Shigeaki Ishizaka

In Vitro Differentiation of Embryonic Stem Cells into Hepatocyte‐Like Cells Identified by Cellular Uptake of Indocyanine Green

Background and Aims. Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently found the emergence of cell clusters that show the cellular uptake of indocyanine green (ICG) in the culture of differentiated ES cells. ICG is clinically used as a test substance to evaluate liver function because it is eliminated exclusively by hepatocytes. The aim of the present study was to investigate the hepatic characteristics of ICG‐stained cells.

Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone.

Background and Aims. Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently identified the emergence of cellular clusters within differentiated ES cell cultures by staining with dithizone (DTZ). DTZ is a zinc‐chelating agent known to selectively stain pancreatic beta cells because of their high zinc content. The aim of the present study was to investigate the characteristics of DTZ‐stained cellular clusters originating from ES cells.

Potential Use of Embryonic Stem Cells for the Treatment of Mouse Parkinsonian Models: Improved Behavior by Transplantation of In Vitro Differentiated Dopaminergic Neurons from Embryonic Stem Cells

Background and Aims. The purpose of the present study was to examine the efficacy of transplantation of mouse embryonic‐stem‐(ES)‐cell‐derived tyrosine hydroxylase‐positive (TH+) cells into Parkinsonian mice using behavioral tests and immunohistochemical evaluation.

Development of hepatocytes from ES cells after transfection with the HNF-3beta gene.

We have attempted to generate embryonic stem (ES) cell‐derived hepatocytes expressing liver‐specific functional properties by use of ES cell technology. It was found that ES cells are allowed to differentiate into hepatocytes possessing high metabolic activities when hepatocyte nuclear factor (HNF)‐3β‐transfected ES cells are cultured in α‐MEM medium supplemented with 10% fetal bovine serum (FBS) and fibroblast growth factor (FGF)‐2 in the three‐dimensional cell culture system at 5% CO2. The differentiated cells induced albumin, triacylglycerol, urea, and glycogen synthesis as well as further expression of metabolic proteins and serum factors as markers of hepatocytic differentiation for at least 4 months. The cells differentiated from HNF‐3β‐transfected ES cells also had hepatocyte‐like ultrastructural characteristics, including several endoplasmic reticula, mitochondrion, and glycogen. Our findings indicate that generation of hepatocytes maintaining high metabolic functions developed from mouse ES cells will facilitate the study of the basic mechanism for hepatogenesis and will certainly provide new opportunities for tissue transplantation.

In vitro functional gut-like organ formation from mouse embryonic stem cells.

Background and Aims. Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently found that ES cells can give rise to a functional gut‐like unit, which forms a three‐dimensional dome‐like structure with lumen and exhibits mechanical activity, such as spontaneous contraction and peristalsis. The aim of the present study was to investigate the electrophysiological and morphological properties of ES cell‐derived contracting clusters.

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.

Effects of glycyrrhizin on immune‐mediated cytotoxicity

Intravenous administration of glycyrrhizin is known to decrease elevated plasma transaminase levels in patients with chronic viral hepatitis, in which immune‐mediated cytotoxicity by cytotoxic T lymphocytes and tumour necrosis factor (TNF)‐α is considered to play an important pathogenic role. However, the immunological interpretation of the transaminase‐lowering action of glycyrrhizin is not known. Studies were performed to elucidate this action immunologically by assessing the effects of glycyrrhizin on immune‐mediated cytotoxicity using an antigen‐specific murine CD4+ T hybridoma line, which exhibits cytotoxicity against antigen‐presenting cells after stimulation with specific antigen, and a murine TNF‐α‐sensitive fibroblast line. Glycyrrhizin inhibited the cytotoxic activity of the T cells against antigen‐presenting cells and also suppressed TNF‐α‐induced cytotoxicity in the TNF‐α‐sensitive cell line in vitro. These results suggest that the decrease of elevated transaminase levels by glycyrrhizin in patients with chronic viral hepatitis is mediated in part by inhibition of immune‐mediated cytotoxicity against hepatocytes.

Transplantation of embryonic stem cell-derived neural stem cells for spinal cord injury in adult mice

Abstract Aims: To investigate the efficacy of embryonic stem cell-derived neural stem cells (NSCs) for spinal cord injury (SCI) in mice and whether a combination treatment with thyroid hormone provides a more effective ES cell-based therapy. Methods: Nestin-positive NSCs were induced from undifferentiated mouse ES cells by a step-by-step culture and used as grafts. Thirty-six mice were subjected to an SCI at Th10 and divided into three groups of 12. Graft cells were transplanted into the injury site 10 days after injury. Group 1 mice were left under observation without receiving graft cells, while mice in Group 2 received 2 × 104 graft cells, and those in Group 3 received 2 × 104 graft cells and were treated with a continuous intraperitoneal injection of thyroxin using osmotic mini-pumps. Behavioral improvement was assessed by a scoring system throughout the experimental period until post-transplantation day (PD) 28. Results: Mice in Groups 2 and 3 demonstrated an improved behavioral function, as compared to those in Group 1 after PD 14. There was no significant difference in behavioral recovery between Groups 2 and 3. Conclusions: Transplantation of ES-NSCs into the injury site was effective for SCI, while thyroxine did not deliver additional effectiveness.

Enhancing effect of polysaccharides from an edible brown alga, hijikia fusiforme (hijiki), on release of tumor necrosis factor‐α from macrophages of endotoxin‐nonresponder C3H/HeJ mice

An enhancing activity for the release of tumor necrosis factor-alpha from macrophages of C3H/HeJ mice was detected in the hot water-soluble extract of an edible brown alga, Hijikia fusiforme (Hijiki in Japanese). This activity was divided into the polysaccharide and nonpolysaccharide fractions, with the former showing much higher activity than the latter. The active components in the polysaccharide fraction were further purified by ion-exchange column chromatography and gel permeation system of high-performance liquid chromatography; they were identified as polysaccharides with apparent molecular mass of about 2,000 and 70 kDa and were designated Hijiki-derived polysaccharides I and II (HPS-I and HPS-II), respectively. They also enhanced macrophage-dependent suppression against the growth of EL-4 tumor cells in an in vitro culture experiment, with HPS-I exhibiting much higher immunologic activity than HPS-II. Furthermore, other comparative experiments confirmed that the immunoenhancing activities of polysaccharides from H. fusiforme are associated with the functions of polysaccharides themselves, but not with the artificial activity induced by contaminated endotoxins. Some biochemical properties of immunoenhancing polysaccharides were partially characterized, and the significance of this finding is discussed from the viewpoint of the protective role of edible seaweeds against carcinogenesis.

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.