Thymic Myoid Cell Research Articles

Thymic myoid cells express high levels of muscle genes

To explore the possible contribution of thymic myoid cells in tolerance induction mechanisms, we quantified by real-time RT-PCR, the expression of 12 muscle genes (the five subunits of acetylcholine receptor, Musk, rapsyn, utrophin, ErbB2, ErbB3, troponin T, and MCK) in a human thymic myoid cell line (MITC), compared to thymic epithelial cells (TEC) and thymocytes. Although expression of all the genes analyzed was detected in TEC and thymocytes, the level of expression in these cells was much lower than in MITC, except for ε-AChR, utrophin and ErbB3 genes. Since myoid cells express high level of most muscle genes and are consistently found in the thymic medulla, they may contribute to the mechanisms involved in the induction and maintenance of immune tolerance.

Differentiation of rat thymic myoid progenitor cell line established by coculture with human T-lymphotropic virus type-I-producing human T cells

Differentiation of rat thymic myoid progenitor cell line established by coculture with human T-lymphotropic virus type-I-producing human T cells

Differentiation of rat thymic myoid progenitor cell line established by coculture with human T-lymphotropic virus type-I producing human T cells.

A thymus-derived myoid precursor cell line (ST1), which differentiates to myoid cells in the growth arrest condition, was established by the cocultivation of F344 rat thymic cells with human T-lymphotropic virus type-I (HTLV-I)-producing human lymphoid cells. No integration of HTLV-I was detected in ST1 cells by Southern blot hybridization. In a differentiation culture condition such as confluent culture or serum starvation, ST1 cells began to fuse, creating multinuclear giant cells, with the induced expression of MyoD1 and various muscle-specific antigens, including alpha-sarcomeric actin, skeletal muscle myosin, myoglobin, desmin, and acetylcholine receptor. Ultrastructural investigation revealed that differentiated ST1B cells created aggregates of thick and thin filaments with Z-band-like composition, then formed sarcomeric structures and tubular honeycomb arrays. Finally, these cells spontaneously contracted with a frequency of 0.5-2.0 Hz and synchronized with adjoining cells. Transplantation of ST1B cells into nude mice produced a small tumor nodule, showing clear differentiation to skeletal muscle cells. ST1B cells did not indicate any colony-forming activities in soft agar, demonstrating that ST1B cells retain some of the physiologically normal phenotypes. This rare cell line is promising for use in various physiological and pathological investigations including functional research of thymic myoid cells and the pathological role in autoimmune diseases, as well as animal model experiments of cell therapy related to muscular degenerative disorders or regeneration of injured muscles.

Establishment of a Human Thymic Myoid Cell Line: Phenotypic and Functional Characteristics

The subset of myoid cells is a normal component of the thymic stroma. To characterize these cells, we immortalized stromal cells from human thymus by using a plasmid vector encoding the SV40 T oncogene. Among the eight cell lines obtained, one had myoid characteristics including desmin and troponin antigens. This new line was designated MITC (myoid immortalized thymic cells). These cells expressed both the fetal and adult forms of muscle acetylcholine receptor (AChR) at the mRNA level, as well as the myogenic transcription factor MyoD1. alpha-Subunit AChR protein expression was detected by flow cytometry and the AChR was functional in patch-clamp studies. In addition, AChR expression was down-modulated by myasthenia gravis sera or by monoclonal antibody anti-AChR on MITC line similarly to TE671 rhabdomyosarcoma cells, making the MITC line an interesting tool for AChR antigenic modulation experiments. Finally, the MITC line expressed LFA-3, produced several cytokines able to act on T cells, and protected total thymocytes from spontaneous apoptosis in vitro. These results are compatible with a role of thymic myoid cells in some steps of thymocyte development. Therefore MITC line appears to be a useful tool to investigate the physiological role of thymic myoid cells.

Myoid cell density in the thymus is reduced duringmdxdystrophy 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.

Myoid cell density in the thymus is reduced during <i>mdx</i> 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.

Thymic myoid cell turnover in myasthenia gravis patients and in normal controls.

