Mamoru Tadokoro

PERINEURIAL CELL TUMOR AND THE SIGNIFICANCE OF THE PERINEURIAL CELLS IN NEUROFIBROMA

The authors attempted to clarify the exact cell components of neurofibroma by immunohistochemical and ultrastructural studies. Materials were randomly selected, 40 cases of neurilemoma and neurofibroma (‐tosis) in addition to 2 cases of tumors composed exclusively of perineurial cells and three cases of normal peripheral nerve. The applied markers included antisera of S‐100 protein for Schwann cells, blood coagulation factor XIIIa for endoneurial fibroblasts or perineurial cells, and laminin and collagen type IV for the basement membrane. S‐100 protein was demonstrated only in normal or neoplastic Schwann cells, but not in perineurial cells. On the other hand, factor XIIIa was often recognized in endoneurial fibroblasts and perineurial cells, but not in Schwann cells. Neurofibroma was basically composed of a mixture of Schwann cells, perineurial cells, and endoneurial fibroblasts, the population of each type of cell differing according to the case and area within a given tumor. Perineurial cell tumor exclusively composed of perineurial cells, though rare, appears to be a definite entity, and its characteristic histological and ultrastructural features were described.

Anatomical and functional recovery by embryonic stem cell-derived neural tissue of a mouse model of brain damage

We have treated undifferentiated mouse embryonic stem (ES) cells with all-trans retinoic acid (RA) to induce differentiation in vitro into neuron-like cells with good cell viability for use as a graft. Furthermore, we asked whether the RA-induced neuron-like cells restored neurological dysfunction. To this end, the cells were transplanted into right hemiplegia model of mice, developed by a cryogenic injury of motor cortex. Motor function of the recipients was gradually improved, whereas little improvement was observed in control mice. The lesion showed clustering of mature and almost mature neuron-like cells in mice transplanted with the RA-treated cells. The grafted cells had synaptic vesicles. This finding may suggest their maturation and synaptic connection in the recipient brain. Even though further study is necessary to elucidate molecular and cellular mechanisms responsible for the functional recovery, we consider that the ES cells may have advantage for use as a donor source in various neurological disorders including motor dysfunction.

Noggin and basic FGF were implicated in forebrain fate and caudal fate, respectively, of the neural tube-like structures emerging in mouse ES cell culture

We developed neural tube-like structures accompanying neural crest-like cells by treating embryonic stem (ES) cells with retinoic acid. The structures contained pseudostratified Nestin+Vimentin+ neuroepithelial cells surrounded by Masson staining+ basement membrane. βIIItubulin+Synaptophysin+ mature neurons and glial fibrillary acidic protein (GFAP)+ glial cells dispersed outside of the membrane. Addition of Noggin to the culture induced prominent proliferation of the neuroepithelial cells, leading to epithelial hyperstratification of the structures. mRNAs of transcription factors essential for forebrain development such as Emx1/2 and Pax6 were specifically expressed and Islet1+Lim1/2- motoneurons appeared by the addition of Noggin. In contrast, basic fibroblast growth factor (bFGF) promoted enlargement of central lumen and elongation of the structures. mRNAs of caudal markers, Gbx2, Cdx2 and Hoxb4/9 were expressed and Lim1/2+ spinal motoneurons appeared by the addition of bFGF. Addition of BMP-4 similarly brought about mild enlargement of central lumen of the structures. Interestingly, the addition of BMP-4 induced Slug+ neural crest-like cells surrounding the tube-like structures. mRNAs of Snail and dHand, other markers for neural crest cells, were also expressed by the addition of BMP-4. These results suggest that Noggin lead the neural-tube like structures to forebrain fate, whereas bFGF was involved in the caudalization. BMP-4 was implicated in emergence of the neural crest-like cells. Differentiation of ES cells by the present methods may mimic neurulation and subsequent neural development of early embryos, and elucidates the opposite effects of Noggin and bFGF for the neural tube development.

Transplantation of motoneurons derived from MASH1-transfected mouse ES cells reconstitutes neural networks and improves motor function in hemiplegic mice.

