Isao Kamo

Expression of markers for both neuronal and glial cells in human amniotic epithelial cells

Human amniotic epithelial (HAE) cells are formed from amnioblasts, separated from the epiblast at about the 8th day after fertilization. We attempted to detect various developmental antigens specific to neural cells by immunocytochemical methods. The cultured HAE cells displayed positive immunoreactivity to RC1, vimentin, A2B5, neurofilament proteins, microtubule-associated protein 2 (MAP2) and MAP2 kinase. In addition, the cells also demonstrated immunoreactivity to glial fibrillary acidic protein, CNPase, myelin basic protein and galactocerebroside. The appearance rate of positive cells was more than 50% in cells positive to RC1, A2B5, vimentin or neuronal markers, and 20-30% to glial cell markers. Double staining showed the heterogeneous appearance of oligodendrocyte lineage cells. These data indicate that HAE cells may have the putative multipotentiality of neurons, astrocytes and oligodendrocytes.

Human amnion mesenchyme cells express phenotypes of neuroglial progenitor cells

Previous studies from our laboratory showed that human amnion epithelial cells (AECs) have multiple functions, such as synthesis and release of catecholamines, acetylcholine, neurotrophic factors, activin, and noggin. In this study, we investigated the identity of neural progenitor cells in human amnion mesenchyme cells (AMCs), which lie immediately adjacent to the AECs. Cryostat sections revealed that vimentin expression was detected in the AMCs and CK19 in AECs. Vimentin‐positive cells made up 97.5% of total cells tested in cultured AMCs. Interestingly, 3.6% of total AMCs expressed the phenotype CK19+/vimentin+, indicating coexpression of epithelial and mesenchyme cell markers. In culturing with bromodeoxyuridine (BrdU) for 24 hr, 66–82% of cells were found to be BrdU positive, suggesting that they have proliferating potency. By using RT‐PCR, AMCs express mRNA of nestin and Musashi1. With a neural cell differentiating protocol, cell bodies extended long bipolar or complex multipolar processes. Nestin (87.7% of total cells tested) and Musashi1 (93.1%) were expressed in undifferentiated cells, and their positively stained cells increased in number slightly after induction. Undifferentiated cells were stained by anti‐Tuj1 and NF‐M, and their positively stained cells increased significantly in number after induction, to 72.8% and 46.0%, respectively. Meanwhile, glial fibrillary acidic protein‐positive cells increased from 25.4% to 43.2% after induction. These studies demonstrate that AMCs have phenotypes of neuroglial progenitor cells and can be differentiated into neuroglial phenotypes by optimal differentiation protocol. Eventually, AMC‐derived stem cells may be a favorable cell vehicle in regenerative medicine.

Haemopoietic biglycan produced by brain cells stimulates growth of microglial cells

We have recently found that soluble biglycan purified from rat thymic myoid cells had haemopoietic activity capable of inducing preferential growth and differentiation of monocytic lineage cells from various haemopoietic sources, including brain microglial cells. In the present study, to understand developmental mechanisms of microglial/monocytic cells in the brain, we have attempted to identify haemopoietic activity of the brain biglycan. The mRNA and the immunological epitope of biglycan were detected in the rat brain homogenates and several rat glial cell lines. Immunohistochemical study showed that several different types of brain cells produced biglycan. During development biglycan synthesis in the brain appeared to be increased. The brain haemopoietic biglycan was easily separated by DEAE-Sepharose chromatography from the macrophage colony stimulating factor (M-CSF) which was concomitantly produced from the brain cells. The brain haemopoietic biglycan, purified through immunoaffinity column, indeed stimulated growth of primarily cultured microglial cells. Taken together, these results suggest that the haemopoietic biglycan plays an important role in generating brain-specific circumstances for development of microglial/monocytic cells.

Clonal heterogeneity of thymic muscle-cell precursors

Three myoid-cell clones were established from the thymuses of two Wistar rats; one thymus yielded two clones, RBISA and R615B2, and the other yielded one clone, R613Ad. The three clones were divided into two subtypes. Both subtypes were able to form myofibrils, expressed AChR on their cell-surface membrane, and contained myofibrillar ATPase characteristic of und ifferentiated type-2C fibers, but they differed from each other in morphology, expression of Thy 1 antigen, spontaneous contractility, and qualitative accumulation of AChR. Immunolocalization studies using antisera against the respective cell subtypes also indicated regional differences in their cellular origin. These results show that the thymus contains heterogenous myoid-cell precursors.

Identification of haemopoietic biglycan in hyperplastic thymus associated with myasthenia gravis

Generally biglycan, a small proteoglycan, has been thought to play a role as an extracellular matrix and/or a reservoir for other factors, such as TGF-beta and collagens. Recently, we have found that a soluble 100 kDa biglycan, produced from the rat thymic myoid cells and the brain glial cells, predominantly stimulates growth and differentiation of monocytic lineage cells from various lymphatic organs, including microglias. In the present study, we attempted to identify biological significance of the corresponding molecules in human, using five myasthenic thymuses (three with hyperplasia and two with thymoma) that had been surgically removed for therapeutic purpose. With immunohistochemistry, many biglycan positive cells were detected in the germinal center of the three hyperplastic thymuses, but not in the two thymuses associated with lymphocytic thymoma. Biglycan purified from the hyperplastic thymuses by an immunoaffinity column was found as a monomer with apparent molecular size of 95-100 kDa and self associated oligomers of greater than 201 kDa. The purified biglycan markedly stimulated the growth and differentiation of monocytic cells from haemopoietic stem cells of the rat bone marrow. These results suggest that particular cells, which produce haemopoietic biglycan, play significant roles in generation and maintenance of the hyperplastic changes associated with myasthenia gravis.

A practical enzyme immunoassay for anti-acetylcholine receptor antibodies in Myasthenia Gravis ☆

A practical enzyme immunoassay (EIA) has been developed for the measurement of anti-acetylcholine receptor (AChR) antibodies in sera from patients with myasthenia gravis (MG). This system is based on the double antibody technique, using denervated rat muscle AChR labeled with horseradish peroxidase-linked alpha-bungarotoxin (HRP-alpha BGT). This method has the following advantages compared to conventional radioimmunoassay (RIA): (1) HRP-alpha BGT is more stable than [125I]alpha BGT and can be used for at least one year without any loss of the binding activity to AChR and enzymatic activity, (2) the procedure avoids the use of radioactive isotopes, and (3) the equipment for our EIA is more economical than that for RIA.

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.

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.

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 microM 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 microM 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.

Therapeutic effects of dipolar aprotic substances on Niemann-Pick cells.

Dimethylsulfoxide (Me2SO) increased acid sphingomyelinase activity in cultured skin fibroblasts (Sakuragawa N et al., Biochem Biophy Res Comm 1985; 126: 756–762). Various drugs were examined to find related compounds showing the same phenomenon as Me2 SO, including other dipolar aprotic substances, various inhibitors of DNA and RNA synthesis, phorbol esters, vitamin E and its derivatives and other miscellaneous agents. Several dipolar aprotic substances, such as Me2SO, dimethylacetamide, tetramethylurea and hexa‐methylene‐bisacetamide, increased the acid and Mg‐independent neutral sphingomyelinase activity in human cultured skin fibroblasts. These dipolar aprotic substances also increased acid and Mg‐independent neutral sphingomyelinase activities in Niemann‐Pick cells, type C, to 300 to 700% of those of untreated controls, repairing their enzyme deficiency. Other lysosomal hydrolases increased to a slightly lesser extent than sphingomyelinase. Dipolar aprotic substances could be used as therapeutic drugs for Niemann‐Pick disease, type C.