Mariko Nishiofuku

Cotransplantation of mouse embryonic stem cells and bone marrow stromal cells following spinal cord injury suppresses tumor development.

Embryonic stem (ES) cells are a potential source for treatment of spinal cord injury (SCI). Although one of the main problems of ES cell-based cell therapy is tumor formation, there is no ideal method to suppress tumor development. In this study, we examined whether transplantation with bone marrow stromal cells (BMSCs) prevented tumor formation in SCI model mice that received ES cell-derived grafts containing both undifferentiated ES cells and neural stem cells. Embryoid bodies (EBs) formed in 4-day hanging drop cultures were treated with retinoic acid (RA) at a low concentration of 5 × 10–9 M for 4 days, in order to allow some of the ES cells to remain in an undifferentiated state. RA-treated EBs were enzymatically digested into single cells and used as ES cell-derived graft cells. Mice transplanted with ES cell-derived graft cells alone developed tumors at the grafted site and behavioral improvement ceased after day 21. In contrast, no tumor development was observed in mice cotransplanted with BMSCs, which also showed sustained behavioral improvement. In vitro results demonstrated the disappearance of SSEA-1 expression in cytochemical examinations, as well as attenuated mRNA expressions of the undifferentiated markers Oct3/4, Utf1, Nanog, Sox2, and ERas by RT-PCR in RA-treated EBs cocultured with BMSCs. In addition, MAP2-immunopositive cells appeared in the EBs cocultured with BMSCs. Furthermore, the synthesis of NGF, GDNF, and BDNF was confirmed in cultured BMSCs, while immunohistochemical examinations demonstrated the survival of BMSCs and their maintained ability of neurotrophic factor production at the grafted site for up to 5 weeks after transplantation. These results suggest that BMSCs induce undifferentiated ES cells to differentiate into a neuronal lineage by neurotrophic factor production, resulting in suppression of tumor formation. Cotransplantation of BMSCs with ES cell-derived graft cells may be useful for preventing the development of ES cell-derived tumors.

Embryonic stem cells reduce liver fibrosis in CCl4-treated mice.

We transplanted undifferentiated embryonic stem (ES) cells into the spleens of carbon tetrachloride (CCl4)‐treated mice to determine their effects on liver fibrosis. Carbon tetrachloride at 0.5 ml/kg of body weight was injected intraperitoneally into C57BL/6 mice twice weekly for up to 20 weeks. Four weeks after the first injection, the mice were divided into two groups and those in group 1 received 1 × 105 ES cells genetically labelled with enhanced green fluorescent protein (GFP) in the spleens, while group 2 mice received 0.1 ml of phosphate‐buffered saline. In group 1, GFP‐immunopositive cells were retained and found in areas of fibrosis in the liver, and reduced liver fibrosis was observed as compared with group 2. Secondary transplantation of ES cells at 12 weeks after the initial transplantation enhanced the reduction in liver fibrosis. No teratoma formation or uncontrolled growth of ES cells in organs, including the liver and spleen, was observed in any of the mice. In the livers of group 1 mice, metalloproteinase 9‐immunopositive cells derived from ES cells as well as those from the recipient were observed. These cells were also found to be immunopositive for the hepatoblast marker Delta‐like (DlK‐1), a member of the DlK‐1 family of transmembrane proteins. These results suggest that ES‐based cell therapy is potentially useful for liver fibrosis treatment and that reduction in CCl4‐induced liver fibrosis by transplantation of ES cells may be related closely to the emergence of metalloproteinase‐producing hepatoblast‐like cells.

A familial case of visceral toxocariasis due to consumption of raw bovine liver

We present 3 adult cases of visceral toxocariasis from the same family, who each consumed thin slices of raw bovine liver weekly, and developed eosinophilia and multiple small lesions in their livers and lungs. Serological examinations using the larval excretory-secretory product of Toxocara canis strongly indicated infection with Toxocara species larvae. The patients responded well to treatment with albendazole. Ingestion of raw liver from paratenic animals is considered to be a common transmission route of human toxocariasis, especially in adults.

