UNASSIGNED: Tendon development requires the coordinated interaction of muscles and tendons. Muscle-derived cells (MDCs), a mixed cell population containing both myogenic and fibroblastic cell subsets, have been found to be ideal seed cells for tendon regeneration. However, the necessity of these cell types for tendon regeneration has not yet been tested. In this study, we aim to explore the possible synergistic effects of myogenic cells and fibroblasts in engineered tendon regeneration.
UNASSIGNED: MDCs were separated into rapidly adhering cell (RAC; fibroblasts) and slowly adhering cell (SAC; myogenic cells) populations. Myogenic- and tenogenic-related molecules were analyzed by immunofluorescent staining, RT-PCR and real-time PCR. The proliferative abilities of MDCs, RACs and SACs were also evaluated. Cell-scaffold constructs were implanted into nude mice, and subsequently evaluated for their histologic, ultrastructure, gene expression, and biomechanical characteristics.
UNASSIGNED: MDCs have better proliferative activity than RAC and SAC population. RACs could express higher levels of tenogenic-related molecules tenomodulin (TNMD) and scleraxis (SCX) than SACs. Whereas SACs only expressed myogenic-related molecules MyoD. In contrast to the tendons engineered using RACs and SACs, the tendons engineered using MDCs exhibited a relatively more mature and well-organized tissue structure and ultrastructure as well as better mechanical properties.
UNASSIGNED: Fibroblasts in muscle may be the primary cell population involved in tendon regeneration and that myogenic cells are an important component of the niche and control the fibroblast activity during tendon regeneration. The synergistic effects between fibroblasts and myogenic cells significantly contribute to efficient and effective regeneration of engineered tendons.

Sarcoglycanopathies are rare limb girdle muscular dystrophies, still incurable, even though symptomatic treatments may slow down the disease progression. Most of the disease-causing defects are missense mutations leading to a folding defective protein, promptly removed by the cell\'s quality control, even if possibly functional. Recently, we repurposed small molecules screened for cystic fibrosis as potential therapeutics in sarcoglycanopathy. Indeed, cystic fibrosis transmembrane regulator (CFTR) correctors successfully recovered the defective sarcoglycan-complex in vitro. Our aim was to test the combined administration of some CFTR correctors with C17, the most effective on sarcoglycans identified so far, and evaluate the stability of the rescued sarcoglycan-complex. We treated differentiated myogenic cells from both sarcoglycanopathy and healthy donors, evaluating the global rescue and the sarcolemma localization of the mutated protein, by biotinylation assays and western blot analyses. We observed the additive/synergistic action of some compounds, gathering the first ideas on possible mechanism/s of action. Our data also suggest that a defective α-sarcoglycan is competent for assembly into the complex that, if helped in cell traffic, can successfully reach the sarcolemma. In conclusion, our results strengthen the idea that CFTR correctors, acting probably as proteostasis modulators, have the potential to progress as therapeutics for sarcoglycanopathies caused by missense mutations.

Muscle stem cells or satellite cells are required for skeletal muscle regeneration. It has been shown that the satellite cell microenvironment, including neighboring cells such as endothelial cells, macrophages or fibroblasts are essential for complete and efficient regeneration. A deficient behavior of these cells compromises regeneration. Therefore, there is a strong interest in understanding the cellular and molecular interactions at work between these cell types during muscle regeneration. Fluorescence-activated cell sorting allows to isolate these four cell types at different time points of regeneration, for further high throughput or behavioral experiments. We present here a method for the concomitant isolation of 4 cell types present in the regenerating skeletal muscle: muscle stem cells, endothelial cells, fibro-adipogenic precursor cells and macrophages.

Regeneration in adult skeletal muscle relies on the activation, proliferation, and fusion of myogenic precursor cells (MPC), mostly resident satellite cells (SC). However, the regulatory mechanism during this process is still under evaluation, with the final aim to manipulate regeneration when the intrinsic mechanism is corrupted. Furthermore, intercellular connections during skeletal muscle regeneration have not been previously thoroughly documented. Our hypothesis was that a direct and close cellular interaction between SC/MPC and invading myeloid cells is a key step to control regeneration. We tested this hypothesis during different steps of skeletal muscle regeneration: (a) the recruitment of activated SC; (b) the differentiation of MPC; (c) myotubes growth, in a mouse model of crush injury. Samples harvested (3 and 5 days) post-injury were screened by light and confocal microscopy. Ultrastructural analysis was performed by conventional transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) followed by 3D modeling of electron tomography (ET) data. This revealed a new type of interaction between macrophages and myogenic cells by direct heterocellular surface apposition over large areas and long linear distances. In the analyzed volume, regions spaced below 20 nm, within molecular range, represented 31% of the macrophage membrane surface and more than 27% of the myotube membrane. The constant interaction throughout all stages of myogenesis suggests a potential new type of regulatory mechanism for the myogenic process. Thus, deciphering structural and molecular mechanisms of SC-macrophage interaction following injury might open promising perspectives for improving muscle healing.