Mouse embryonic stem (ES) cells were transfected with a MASH1 expression vector and G418-resistant cells were selected. The MASH1-transfected cells became neuron-like appearance and expressed betaIIItubulin and panNCAM. Glial fibrillary acidic protein (GFAP) and galactocerebroside (GalC)-expressing cells were rarely detected. Half of the neural cells differentiated into the Islet1+ motoneuron lineage. Thus, we obtained motoneuron lineage-enriched neuronal cells by transfection of ES cells with MASH1. A hemiplegic model of mice was developed by cryogenic injury of the motor cortex, and motoneuron lineage-enriched neuronal cells were transplanted underneath the injured motor cortex neighboring the periventricular region. The motor function of the recipients was assessed by a beam walking and rotarod tests, whereby the results gradually improved, but little improvement was observed in vehicle injected control mice. We found that the grafted cells not only remained close to the implantation site, but also exhibited substantial migration, penetrating into the damaged lesion in a directed manner up to the cortical region. Grafted neuronal cells that had migrated into the cortex were elongated axon-positive for neurofilament middle chain (NFM). Synaptophysin immunostaining showed a positive staining pattern around the graft, suggesting that the transplanted neurons interacted with the recipient neurons to form a neural network. Our study suggests that the motoneuron lineage can be induced from ES cells, and grafted cells adapt to the host environment and can reconstitute a neural network to improve motor function of a paralyzed limb.

Overexpression of heat shock protein 27 in squamous cell carcinoma of the uterine cervix: a proteomic analysis using archival formalin-fixed, paraffin-embedded tissues.

Proteomic analysis of squamous cell carcinoma of the uterine cervix was performed using total protein from archival formalin-fixed, paraffin-embedded tissues. A wide range of proteins with molecular weights of 10 to greater than 200 kd was extracted from formalin-fixed, paraffin-embedded tissues using a recently developed protocol based on the heat-induced antigen retrieval technique. The extracted proteins from normal squamous epithelium (n = 53) and squamous cell carcinoma (n = 21) were fluorescently labeled and separated using 2-dimensional gel electrophoresis. We identified 728 differentially expressed proteins, with 144 up-regulated and 584 down-regulated as compared with normal squamous epithelial tissue samples. Nine proteins showing pronounced up-regulation in squamous cell carcinoma were analyzed on liquid chromatography-tandem mass spectrometry. Among the candidate proteins identified, minichromosome maintenance 8, a disintegrin and metalloproteinase domain 18, and heat shock protein 27 were analyzed in Western blotting, resulting in significant overexpression of heat shock protein 27 in squamous cell carcinoma over normal mucosa (P < .05). Furthermore, immunostaining revealed heat shock protein 27 overexpression not only in squamous cell carcinoma but in various stages of cervical intraepithelial neoplasia (grades 1-3, n = 90), including dysplasia and carcinoma in situ. The expression levels of heat shock protein 27 in cervical intraepithelial neoplasia grades 1 to 3 and squamous cell carcinoma were significantly higher than that in normal mucosa (P < .05). In the neoplastic lesions, heat shock protein 27 expression levels in cervical intraepithelial neoplasia grade 3 and squamous cell carcinoma were significantly higher than that in cervical intraepithelial neoplasia grade 1 (P < .05). These results may suggest a role of heat shock protein 27 in tumor development and progression in the cervical intraepithelial neoplasia-squamous cell carcinoma sequence. Future experiments using formalin-fixed, paraffin-embedded tissue-based proteomic analysis will be a powerful tool for various pathologic studies.

Pathologic changes of glial cells in murine model of Niemann–Pick disease type C: Immunohistochemical, lectin-histochemical and ultrastructural observations

Abstract Background : In recent years, morbid states of glial cells have been reported in several neurodegenerative diseases. We studied neuropathologically the glial cells in a murine model of Niemann–Pick disease type C (NPC) to clarify involvement of glias, the most important supportive cells in the central nervous system, by the disease.

Biochemical and morphological characterization of spontaneously hypertensive hyperlipidaemic rats

1. To develop and characterize a new animal model of hypertension and hyperlipidaemia, we cross‐bred spontaneously hypertensive rats (SHR) with spontaneously hyperlipidaemic rats (HLR).

