Masahiro Tanemura

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.

Circulating microRNA-21 as a novel biomarker for hepatocellular carcinoma.

BACKGROUND & AIMS Several groups have reported the significance of circulating microRNA as a biochemical marker of cancer. To our knowledge, however, there are no reports on the significance of circulating microRNA in hepatocellular carcinoma. The aim of this study was to evaluate the significance of plasma microRNA-21 level as a biochemical marker for hepatocellular carcinoma. METHODS Plasma microRNA-21 level was measured by qRT-PCR in 10 patients before and after curative resection of hepatocellular carcinoma. Plasma microRNA-21 was also compared in other groups of: 126 patients with hepatocellular carcinoma, 30 patients with chronic hepatitis, and 50 healthy volunteers. The power of microRNA-21 in differentiating hepatocellular carcinoma from chronic hepatitis or from healthy volunteers was compared to that of α-fetoprotein. RESULTS In the 10-patient group, plasma microRNA-21 levels significantly diminished after surgery compared with the pre-operative values (p=0.0125). Plasma microRNA-21 level in the 126 patients with hepatocellular carcinoma was significantly higher than in patients with chronic hepatitis and healthy volunteers (p<0.0001, p<0.0001, respectively). ROC analysis of plasma microRNA-21 yielded an AUC of 0.773 with 61.1% sensitivity and 83.3% specificity when differentiating hepatocellular carcinoma from chronic hepatitis, and an AUC of 0.953 with 87.3% sensitivity and 92.0% specificity when differentiating hepatocellular carcinoma from healthy volunteers. Both sets of values were superior to α-fetoprotein and improved for the combination of microRNA-21 and α-fetoprotein. CONCLUSIONS Plasma microRNA-21 level is a promising biochemical marker for hepatocellular carcinoma.

MicroRNA-21 induces resistance to the anti-tumour effect of interferon-α/5-fluorouracil in hepatocellular carcinoma cells

Background:We reported recently the clinical efficiency of interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy in advanced hepatocellular carcinoma (HCC). However, prediction of the response to the combination therapy remains unsatisfactory. The aim of this study was to investigate the anti-tumour effects of microRNA (miR)-21 on the sensitivity of HCC cells to IFN-α/5-FU and whether miR-21 can be used as a predictor of the response to such therapy in HCC.Methods:Changes in the sensitivity of HCC cells (PLC/PRF/5 and HepG2) to IFN-α/5-FU were examined after transfection with pre-miR-21 or anti-miR-21. The correlation between miR-21 expression level, evaluated by qRT–PCR, and response to the therapy was also investigated in clinical HCC specimens.Results:Hepatocellular carcinoma cells transfected with pre-miR-21 were significantly resistant to IFN-α/5-FU. Annexin V assay showed that the percentage of apoptotic cells was significantly lower in cells transfected with pre-miR-21 than control cells. Transfection of anti-miR-21 rendered HCC cells sensitive to IFN-α/5-FU, and such sensitivity was weakened by transfection of siRNAs of target molecules, PETN and PDCD4. miR-21 expression in clinical HCC specimens was significantly associated with the clinical response to the IFN-α/5-FU combination therapy and survival rate.Conclusions:The miR-21 in HCC cell lines and clinical HCC samples is a significant modulator of the anti-tumour effect of IFN-α and 5-FU. This suggests that miR-21 is a potentially suitable marker for the prediction of the clinical response to the IFN-α/5-FU combination therapy.

Adipose-derived mesenchymal stem cells and regenerative medicine

Adipose tissue‐derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β‐cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow‐derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.

Differential expression of alpha-GAL epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) on pig and mouse organs.

