Myoid cell density in the thymus is reduced during mdx dystrophy and after muscle crush.

Thymic myoid cells share structural and behavioural features with cells of the skeletal muscle lineage: they express regulatory genes and contractile proteins, and they can form myofibers in culture. Historically, those features suggested that myoid cells could be precursors for muscle repair in addition to the satellite cells in muscle that are typically designated as the only muscle precursors. Muscles of the mutant mdx dystrophic mouse strain have a large demand for precursors, which is greatest at a young age. In the present study, immunostaining for troponin T was used to localize myoid cells. We tested the hypothesis that the myoid cell population changes when there is a demand for muscle precursors and that these changes would be anticipated if myoid cells have a role as myogenic precursors or stem cells in muscle. Chronic demands for muscle precursors in mdx dystrophic mice were accompanied by lower myoid cell density in comparison with density in two normal strains (C57BL10/ScSn and Swiss Webster). Acute demand for precursors was accompanied by a sharp decline in thymic myoid cell density within 2 days after a crush injury to one tibialis anterior muscle in normal but not dystrophic animals. To standardize the developmental age of the thymus, density was determined in all animals at 28 days of age. Given the current interest in nonmuscle sources of myogenic stem cells, these data suggest that changes in the density of thymic myoid cells may accompany acute and chronic demands for muscle precursors. Further experiments are required to determine whether thymic myoid cells are participants in distant muscle cell proliferation, new fiber formation, or the establishment of new stem cells in regenerated muscle.     

Ultrastructure of thymic myoid cells

Mature myoid cells in the parenchyma of reptilian thymus contain all the organelles typical of striated muscle. The presence of both immature and degenerating stages indicates a turnover of myoid cells in the adult thymus. In the earlier stages of differentiation myoid cells resemble thymic epithelial cells. A close parallel exists between developing myoid cells, skeletal muscle differentiating in vitro, rhabdomyoma and rhabdomyosarcoma. Elaborate lattice-like structures are formed by transverse tubules. These structures are compared with similar configurations which have been described in muscle and mitochondrial cristae.     

Phenotypic and functional characterization of human thymic stromal cell lines.

To establish new tools for studying human thymic stromal cells, we transfected adherent cells from a human postnatal thymus using a plasmid encoding SV40 large T antigen. Among the cell lines obtained, we characterized four epithelial cell lines (LT-TEC1 to LT-TEC4) and one thymic myoid cell line (MITC). Several morphological, functional and phenotypic differences were observed between these 2 cell types. Epithelial cells were heterogeneous and larger than myoid cells. Untreated LT-TEC lines expressed MHC class I, ICAM-1 and LFA-3 antigens and not MHC class II antigens, similarly to primary thymic epithelial cells (PTEC), while MITC line expressed only class I and LFA-3 antigens. After IFN-gamma treatment, MHC class II and ICAM-1 antigens were markedly upregulated in LT-TEC lines but not in MITC, indicating the absence or a dysfunction of regulatory factors in MITC line. Myoid cells expressed mRNA for all the subunits of the acetylcholine receptor (AChR) while epithelial cells expressed only the alpha, beta and epsilon subunits. Strikingly, LT-TEC produced much more C-C chemokines and IL-6 than MITC cells, while these latter produced higher levels of IL-8 and TNF-alpha. Altogether, these results reveal phenotypic and functional differences between these two stromal cell types, suggesting a potential involvement of myoid cells in the thymic function.     

On the character of the monolayer outgrowth and the fate of the peritubular myoid cells in cultured mouse testis

Postnatal differentiation of the peritubular myoid cells in mouse testis is hormone dependent. In order to analyse the differentiation of the peritubular tissue, an attempt was made to develop an experimental model system utilizing an in vitro method. Fragments obtained from adult, 7- or 10-day-old mice, were cultured in McCoy's modified 5a medium for 9–19 days. The fragments and monolayers that grew from them were examined with the electron microscope at the end of the culture period. Monolayers originating from either mature or immature testicular expiants were comparable in appearance. They were composed of spindle-shaped cells that contained abundant profiles of granular endoplasmic reticulum and free ribosomes, as well as arrays of 40–60 Å thick filaments and associated dense bodies. In these respects they resembled smooth muscle cells in culture, in developmental, and in pathological conditions. Examination of the peritubular tissue in the testicular explants indicated that the monolayer of myoid cells originated from the fibroblasts rather than the peritubular myoid cells. Peritubular cells in explants from mature rats retained their myoid features at the end of the culture period but myoid cell differentiation failed to progress in expiants obtained from immature animals. Additional work is necessary in order to establish the suitability of these preliminary culture attempts to support normal development before conclusions may be drawn concerning the role of hormones in myoid cell differentiation. The role of microfilaments as a contractile organelle of cells is discussed.     

