Tsunekazu Mizushima

Reprogramming of Mouse and Human Cells to Pluripotency Using Mature MicroRNAs

Induced pluripotent stem cells (iPSCs) can be generated from differentiated human and mouse somatic cells using transcription factors such as Oct4, Sox2, Klf4, and c-Myc. It is possible to augment the reprogramming process with chemical compounds, but issues related to low reprogramming efficiencies and, with a number of protocols, residual vector sequences, remain to be resolved. We show here that it is possible to reprogram mouse and human cells to pluripotency by direct transfection of mature double-stranded microRNAs (miRNAs). Our approaches use a combination of mir-200c plus mir-302 s and mir-369 s family miRNAs. Because this reprogramming method does not require vector-based gene transfer, it holds significant potential for biomedical research and regenerative medicine.

A Novel Sphingosine 1-Phosphate Receptor Agonist, 2-Amino-2-propanediol Hydrochloride (KRP-203), Regulates Chronic Colitis in Interleukin-10 Gene-Deficient Mice

Current treatments for patients with Crohns disease (CD) are based on recent advances in elucidating the pathophysiology of the disease. A satisfactory therapeutic strategy has not been well established. A new sphingosine 1-phosphate (S1P) receptor agonist, 2-amino-2-propanediol hydrochloride (KRP-203), has been developed for immunomodulation in autoimmune diseases and organ transplantation. We aimed to evaluate the efficacy and potency of KRP-203 on the treatment of chronic colitis in an interleukin (IL)-10 gene-deficient (IL-10–/–) mouse model. KRP-203 agonistic activity on S1P receptor was assessed in vitro. KRP-203 was administered for 1 or 4 weeks to IL-10–/– mice with clinical signs of colitis. The histological appearance of the colon and the numbers, phenotype, and cytokine production of lymphocytes were compared with a control group. KRP-203 treatment was effective in preventing body weight loss in the IL-10–/– colitis model. One-week administration resulted in the sequestration of circulating lymphocytes within the secondary lymphoid tissues. After 4 weeks of treatment, highly significant reductions were observed in number of CD4+ T cell and B220+ B cell subpopulations in the lamina propria of the colon and peripheral blood. KRP-203 obviously inhibited the production of interferon-γ, IL-12, and tumor necrosis factor-α by the colonic lymphocytes, but had no influence on IL-4 production. KRP-203 significantly inhibits ongoing IL-10–/– colitis in part through decreasing the infiltration of lymphocytes at inflammatory sites and by blocking T-helper 1 cytokine production in the colonic mucosa. Therefore, the possibility arises that KRP-203 plays a potential role in control of chronic colitis.

Jumonji Domain Containing 1A Is a Novel Prognostic Marker for Colorectal Cancer: In vivo Identification from Hypoxic Tumor Cells

Purpose: This study aimed to identify novel hypoxia-inducible and prognostic markers in vivo from hypoxic tumor cells. Experimental Design: Using carbonic anhydrase 9 and CD34 as a guide for hypoxic tumor cells, laser capture microdissection was used to isolate colorectal cancer (CRC) liver metastases. The samples were analyzed by microarray analysis, in parallel with five CRC cell lines cultured under hypoxic conditions. To evaluate the prognostic impact of the expression of certain genes, samples from a total of 356 CRC patients were analyzed by microarray or quantitative reverse transcription-PCR. In vitro mechanistic studies and in vivo therapeutic experiments were also done about a histone H3 Lys9 demethylase, Jumonji domain containing 1A (JMJD1A). Results: Several candidate genes were identified by microarray analysis of liver metastases and culturing of CRC cells under hypoxic conditions. Among them, we found that JMJD1A was a novel independent prognostic factor for CRC (P = 0.013). In vitro assays revealed that loss of JMJD1A by small interfering RNA treatment was associated with a reduction of proliferative activity and decrease in invasion of CRC cell lines. Furthermore, treatment with an adenovirus system for antisense JMJD1A construct displayed prominent therapeutic effects when injected into established tumor xenografts of the CRC cell lines HCT116 and DLD1. Conclusions: JMJD1A is a useful biomarker for hypoxic tumor cells and a prognostic marker that could be a promising therapeutic target against CRC. Clin Cancer Res; 16(18); 4636–46. ©2010 AACR.

