼠仍有表达,在正常同龄C57鼠不表达。结论MyoD与Myogenin在肌肉损伤后的再生修复过程中起作用,可作为鉴定肌肉前体细胞和反映肌肉再生的指标。     

TRAF6 regulates proliferation and differentiation of skeletal myoblasts

We could recently demonstrate an important role of receptor interacting protein-2 (RIP2), an activator of nuclear factor kappa B (NF-κB) and a target of activated receptors of the tumor necrosis factor receptor (TNFR) type, in myogenic differentiation and regeneration. Here, we analyze a potential role of TNFR associated factor 6 (TRAF6), which also associates with the cytoplasmic domain of TNFR type, but also IL-1-R and TLR type receptors, and activates NF-κB, in these processes. Specifically, we show that during myogenic differentiation in vitro, traf6 gene expression is downregulated in normal myoblasts, but not in rhabdomyosarcoma cells, suggesting a role of the TRAF6 protein in this process. Inhibition of traf6 expression using specific siRNAs led to an inhibition of both myoblast proliferation and differentiation, whereas inhibition of the TRAF6 effector NF-κB alone in our system only blocked proliferation. Finally, we demonstrate that the traf6 gene is downregulated in skeletal muscle tissue of the dystrophic mdx mouse. Taken together, these data argue for a role of TRAF6 in the regulation of skeletal muscle differentiation and regeneration.     

Decorin promotes myogenic differentiation and mdx mice therapeutic effects after transplantation of rat adipose-derived stem cells

Background aimsAdipose-derived stem cells (ADSC) have been considered as attractive candidates for the treatment of Duchenne muscular dystrophy (DMD), but the rate of ADSC myogenesis is very low. Myostatin (Mstn), a negative regulator of myogenesis, is known to be responsible for limiting skeletal muscle regeneration. Decorin could bind Mstn and deactivate it. Decorin has been shown to improve myogenic differentiation in mdx mice. We hypothesized that inhibition of Mstn by using decorin may ameliorate myogenic differentiation of ADSC.MethodsRat ADSC were transfected with the lentivirus-containing green fluorescence protein (GFP) and human decorin gene. The transfected ADSC were induced by 5-azacytidine (5-AzaC). The rates of myogenic differentiation and adipogenesis were detected. The transfected ADSC were injected into mdx mice and the expression of Mstn and decorin detected by Western blot. Dystrophin was detected after transfected ADSC transplantation by immunofluorescence staining and Western blot. Serum creatine kinase (CK) and histologic changes were also evaluated.ResultsThe optimal multiplicity of infection of ADSC was 10. Decorin improved muscle mass. In accordance with the increased muscle mass, dystrophin expression increased. Following the level of decorin increase, the Mstn expression decreased. Furthermore, serum CK and histologic changes in centrally nucleated fiber (CNF) decreased.ConclusionsImproved myogenic differentiation of ADSC was observed by using decorin. This process was probably the result of decorin inhibiting Mstn. A new method of DMD therapy combining Mstn inhibition (using decorin) and ADSC transplantation is probably feasible.     

Skeletal myogenesis by human primordial germ cell-derived progenitors

We have isolated and cultured human primordial germ cells (PGCs) from early embryos. The PGCs expressed embryonic germ (EG) cell-specific surface markers, including Oct4 and Nanos. We derived a cell population from these PGCs that we termed embryoid body-derived (EBD) cells. EBD cells can be extensively expanded in vitro for more than 50 passages and express lineage markers from all three primary germ layers. The myogenic potential of the EBD cells was examined both in vitro and in vivo.In vitro, the EBD cells can be induced to form multinucleated myotubes, which express late skeletal muscle-specific markers, including MHC and dystrophin, when exposed to human galectin-1. In vivo, the EBD cells gave rise to all the myogenic lineages, including the skeletal muscle stem cells known as satellite cells. Strikingly, these cells were able to partially restore degenerated muscles in the SCID/mdx mouse, an animal model of the Duchenne’s muscular dystrophy. These results indicate the EBD cells may be a promising source of myogenic stem cells for cell-based therapies for muscle degenerative disorders.     

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.