Changes of subcellular localization of Thy-1 antigen during thymic myoid cell differentiation

Using a quantitative enzyme immunoassay, Thy-1 antigen expressed by a rat myoid cell line R615B2 was detected mainly on the cell surface at a single cell stage, whereas at the stage of forming myotubes, Thy-1 was found predominantly in the cytoplasm. The muscle specific creatine kinase activity also increased in association with the shift of Thy-1 from the cell surface to the cytoplasm, suggesting biological significance of Thy-1 redistribution in muscle differentiation from single cells to multinucleated cells.     

Appearance of alpha-smooth muscle actin in human eye lens cells of anterior capsular cataract and in cultured bovine lens-forming cells

Using light and electron-microscopic immunolocalization techniques, and gel electrophoresis combined with immunoblotting, we have examined the expression of cytoskeletal proteins in normal human fetal, child and adult lenses, in human anterior capsular cataract and in bovine lens cells in vivo and in vitro. In this report, we focus our observations on the pattern of actin-isoform expression during normal and pathological situations in vivo and culture conditions. We have noted that cells of developing and mature human lenses as well as bovine lens cells in situ contain only beta- and gamma-actins. In contrast, alpha-smooth muscle (alpha-sm) actin, an isoform typical of smooth muscle differentiation, was demonstrated in bovine lens cells at different times of culture. Moreover, the multilayered cells observed in the subcapsular zone of human anterior capsular cataract were characterized by the presence of alpha-sm actin. Thus, extensive changes in actin-isoform expression take place in lens cells growing in culture and may also occur during cataractogenesis. The biological meaning of the appearance of a marker of myoid differentiation in the ectodermally derived lens-forming cells is discussed.     

Myoepithelial cells in human thymus: staining, polarization and fluorescence microscopic studies.

Myoid cells in human thymus were studied around the turn of the century, and alterations in patients with cardiovascular disease were reported. It was therefore deemed of interest to reinvestigate these long forgotten cells. The configurational staining, polarization and fluorescence microscopic properties of smooth myofibrils in thymic epithelial cells were identical with those of classical myoepithelial cells, smooth muscle, and A bands of striated muscle. Cross-striated myoid cells could not be found in thymus of children. Myoepithelial cells formed a layer at the surface of thymic lobules; others were scattered throughout the cortex and medulla. In addition, the medulla contained seemingly hypertrophic myoepithelial cells. Hassall's corpuscles consisted of layers of myoepithelial cells. Hammar (1905) regarded epithelial cells with smooth myofibrils in human thymus as equivalents of the cross-striated myoid cells in lower vertebrates. The myoepithelial cells observed in this study are apparently identical with the smooth myoid cells of early anatomists; the hypertrophic myoepithelial cells correspond to the unicellular Hassall's corpuscles. The functions of these cells are not yet clear; the wide variations from case to case in the same age group indicate that the myoepithelial cells are affected by a variety of diseases.     

Optimization of human skeletal muscle precursor cell culture and myofiber formation in vitro

Muscle bioengineering is proposed as a treatment option for various conditions requiring restoration of muscle function. In order to allow for rapid clinical translation culture conditions have to be optimized for human application. The optimal isolation and culture technique should be able to support cell growth and differentiation using defined media only. Therefore, we have evaluated alternative culture conditions to determine the optimal growth condition for the engineering of human skeletal muscle. In this research, we present protocols for consistent isolation and growth of human muscle precursor cells (MPCs). MPCs were grown from human biopsies and expanded in culture using defined media and collagen coated dishes only. The best results were achieved using a one-step pre-plating and by supplementing the growth medium with insulin, dexamethasone, human basic fibroblast growth factor (hFGF) and human epithelial growth factor (hEGF). Detailed cell characterization using fluorescence-activated cell-sorting analysis and morphological analysis at different passages were performed. Further, the applicability of these cells for tissue engineering purposes was assessed by measuring expansion potential, formation of myofibers and fused myotubes. We have established a culture technique for human MPCs that allows for reliable cell growth and expansion using collagen coated dishes and defined media only. Cell characterization demonstrated a muscle phenotype and the ability to form myofibers in vitro.     

Thymic myoid cells protect thymocytes from apoptosis and modulate their differentiation: implication of the ERK and Akt signaling pathways

Thymic myoid cells correspond to a muscle-like cell population present in the thymic medulla. They are well conserved throughout species evolution, but their biological role is not known. We demonstrated that myoid cells protected thymocytes from apoptosis as evidenced by a strong decrease of annexin-V-FITC positive thymocytes. This effect was (1) specific of myoid cells compared to thymic epithelial cells; (2) dependent on direct cell-to-cell contacts and (3) triggered rapidly after 2 h in cocultures. This protective phenomenon was due to the activation of prosurvival mechanisms. Indeed, myoid cells activated extracellular-regulated kinases (ERK1/2) and Akt in thymocytes. Myoid cells also influenced thymocyte maturation. We observed an increase in CD4(+) and a decrease in CD8(+) single positive (SP) thymocytes when cocultured with myoid cells, independently of a CD8(+)SP increased death or a CD4(+)SP overproliferation. Consequently, thymic myoid cells protect thymocytes from apoptosis and could also modulate their differentiation process.     