Increased Th17-Inducing Activity of CD14+ CD163low Myeloid Cells in Intestinal Lamina Propria of Patients With Crohn’s Disease

BACKGROUND & AIMS Abnormal activity of innate immune cells and T-helper (Th) 17 cells has been implicated in the pathogenesis of autoimmune and inflammatory diseases, including Crohns disease (CD). Intestinal innate immune (myeloid) cells have been found to induce development of Th17 cells in mice, but it is not clear if this occurs in humans or in patients with CD. We investigated whether human intestinal lamina propria cells (LPCs) induce development of Th17 cells and whether these have a role in the pathogenesis of CD. METHODS Normal intestinal mucosa samples were collected from patients with colorectal cancer and noninflamed and inflamed regions of mucosa were collected from patients with CD. LPCs were isolated by enzymatic digestion and analyzed for expression of HLA-DR, lineage markers CD14 and CD163 using flow cytometry. RESULTS Among HLA-DR(high) Lin(-) cells, we identified a subset of CD14(+) CD163(low) cells in intestinal LPCs; this subset expressed Toll-like receptor (TLR) 2, TLR4, and TLR5 mRNAs and produced interleukin (IL)-6, IL-1β, and tumor necrosis factor in response to lipopolysaccharide. In vitro co-culture with naïve T cells revealed that CD14(+) CD163(low) cells induced development of Th17 cells. CD14(+) CD163(low) cells from inflamed regions of mucosa of patients with CD expressed high levels of IL-6, IL-23p19, and tumor necrosis factor mRNAs, and strongly induced Th17 cells. CD14(+) CD163(low) cells from the noninflamed mucosa of patients with CD also had increased abilities to induce Th17 cells compared with those from normal intestinal mucosa. CONCLUSIONS CD14(+) CD163(low) cells in intestinal LPCs from normal intestinal mucosa induce differentiation of naive T cells into Th17 cells; this activity is increased in mucosal samples from patients with CD. These findings show how intestinal myeloid cell types could contribute to pathogenesis of CD and possibly other Th17-associated diseases.

IgG Oligosaccharide Alterations Are a Novel Diagnostic Marker for Disease Activity and the Clinical Course of Inflammatory Bowel Disease

BACKGROUND AND AIMS:Patients with inflammatory bowel disease (IBD) share several immunologic similarities with rheumatoid arthritis (RA). Patients with RA have significantly increased levels of serum agalactosyl immunoglobulin G (IgG). Our aim was to investigate the clinical significance of analyzing the oligosaccharide structure of serum IgG in patients with IBD.METHODS:Serum IgG oligosaccharide structures were analyzed using high-performance liquid chromatography in 60 patients with Crohns disease (CD), 58 patients with ulcerative colitis (UC), 27 healthy volunteers (HV), and 15 disease controls (DC). The activity and mRNA level of beta-1,4-galactosyltransferase (Beta4GalT) in antibody-secreting cells were investigated in these subjects.RESULTS:The agalactosyl fraction of the fucosylated IgG oligosaccharides (G0F/G2F) in CD and UC was significantly greater than that in HV and DC (P < 0.001). The percentage of subjects with a high G0F/G2F in CD, UC, HV, and DC was 72%, 33%, 0%, and 0%, respectively. G0F/G2F, which is significantly correlated with disease severity in both CD and UC, had higher sensitivity to diagnose IBD compared with anti-Saccharomyces cerevisiae antibody. Moreover, G0F/G2F was significantly correlated with the prognosis of UC patients: patients with a high G0F/G2F did not maintain long-term remission. The activity and mRNA level of Beta4GalT were significantly elevated in UC but not in CD.CONCLUSIONS:G0F/G2F is a potentially effective diagnostic marker of disease activity in both CD and UC, and of the clinical course in UC. A pathophysiologic difference between CD and UC was also demonstrated.

Gastroenterological surgery in Japan: The past, the present and the future

In the last two centuries, there has been remarkable progress in the field of gastroenterological surgery, including the curative resection of cancers, replacement of failed organs through transplantation, increased safety of undergoing major surgeries and decreased operative morbidity through developments in minimal access surgery. Japan has very much been at the forefront of these advances, as is evident from the present review, from advancing the surgical management of gastric cancer to the pioneering work in live‐donor transplantation. This review also highlights many instances where surgical management of the same pathologies has evolved differently between Japan and the West. It is encouraging that many procedures established in Japan are eventually taken up by the West, often after rigorous assessment affirming the quality and applicability of such techniques. In Japan, many of the crucial issues in gastroenterological surgery are increasingly addressed through large multi‐institutional prospective control trials, ensuring that Japanese surgeons continue to contribute to the advances in gastroenterological surgery.

Therapeutic effects of novel sphingosine-1-phosphate receptor agonist W-061 in murine DSS colitis.