The migratory and organogenetic capacities of muscle cells at different stages of differentiation were tested in heterospecific chick/quail recombinants. Grafts containing muscle cells were taken from the premuscular masses from 4- to 5-day quail embryos, from the limb or trunk muscles of 12-day embryonic and 4-day post-natal quails, and from experimentally produced bispecific premuscular masses in which the myoblasts are of quail origin and the connective tissue cells of chick origin. Grafts were implanted into 2-day chick embryos in place of the somitic mesoderm at the limb level. Hosts were examined 4 to 7 days after operation.After implantation of a piece of premuscular mass, quail cells were found at and around the site of the graft in the truncal region and within the limb as far as the autopod. Quail cells participated predominantly in the trunk and limb musculature, which contained a number of quail myotubes and of bispecific quail/chick myotubes. Apart from skeletal muscles, quail cells contributed sporadically to nerve envelopes and blood vessel walls in the limb.When the graft was of bispecific constitution, quail nuclei in the limb and the trunk were found exclusively in monospecific and bispecific myotubes.After implantation of differentiated embryonic or post-natal muscle tissue, quail cells in the limb contributed only sporadically to nerve envelopes and blood vessel walls, while in the trunk they also participated in the formation of muscles and tendons.It is concluded that the myogenic cells in 4 to 5-day quail premuscular masses are still able to undergo an extensive migration into the limb buds and there participate in the formation of myotubes and anatomically normal muscles. They display developmental potentialities equivalent to those of the somitic myogenic stem cells. These capacities are lost in 12-day embryonic muscles.

To understand the mechanism of muscle remodeling during Xenopus laevis metamorphosis, we examined the in vitro effect of insulin-like growth factor 1 (IGF-1) on growth and differentiation of three different-fate myogenic cell populations: tadpole tail, tadpole dorsal, and young adult leg muscle. IGF-1 promoted growth and differentiation of both tail and leg myogenic cells only under conditions where these cells could proliferate. Inhibition of cell proliferation by DNA synthesis inhibitor cytosine arabinoside completely canceled the IGF-1\'s cell differentiation promotion, suggesting the possibility that IGF-1\'s differentiation-promotion effect is an indirect effect via IGF-1\'s cell proliferation promotion. IGF-1 promoted differentiation dose dependently with maximum effect at 100-500 ng/ml. RT-PCR analysis revealed the upregulation (11-fold) of ifg1 mRNA expression in developing limbs, suggesting that IGF-1 plays a role in promoting muscle differentiation during limb development. The combined effect of triiodo-L-thyronine (T3) and IGF-1 was also examined. In adult leg cells, IGF-1 promoted growth and differentiation irrespective of the presence of T3. In larval tail cells, cell count was 76% lower in the presence of T3, and IGF-1 did not promote proliferation and differentiation in T3-containing medium. In larval dorsal cells, cell count was also lower in the presence of T3, but IGF-1 enhanced proliferation and differentiation in T3-containing medium. This result is likely due to the presence among dorsal cells of both adult and larval types (1:1). Thus, IGF-1 affects only adult-type myogenic cells in the presence of T3 and helps accelerate dorsal muscle remodeling during metamorphosis.