The Ontogeny of Thymic Myoid Cells in the Chicken

The ontogeny of thymic myoid cells in the chick was studied electron microscopically and immunohistochemically. An anticreatine kinase antibody which reacts specifically to skeletal muscle cells was used. This antibody reacts only to myoid cells in the thymus. Myoid cells were found in the medulla or in the interlobular region, though the number of the myoid cells was small. Immunohistochemically, myoid cells were detected on the 18th day of incubation. Mature myoid cells showed clear cross striations after immunohistochemical staining around the time of hatching. Electron microscopically, myoid cells were detectable on the 19th day of incubation. The discrepancy between immunohistochemical and electron microscopical detection may be due to the low number of myoid cells.     

Human T cell leukemia virus type I induces DNA synthesis and immunoglobulin secretion in human lymphocyte cultures

Viral particles obtained from HTLV-I (human T cell leukemia virus, type I)-transformed T cell lines induced immunoglobulin production by normal peripheral blood lymphocytes. Conversely, no immunoglobulin could be detected in the supernatant medium in purified B cells cultivated with HTLV-I, suggesting that the presence of T cells is mandatory for HTLV-I to induce B cell polyclonal activation. The T cell help was mediated by soluble factors, as indicated in experiments showing that cell-free conditioned medium from T lymphocytes activated by HTLV-I was able to induce B cell proliferation and differentiation. Furthermore, a direct effect of HTLV-I on B cell proliferation was demonstrated when viral particles were added to purified B cells together with suboptimal doses of Staphylococcus aureus Cowan strain I (SAC). These observations show that an immediate early effect of HTLV-I infection was exerted on B cells, mainly in a T cell-dependent manner. Such an effect may account for the hypergammaglobulinemia observed in HTLV-I seropositive individuals, and in patients with HTLV-I-associated neurological disorders.     

Spontaneous myogenic differentiation of Flk-1-positive cells from adult pancreas and other nonmuscle tissues

At the embryonic or fetal stages, autonomously myogenic cells (AMCs), i.e., cells able to spontaneously differentiate into skeletal myotubes, have been identified from several different sites other than skeletal muscle, including the vascular compartment. However, in the adult animal, AMCs from skeletal muscle-devoid tissues have been described in only two cases. One is represented by thymic myoid cells, a restricted population of committed myogenic progenitors of unknown derivation present in the thymic medulla; the other is represented by a small subset of adipose tissue-associated cells, which we recently identified. In the present study we report, for the first time, the presence of spontaneously differentiating myogenic precursors in the pancreas and in other skeletal muscle-devoid organs such as spleen and stomach, as well as in the periaortic tissue of adult mice. Immunomagnetic selection procedures indicate that AMCs derive from Flk-1(+) progenitors. Individual clones of myogenic cells from nonmuscle organs are morphologically and functionally indistinguishable from skeletal muscle-derived primary myoblasts. Moreover, they can be induced to proliferate in vitro and are able to participate in muscle regeneration in vivo. Thus, we provide evidence that fully competent myogenic progenitors can be derived from the Flk-1(+) compartment of several adult tissues that are embryologically unrelated to skeletal muscle.     

Effects of thymic myoid cell culture supernatant on cells from lymphatic tissues

Conditioned media (MCM) of cloned thymic myoid cells (IT45R92, R613Ad, and R615B2) were used to investigate their possible involvement in thymic biological events. Those myoid cells produced in a culture medium biological activities capable of stimulating the growth of thymocytes, spleen cells, and bone marrow cells of mice and rats. Surface markers detected on spleen cells proliferating in MCM were characteristic of monocyte-macrophage lineages (C3R, Fc gamma R, asialo GM1) and T-cell lineages (Thy 1) but not B cells (sIgG). Chromatographic studies also suggested that the biological activities of MCM could be separated into two different molecular entities, such as a colony-stimulating activity and an interleukin 1-like activity which supported the growth of monocyte-macrophage lineages and T-cell lineages, respectively. These results indicate that thymic myoid cells produce cytokines important for the regulation of intrathymic interleukin cascade by which clonally differentiated thymic lymphocytes may be expanded into a sizable pool.     

CD4-independent infection by human immunodeficiency virus type 1 after phenotypic mixing with human T-cell leukemia viruses.