Transfection with pax6 Gene of Mouse Embryonic Stem Cells and Subsequent Cell Cloning Induced Retinal Neuron Progenitors, Including Retinal Ganglion Cell-Like Cells, in vitro

Objective: It is theoretically possible to induce various cell types, including retinal neurons, from embryonic stem cells (ESCs). pax6 regulates early events in eye development, including the generation of retinal ganglion cells (RGCs). We previously reported the successful induction of corneal epithelial cells from ESCs transfected with the pax6 gene. Here, we attempted to establish cloned RGC-like cells from ESCs transfected with the pax6 gene. Methods: Undifferentiated mouse ESCs were transfected with pax6 cDNA by electroporation, followed by selection with G418. We conducted limiting-dilution culture of pax6-transfected cells. We expanded the cloned pax6-transfected cells, which expressed nestin and musashi-1, for further characterization in culture media containing fibronectin. The cells were characterized using RT-PCR, immunostaining, electron microscopy, renal subcapsular transplantation assay and Ca imaging. Results: We obtained clonally expanding pax6-transfected cells, all of which were positive for six3, sonic hedgehog (shh), math5, brn3, thy1 and melanopsin, by using several ESCs. When transplanted into a mouse renal capsule, they differentiated into neurons with elongated axons, expressing βIII tubulin and neurofilament middle chain, and were free from teratoma development. Electron-microscopic examination showed neurotubules and neurofilaments in the axon-like processes of the cloned pax6-transfected cells. High KCl stimulation increased free Ca influx on Ca2+ imaging. Conclusions: ESCs were applicable for the induction of retinal progenitor cells, including RGC-like cells, by transfection with the pax6 gene and subsequent limiting-dilution culture. Cloned cell lines may be useful to analyze the requirements for retinal progenitor cell differentiation, and our study suggests the clinical application of this cell type.

E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis.

Background and Aim Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis. Methods The expression and localization of synoviolin in the liver were analyzed in CCl4-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno+/− mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno+/− mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno−/− mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno−/− MEF cells. Results In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno+/− mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno+/− mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno−/− MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum. Conclusion Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.

Evaluation of Thermoreversible Gelation Polymer for Regeneration of Focal Liver Injury

Liver injuries are often associated with complications including infection of the dead space, bleeding, leakage of bile and so on. We have recently developed a thermoreversible gelation polymer (TGP) which provides a good healing environment for wounds and possibly reduces complications. The purpose of this study was to evaluate whether adequate regeneration occurred with a liver defect by using TGP. The sol-gel transition of TGP is reversibly controlled by temperature; TGP is soluble below a lower critical solution temperature (LCST) of 22°C, and becomes solid above the LCST. Soluble TGP can reach anywhere, and gelation of TGP occurs at the wound surface by body temperature to fill the wound/cavity. A section of median part of the left lobe comprising 3% (2 × 2 cm wide and 1 cm deep) of the liver was resected, and the Beagle dogs were assigned to three groups: ‘resection alone group’, ‘resection + fibrin glue (FG) group’ and ‘resection + TGP group’. The resection alone group and the resection + FG group showed severe fibrosis at week 12, and a scarring was clearly visible. The resection + TGP group showed almost complete healing by week 4, with no adhesion and recession of the wound; the resection site was completely filled with TGP, liver-like capsule organoids emerged to cover the wound and neovascularization was observed within the organoids. Furthermore, the resected liver regenerated completely by week 12, TGP was replaced by hepatocytes, and the presence of hepatic lobules confirmed structural reorganization. The number of RCA-1-positive macrophages accumulating around the wound was significantly reduced in the resection + TGP group compared to the other two groups. In the early stage of liver resection and regeneration, TGP seemed to suppress the accumulation of macrophages and stellate cells. In the late stage, when massive inflammatory cell accumulation had subsided, TGP was degraded, that may contribute to avoid unnecessary inhibition of the liver regeneration process. Collectively, TGP may induce efficient regeneration by reducing the fibrosis and enhancing proliferation, even with a minor liver defect. Because TGP has good biocompatibility, it may become useful as an ideal biomaterial for the treatment of liver injuries.