Effects of Wnt-10b on proliferation and differentiation of adult murine skin-derived CD34 and CD49f double-positive cells

Although mouse Wnt-10b has been shown to play various roles in a wide range of biological actions, the effects on epithelial stem/progenitor cells in the skin have not been reported. In the present study, we investigated the effects of Wnt-10b on proliferation and differentiation of murine skin-derived CD34 and CD49f double-positive (CD34(+)CD49f(+)) cells, a supposed fraction as enriched epithelial stem/progenitor cells. The cells were prepared from dorsal skin samples obtained from young adult mice as alpha6 integrin (CD49f) and CD34 double-positive cells by fluorescent activated cell sorting (FACS), and they were cultured with or without Wnt-10b to investigate its effects on proliferation and differentiation. Involvement of canonical Wnt signaling pathway was confirmed by TOPFLASH assay, and differentiation of the CD34(+)CD49f(+) cells was assessed by RT-PCR analysis and immunocytochemical examinations. The skin-derived CD34(+)CD49f(+) cells were immunopositive for Lhx2 and expressed mRNA of classical markers for bulge stem cells, including Lhx2, keratin15, Sox9, S100a6, and NFATc1. Their proliferation was suppressed by Wnt-10b, and the markers for differentiated epithelial cells became to be expressed in the culture with Wnt-10b. These results suggest that Wnt-10b promotes differentiation of epithelial stem/progenitor cells in the skin.

CoCl2 Inhibits Neural Differentiation of Retinoic Acid-Treated Embryoid Bodies

The effects of CoCl(2) on retinoic acid (RA)-treated embryoid bodies (EBs) were investigated. Four-day EBs were treated with 5x10(-6) M of RA for 4 d, then subjected to attached culturing for 7 d in the presence of CoCl(2) at 0, 20, and 100 microM. Differentiation into MAP2- and GFAP-immunopositive cells was inhibited by CoCl(2) in a dose-dependent manner. Next, RA-treated EBs were dissociated into single cells and cultured for 7 d at an initial cell density of 1x10(3)/cm(2). The number of cells increased in a CoCl(2)-dose dependent fashion. In cultures with 100 microM of CoCl(2), more than 90% of the cells were immunopositive for nestin and nestin-immunopositive cells formed clusters, while there were few cells immunopositive for MAP2 or GFAP. These results suggest that CoCl(2) inhibits neural differentiation of RA-treated EB cells and promotes the proliferation of nestin-immunopositive cells, i.e., embryonic stem (ES)-derived neural stem-like cells.

Modulated differentiation of embryonic stem cells into hepatocyte-like cells by coculture with hepatic stellate cells.

We investigated the effects of coculture with hepatic stellate cells (HSCs) on the differentiation of embryonic stem (ES) cells and embryoid bodies (EBs). Rat HSCs were incubated until becoming semi-confluent and adherent to the dish. Undifferentiated mouse ES cells and 4-day EBs were cultured in gelatin-coated or HSC-feeder dishes, then induced hepatocyte-like cells and the remaining undifferentiated ES cells were examined using immunocytochemical and RT-PCR methods. HSCs promoted the differentiation of EBs into hepatocyte-like cells, whereas they inhibited the differentiation of undifferentiated ES cells. Among EB outgrowths cocultured with HSCs, albumin-immunopositive cells were clearly and abundantly observed, while they were faintly and scarcely seen in EB outgrowths without HSCs. mRNA expressions of the hepatocyte-related markers such as albumin, transthyretin, alpha-1-antitrypsin, tryptophan-2,3-dioxygenase, phosphoenolpyruvate carboxykinase, hepatocyte nuclear factor 4α and cytochrome P4507a1 were clearly detected in EB outgrowths cocultured with HSCs, while they were only weakly detected or undetected in spontaneous EB outgrowths without HSCs. In contrast to the promoted hepatic differentiation of EBs by HSCs, undifferentiated ES cells formed cellular colonies in HSC-feeder dishes that were similar to the colonies of undifferentiated ES cells kept in maintenance medium containing leukemia inhibitory factor. In addition, ES cell colonies were immunopositive for Oct-3/4, markers of an undifferentiated state, and there were few ALB-immunopositive cells in the colonies. Thus, HSCs have contrasting effects on EBs undergoing differentiation and undifferentiated ES cells, i.e., positive and negative modulation, respectively.

Diagnostic problems in a patient with amicrofilaremic Loa loa.

We report a Japanese patient with loiasis who became infected in Cameroon. Despite the clinical history and laboratory data providing adequate evidence for suspecting loiasis, microfilariae were not detected in the blood. It is important to note that most infected travelers whose home countries are in nonendemic regions are amicrofilaremic.

Isolation and characterization of murine hepatocytes following collagenase infusion into left ventricle of heart

A method for obtaining mouse hepatocytes by infusing collagenase solution into the left ventricle was established. This technique was shown to be equivalent to the intra-portal infusion method and more practical, especially in postnatal mice with a small body size.