Although IL-17 is a pro-inflammatory cytokine reportedly involved in various autoimmune inflammatory disorders, its role remains unclear in murine models of colitis. Acute colitis was induced by 2.5% dextran sodium sulfate (DSS) treatment for 5 days. A novel sphingosine-1-phosphate receptor agonist W-061, a prototype of ONO-4641, was orally administered daily, and histopathological analysis was performed on the colon. The number of lymphocytes and their cytokine production were also evaluated in spleen, mesenteric lymph node, Peyers patch and lamina propria of the colon. Daily administration of W-061 resulted in improvement of DSS-induced colitis, and significantly reduced the number of CD4+ T cells in the colonic lamina propria. Numbers of both Th17 and Th1 cells were reduced by W-061 treatment. W-061, however, had no influence on the number of Treg cells in lamina propria. Thus, Th17 and Th1 cells in lamina propria were thought to be the key subsets in the pathogenesis of DSS-induced colitis. In conclusion, W-061 may be a novel therapeutic strategy to ameliorate acute aggravation of inflammatory bowel diseases.

Decreased miR-340 expression in bone marrow is associated with liver metastasis of colorectal cancer

Studies have shown the prognostic significance of disseminated tumor cells (DTC) in bone marrow of patients with colorectal cancer. However, the molecular characteristics of DTCs, including their miRNA expression profiles, remain mostly unknown. In this study, we analyzed the miRNA expression of DTCs in bone marrow. EpCAM+ bone marrow cells were collected using immunomagnetic beads after exclusion of CD14+ and CD45+ cells, then subjected to miRNA microarray analysis. Cluster analysis (7 colorectal cancer patients with liver metastasis and 12 colorectal cancer patients without liver metastasis) indicated that miR-340 and miR-542-3p expressions were significantly decreased in EpCAM+ bone marrow cells of patients with liver metastasis (P = 0.019 and 0.037, respectively). We demonstrated that pre-miR-340 administration inhibited growth of colon cancer cells and suppressed c-Met expression in vitro. In clinical samples of colorectal cancer, miR-340 was expressed at significantly lower levels in tumor tissues compared with normal mucosa. Survival analysis in 136 patients with colorectal cancer indicated that low miR-340 expression was correlated with shorter 5-year disease-free survival (P = 0.023) and poor 5-year overall survival (P = 0.046). It was of note that the colorectal cancer group with low miR-340 and high c-Met expression had the worst prognosis. We further demonstrated that systemic pre-miR-340 administration suppressed growth of pre-established HCT116 tumors in animal therapeutic models. These findings indicate that miR-340 may be useful as a novel prognostic factor and as a therapeutic tool against colorectal cancer. Our data suggest that miR-340 in bone marrow may play an important role in regulating the metastasis cascade of colorectal cancer. Mol Cancer Ther; 13(4); 976–85. ©2014 AACR.

Immunotherapy of murine colon cancer using receptor tyrosine kinase EphA2-derived peptide-pulsed dendritic cell vaccines

Further optimization of dendritic cell (DC)‐based vaccines is required clinically against advanced stage cancer. Given the broad range of expression levels observed in the recently defined tumor antigen EphA2 in a diverse types of cancers, especially in advanced stage or metastatic cancers, the authors evaluated the effectiveness of vaccination using DCs pulsed with EphA2‐derived peptides (Eph‐DCs) in a murine colon cancer model.

Tumour-suppressive function of SIRT4 in human colorectal cancer.

Background:SIRT4, which is localised in the mitochondria, is one of the least characterised members of the sirtuin family of nicotinamide adenine dinucleotide-dependent enzymes that play key roles in multiple cellular processes such as metabolism, stress response and longevity. There are only a few studies that have characterised its function and assessed its clinical significance in human cancers.Methods:We established colorectal cancer cell lines (SW480, HCT116, and HT29) overexpressing SIRT4 and investigated their effects on proliferation, migration and invasion, as well as E-cadherin expression, that negatively regulates tumour invasion and metastases. The associations between SIRT4 expression in colorectal cancer specimens and clinicopathological features including prognosis were assessed by immunohistochemistry.Results:SIRT4 upregulated E-cadherin expression and suppressed proliferation, migration and invasion through inhibition of glutamine metabolism in colorectal cancer cells. Moreover, SIRT4 expression in colorectal cancer decreased with the progression of invasion and metastasis, and a low expression level of SIRT4 was correlated with a worse prognosis.Conclusions:SIRT4 has a tumour-suppressive function and may serve as a novel therapeutic target in colorectal cancer.