Although human immunodeficiency virus (HIV) is the causative agent of the acquired immunodeficiency syndrome and related disorders, it has been suggested that viral cofactors may accelerate the progression of the disease. We present evidence that human T lymphoid cells productively coinfected by HIV type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) or HTLV-II generate a progeny of phenotypically mixed viral particles that allow the penetration of HIV-1 into previously nonsusceptible CD4- human cells, including mature CD8+ T lymphocytes, B lymphoid cells, epithelial cells, and skeletal muscle cells. The infection is independent of the major HIV-1 receptor, (i.e., the CD4 glycoprotein) since OKT4a, a neutralizing anti-CD4 monoclonal antibody, fails to block the penetration of HIV-1. Similarly, infection is not inhibited by monoclonal antibody M77, directed toward the neutralizing loop of the gp120 envelope glycoprotein of HIV-1. In contrast, pretreatment of the virus stock with HTLV-I-neutralizing human serum completely abolishes the penetration of phenotypically mixed HIV-1 into CD4- cells. These results suggest that HTLV-I or HTLV-II may increase the pathogenicity of HIV-1 by broadening the spectrum of its cellular tropism and, thus, favoring its spread within the organism of coinfected hosts.     

A human skeletal muscle cell line obtained from an adult donor.

A cell line (RCMH) in permanent culture was established from surgically removed adult normal human skeletal muscle by exposure to conditioned media obtained from thyroid cells. Cells proliferated indefinitely but displayed density inhibition of growth while maintaining some differentiated markers. Under certain incubation conditions, cells fused into myotube-like structures, with a concomitant increase in muscle specific proteins, such as human myoglobin, skeletal muscle myosin, desmin and dystrophin, as identified using immunocytochemical procedures. In addition, RCMH cells displayed high affinity receptors for alpha-bungarotoxin (Bmax = 0.7 pmol/mg protein, Kd = 1.5 nM) and dihydropyridines (Bmax = 0.3 pmol/mg protein, Kd = 0.5 nM for [3H]PN200-110); these values are comparable to those reported for muscle cells in primary culture. Patch-clamp studies showed the presence of 42 pS carbachol gated channels and of 5 pS calcium channels (current carried by barium); chloride and potassium channels were also seen. This new cell line appears to be a convenient model system to study skeletal muscle function.     

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.     

Establishment of a leukemic cell model for studying human pre-B to B cell differentiation

Reproducible models for examining early stages of human B cell differentiation are poorly developed. We now describe the establishment and characterization of a novel human leukemic cell line that recapitulates the pre-B to B cell stage of differentiation. This cell line, designated BLIN-1, was initially established in tissue culture medium containing low m.w. B cell growth factor, and consistently shows a dependency on this cytokine for optimal growth at low density. BLIN-1 cells have a 9p chromosomal abnormality, identical to the abnormality present in the leukemic blasts from the patient's original bone marrow aspirate. The immunologic phenotype of BLIN-1 is consistent with a cell arrested at the pre-B cell stage of development. Analysis of Ig gene rearrangement and Ig expression in a series of BLIN-1 subclones show that the cells spontaneously rearrange kappa light chain genes, leading to the differentiation of surface kappa-negative pre-B cells into surface kappa-positive B cells. The BLIN-1 cell line is, to our knowledge, the first defined human model for examining this critical developmental stage in human B cell ontogeny. As such, it offers a unique resource for examining variables influencing onset of kappa L chain gene rearrangement and expression.     

The effect of ST2 gene product on anchorage-independent growth of a glioblastoma cell line, T98G.

The ST2 gene, which is specifically induced by growth stimulation in fibroblasts, encodes interleukin-1 receptor-related proteins and is widely expressed in hematopoietic, helper T, and various cancer cells. However, the physiological as well as pathological functions of the ST2 gene products are not yet fully understood. In this study, we analyzed the expression of the ST2 gene in human glioma cell lines and human brain tumor samples with real-time polymerase chain reaction method, the results of which revealed that the expression level of the ST2 gene in glioma cell lines and glioblastoma samples is significantly lower than that in a fibroblastic cell line, TM12, and benign brain tumors, suggesting the reverse relationship between malignancy and ST2 expression. As we could not detect the soluble ST2 protein in the culture fluid of the T98G glioblastic cell line by ELISA, we established stable transformants of T98G that continuously produce and secrete the ST2 protein, in order to study the effect of the ST2 protein on malignancy. Although we could not detect a remarkable difference in proliferation between transformants and control cells in conventional tissue culture dishes, the efficiency of colony formation in soft agar was significantly decreased in the case of cells that continuously produce the ST2 protein. Furthermore, inhibition of colony formation in soft agar was observed in wild-type T98G cells when purified soluble ST2 protein was added to the culture, in a dose-dependent manner. Taken together, the results suggest that the expression of ST2 suppressed the anchorage-independent growth and malignancy.