Pax3 and Foxc2 have been shown genetically to mutually repress each other in the mouse somite. Perturbation of this balance in multipotent cells of the dermomyotome influences cell fate; upregulation of Foxc2 favours a vascular fate, whereas higher levels of Pax3 lead to myogenesis. Foxc1 has overlapping functions with Foxc2. In Foxc1/2 double-mutant embryos, somitogenesis is severely affected, precluding analysis of somite derivatives. We have adopted a conditional approach whereby mutations in Foxc1 and Foxc2 genes were targeted to Pax3-expressing cells. Inclusion of a conditional reporter allele in the crosses made it possible to follow cells that had expressed Pax3. At the forelimb level, endothelial and myogenic cells migrate from adjacent somites into the limb bud. This population of endothelial cells is compromised in the double mutant, whereas excessive production of myogenic cells is observed in the trunk. However, strikingly, myogenic progenitors fail to enter the limbs, leading to the absence of skeletal muscle. Pax3-positive migratory myogenic progenitors, marked by expression of Lbx1, are specified in the somite at forelimb level, but endothelial progenitors are absent. The myogenic progenitors do not die, but differentiate prematurely adjacent to the somite. We conclude that the small proportion of somite-derived endothelial cells in the limb is required for the migration of myogenic limb progenitors.

BACKGROUND: Muscular dystrophies (MDs) are genetic diseases that produce progressive loss of skeletal muscle fibers. Cell therapy (CT) is an experimental approach to treat MD. The first clinical trials of CT in MD conducted in the 1990s were based on myoblast transplantation (MT). Since they did not yield the expected results, several researchers sought to discover other cells with more advantageous properties than myoblasts whereas others sought to improve MT.
METHODS: We explain the properties that are required for a cell to be used in CT of MD. We briefly review most of the cells that were proposed for this CT, and to what extent these properties were met not only in laboratory animals but also in clinical trials.
CONCLUSIONS: Although the repertoire of cells proposed for CT of MD has been expanded since the 1990s, only myoblasts have currently demonstrated unequivocally to significantly engraft in humans. Indeed, MT for MD involves significant technical challenges that need be solved. While it would be ideal to find cells involving less technical challenges for CT of MD, there is so far no clinical evidence that this is possible and therefore the work to improve MT should continue.

The common killifish or mummichog (Fundulus heteroclitus) is an estuarine teleost increasingly used in comparative physiology, toxicology and embryology. Their ability to withstand extreme environmental conditions and ease of maintenance has made them popular aquatic research organisms. Scientific advances with most popular model organisms have been assisted with the availability of continuous cell lines; however, cell lines from F. heteroclitus appear to be unavailable. The development of a killifish cell line, KFE-5, derived from the mid trunk region of a late stage embryo is described here. KFE-5 grows well in Leibovitz\'s L-15 media with 10% fetal bovine serum (FBS). This cell line has been passaged over 60 times in a span of three years, and cells at various passages have been successfully cryopreserved and thawed. The cells are mostly fibroblastic but contain myogenic cells that differentiate into mono-, bi- and multi-nucleated striated myocytes. Immunofluorescence detection of muscle specific antigens such as α-actinin, desmin, and myosin confirms KFE-5 as a myogenic cell line. KFE-5 has a temperature preference for 26-28°C and has been shown to withstand temperatures up to 37°C. The cell line responds to chemical signals including growth factors, hormones and extracellular matrix components. KFE-5 could thus be useful not only for mummichog\'s thermobiology but also for studies in fish muscle physiology and development.

To assess the effects of three methods of inducing myogenic cells differentiation, umbilical blood mesenchymal stem cells (UCMSCs) from nearly full-term pregnancy mongrel dogs were purified and cultured. Fourth-passage UCMSCs were used to detect surface antigens, including CD11a, CD11b, CD29, CD34 and CD71. The cells were induced by 5-azacytidine (5-aza), myocardial lysates and myocardial induced fluid. Positive expression of Nkx2.5, α-actin, desmin, β-MHC and troponin-I (TN I) were detected after 3 weeks. The immunohistochemical results were CD11a (-), CD11b (-), CD34 (-), CD29 (+) and CD71 (+). Nkx2.5 was detected in 5-aza group, myocardial lysates group and myocardial induced fluid group. Semi-quantitative analysis showed Nkx2.5 expression significantly higher in myocardial lysates group than in the 5-aza group or myocardial-induced fluid group (P < 0.05), but there was no significant difference between the 5-aza and myocardial-induced fluid groups for Nkx2.5 expression (P > 0.05). MSCs did not express myocardial structural proteins before differentiation, but α-actin, desmin, β-MHC and troponin-I were present after differentiation. The positive expression of four proteins differed with the differentiation conditions. The UCMSCs can be differentiated into myogenic cells by three methods, but the degrees of differentiation are inconsistent. Our results show that the effects of 5-aza and myocardial lysates are better than that of myocardial induced fluid.