Thymic myoid cells (TMC) are sparse muscle-like cells in the thymic medulla, which are believed to trigger the autoimmune response in myasthenia gravis (MG). Ultrastructural investigations have revealed mature, degenerating, and immature TMC, but the number of TMC in MG patients does not differ from that in controls. We examined the turnover of TMC at the subcellular level, performing an immunocytochemical study with muscle-specific anti-desmin labelling of 10 thymuses derived from MG patients with lymphofollicular hyperplasia and from 8 normal controls. All thymuses examined revealed mature, immature, and degenerating TMC. Mature TMC contained desmin filaments in between Z-discs provided the sarcomeres were arranged in register. Morphological features of degenerating TMC included hypercontracted sarcomeres, cytoplasmic granular debris, chromatin clumping and, occasionally, membrane-bound bodies. Macrophages were not involved in the process. Immature TMC were of small diameter and contained myofilaments not arranged in myofibrils. In an MG thymus, small immature TMC were found clustered with dying TMC. It may be that degeneration of TMC is a stimulus for the generation of new TMC with faster turnover. This mechanism may mean that more antigen is available in MG patients than in normal controls, despite constant TMC numbers.

Muscle inactivity reduces the content of parvalbumin in rat thymic myoid cells in vitro

We investigated the influence of muscle inactivity by the addition of 1 μM tetrodotoxin in culture medium (complete abolishment of spontaneous firings); feeding with 50 mM KCl in the culture medium (inactivation of action potentials by depolarization) and the addition of 50 μM dantrolene sodium in culture medium (excitation-contraction uncoupling) on the content of parvalbumin in a cultured rat thymic myoid cell line (R615B2: M cells) using a highly sensitive enzyme immunoassay for rat parvalbumin. This is the first report of the content of parvalbumin in thymic myoid cells. Our studies clearly show that the content of parvalbumin is significantly decreased in paralyzed M cells compared with controls which had continuous muscle contractions. It is strongly suggested that muscle contractile activity plays an important role on the expression of parvalbumin in thymic myoid cells.

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.

Semi-quantitative immunohistochemical studies on Thy-1 antigen expressed by thymic myoid cells

Thy-1 antigen expression in the rat thymic myoid cell line R615B2 (Thy-1(+)) and R613Ad (Thy-1(−)) was studied with a semi-quantitative immunohistochemical assay without disrupting the cell cultures. With this assay, the quantity of Thy-1 antigen on R615B2 cells was detected separately in the cytoplasm and on the cell surface by the use of appropriate fixatives such as chilled ethanol and methanol +0.3% H 2O 2. Extracellular Thy-1 antigen was also found in the culture supernatant of R615B2 cells. More than half of extracellular Thy-1 antigen remained in the supernatant even after 100 000 × g centrifugation. No form of Thy-1 antigen was detected at significant levels in R613Ad cells.

Mitogenic heparin-binding lectin-like protein from cloned thymic myoid cells.

A mitogenic heparin-binding (reactive) lectin-like protein (HBP) was purified from the extract of a cloned rat thymic myoid cell R615B2 by a one-step procedure of affinity chromatography on a heparin--Sepharose CL-6B column. Four distinct peptide bands with molecular weights of 10,000, 13,000, 13,700, and 14,600 were detected on SDS-polyacrylamide gel electrophoresis. This protein is mitogenic at concentrations of as low as 1.1-70.0 ng/ml for peanut lectin-nonagglutinated thymocytes and splenocytes from euthymic mice and rats but not for splenocytes from nude mice. These results indicate that thymic myoid cell-derived HBP is an important signal for one particular step in T-cell differentiation.

Astrocyte growth enhanced by culture supernatant of a cloned rat thymic myoid cell

A conditioned medium (CM) from a cloned rat thymic myoid cell (IT-45R92) was used to investigate a possible relationship between the thymus and brain. CM stimulated the DNA synthesis of the cultured perinatal rat brain cells and spleen myelo-lymphoid cells but not that of the other types of cells including dorsal root ganglia, spinal cord, heart muscle, skeletal muscle, kidney, liver, skin and thymic fibroblasts. CM appeared to stimulate glioblasts to differentiate into mature astrocytes that have a characteristic intracellular fibrous structure and a glial fibrillary acidic protein. These results indicated that thymic myoid cells release (a) ‘cytokine(s)’ for the astrocytes and myelo-lymphoid cells, similar to a ‘lymphokine’ (glia cell stimulating factor, GSF) produced by murine and human lymphocytes stimulated with mitogens.

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, FcγR, 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.