BACKGROUND Expression of the alpha-gal epitope in mice can be completely eliminated by disruption of the alpha1,3 galactosyltransferase gene. As an initial step for assessing the feasibility of this approach in the pig, it was of interest to compare the expression of alpha-gal epitopes in pig and mouse organs. METHODS Membranes from pig and mouse organ homogenates were analyzed for alpha-gal epitope expression by Western blots, enzyme-linked immunosorbent assay (ELISA), immunostaining of tissues, and ELISA inhibition assay. RESULTS Immunostaining of Western blots with human anti-Gal detected alpha-gal epitopes on glycoproteins from pig organs but not on glycoproteins from the corresponding mouse organs. ELISA with membrane homogenates and immunostaining of tissue sections demonstrated a much higher binding of human anti-Gal to alpha-gal epitopes on pig membranes than on mouse membranes. ELISA inhibition assay with monoclonal anti-Gal indicated that alpha-gal epitope expression in pig organs is up to 500-fold higher than in mouse organs. CONCLUSION Expression of alpha-gal epitopes in pig organs is many fold higher than in mouse organs. The abundance of these epitopes in pigs raises the question of whether pigs can properly develop without expression of alpha-gal epitopes.

miR-146a suppresses the sensitivity to interferon-α in hepatocellular carcinoma cells

BACKGROUND Interferon-based (IFN-based) therapy is effective in the treatment of advanced hepatocellular carcinoma (HCC). However, the issue of resistance to this therapy remains to be solved. The aim of this study was to identify microRNAs (miRNAs) that govern the sensitivity to IFN-α in HCC cells. METHODS miRNA microarray analysis using IFN-α-resistant clones of PLC/PRF/5 (PLC-Rs) and their parental cells (PLC-P) was conducted. Changes in the anti-cancer effects of IFN-α were studied after gain-of-function and loss-of-function of the candidate miRNA. RESULTS miR-146a expression was significantly higher in PLC-Rs than in PLC-P. miR-146a decreased the sensitivity to IFN-α through the suppression of apoptosis. Further experiments showed that miR-146a-related resistance to IFN-α was mediated through SMAD4. CONCLUSIONS The results indicated that miR-146a regulated the sensitivity of HCC cells to the cytotoxic effects of IFN-α through SMAD4, suggesting that this miRNA could be suitable for prediction of the clinical response and potential therapeutic target in HCC patients on IFN-based therapy.

Rapamycin Causes Upregulation of Autophagy and Impairs Islets Function Both In Vitro and In Vivo

Autophagy is a lysosomal degradation process of redundant or faulty cell components in normal cells. However, certain diseases are associated with dysfunctional autophagy. Rapamycin, a major immunosuppressant used in islet transplantation, is an inhibitor of mammalian target of rapamycin and is known to cause induction of autophagy. The objective of this study was to evaluate the in vitro and in vivo effects of rapamycin on pancreatic β cells. Rapamycin induced upregulation of autophagy in both cultured isolated islets and pancreatic β cells of green fluorescent protein–microtubule‐associated protein 1 light chain 3 transgenic mice. Rapamycin reduced the viability of isolated β cells and down‐regulated their insulin function, both in vitro and in vivo. In addition, rapamycin increased the percentages of apoptotic β cells and dead cells in both isolated and in vivo intact islets. Treatment with 3‐methyladenine, an inhibitor of autophagy, abrogated the effects of rapamycin and restored β‐cell function in both in vitro experiments and animal experiments. We conclude that rapamycin‐induced islet dysfunction is mediated through upregulation of autophagy, with associated downregulation of insulin production and apoptosis of β cells. The results also showed that the use of an autophagy inhibitor abrogated these effects and promoted islet function and survival. The study findings suggest that targeting the autophagy pathway could be beneficial in promoting islet graft survival after transplantation.

Reduction of the Major Swine Xenoantigen, the α-Galactosyl Epitope by Transfection of the α2,3-Sialyltransferase Gene

α2,3-Sialyltransferase represents a putative enzyme that reduces the Galα1-3Gal β1-4GlcNAc-R (the α-galactosyl epitope) by intracellular competition with α1,3-galactosyltransferase for a common acceptor substrate. This study demonstrates that the overexpression of the α2,3-sialyltransferase gene suppresses the antigenicity of swine endothelial cells to human natural antibodies by 77% relative to control cells and by 30% relative to cells transfected with α1,2-fucosyltransferase, and in addition, it reduces the complement-mediated cell lysis by 75% compared with control cells and by 22% compared with cells transfected with α1,2-fucosyltransferase. The mechanism by which the α-galactosyl epitope was reduced was also studied. Suppression of α1,3-galactosyltransferase activity by 30–63% was observed in the transfectants with α2,3-sialyltransferase, and mRNA expression of the α1,3-galactosyltransferase gene was reduced as well. The data suggest that the α2,3-sialyltransferase effectively reduced the α-galactosyl epitope as well as or better than the α1,2-fucosyltransferase did and that the reduction of the α-galactosyl epitope is due not only to substrate competition but also to an overall reduction of endogenous α1,3-galactosyltransferase enzyme activity.

Combined transplantation of pancreatic islets and adipose tissue-derived stem cells enhances the survival and insulin function of islet grafts in diabetic mice.

Background. Overcoming significant loss of transplanted islet mass is important for successful islet transplantation. Adipose tissue-derived stem cells (ADSCs) seem to have angiogenic potential and antiinflammatory properties. We hypothesized that the inclusion of ADSCs with islet transplantation should enhance the survival and insulin function of the islet graft. Methods. Syngeneic ADSCs and allogeneic islets were transplanted simultaneously under the kidney capsules of diabetic C57BL/6J mice. Rejection of the graft was examined by measurement of blood glucose level. Revascularization and inflammatory cell infiltration were examined by immunohistochemistry. Results. Transplantation of 400 islets only achieved normoglycemia with graft survival of 13.6±1.67 days (mean±standard deviation), whereas that of 100 or 200 allogeneic islets never reversed diabetes. Transplantation of 200 islets with 2×105 ADSCs reversed diabetes and significantly prolonged graft survival (13.0±5.48 days). Results of glucose tolerance tests performed on day 7 were significantly better in islets-ADSCs than islets-alone recipients. Immunohistochemical analysis confirmed the presence of insulin-stained islet grafts with well-preserved structure in islets-ADSCs transplant group. Significant revascularization (larger number of von Willebrand factor-positive cells) and marked inhibition of inflammatory cell infiltration, including CD4+ and CD8+ T cells and macrophages, were noted in the islets-ADSCs transplant group than islets-alone transplant group. Conclusions. Our results indicated that cotransplantation of ADSCs with islet graft promoted survival and insulin function of the graft and reduced the islet mass required for reversal of diabetes. This innovative protocol may allow “one donor to one recipient” islet transplantation.

Mural nodule in branch duct–type intraductal papillary mucinous neoplasms of the pancreas is a marker of malignant transformation and indication for surgery

BACKGROUND The management of branch duct-type intraductal papillary mucinous neoplasms (IPMNs) remains controversial. This study aimed to elucidate the preoperative clinical factors that identify high-risk malignant transformation in branch duct-type IPMN. METHODS We retrospectively evaluated 38 patients diagnosed with branch duct-type IPMN who underwent pancreatectomy, identifying different preoperative factors between adenoma (intraductal papillary mucinous adenoma [IPMA]) and carcinoma (intraductal papillary mucinous carcinoma [IPMC]). RESULTS Twelve patients were diagnosed with IPMC. The mean tumor size was 31.9 ± 11.8 mm for IPMA and 35.7 ± 17.1 mm for IPMC (P = .467). No significant differences were found between IPMA and IPMC patients with regard to age, sex, symptoms, and tumor number. The mean diameter of the main pancreatic duct was significantly larger in IPMCs (8.3 ± 5.9 mm) compared with IPMAs (4.7 ± 2.3 mm; P = .011). The mural nodule was a good predictor of malignancy (P = .0002) and was identified as the only independent and significant marker of IPMC in multivariate analysis. CONCLUSIONS The presence of mural nodules is a potentially suitable marker for differentiating IPMC from IPMA, and is important for making decisions about surgical interventions.