Publications by authors named "Katsuhisa Horimoto"

59 Publications

Application of multiple omics and network projection analyses to drug repositioning for pathogenic mosquito-borne viruses.

Sci Rep 2021 May 12;11(1):10136. Epub 2021 May 12.

Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, 135-0064, Japan.

Pathogenic mosquito-borne viruses are a serious public health issue in tropical and subtropical regions and are increasingly becoming a problem in other climate zones. Drug repositioning is a rapid, pharmaco-economic approach that can be used to identify compounds that target these neglected tropical diseases. We have applied a computational drug repositioning method to five mosquito-borne viral infections: dengue virus (DENV), zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and Chikungunya virus (CHIV). We identified signature molecules and pathways for each virus infection based on omics analyses, and determined 77 drug candidates and 146 proteins for those diseases by using a filtering method. Based on the omics analyses, we analyzed the relationship among drugs, target proteins and the five viruses by projecting the signature molecules onto a human protein-protein interaction network. We have classified the drug candidates according to the degree of target proteins in the protein-protein interaction network for the five infectious diseases.
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http://dx.doi.org/10.1038/s41598-021-89171-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8115341PMC
May 2021

Author Correction: "Dysfunctions" induced by Roux-en-Y gastric bypass surgery are concomitant with metabolic improvement independent of weight loss.

Cell Discov 2020 Apr 28;6(1):25. Epub 2020 Apr 28.

Key Laboratory of Systems Biology, Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy Sciences, Shanghai, 200031, China.

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http://dx.doi.org/10.1038/s41421-020-0163-1DOI Listing
April 2020

Medical database analysis of japanese multiple myeloma patients with planned stem cell transplantation (MEDALIST) - a focus on healthcare resource utilization and cost.

Int J Hematol 2021 Feb 15;113(2):271-278. Epub 2020 Oct 15.

Department of Hematology, Faculty of Medicine, Hokkaido University, Sapporo, Japan.

This study explored the burden associated with stem cell mobilization, with or without cyclophosphamide (CPA), in patients who intended to receive autologous stem cell transplantation (ASCT) for multiple myeloma (MM). A Japanese health care claims database (MDV) was used to analyze the health care resource utilization patterns and medical cost between 2013 and 2016 (pre-plerixafor launch). The patients were further categorized into groups who received granulocyte-colony stimulating factor (G-CSF) alone or G-CSF + CPA group and analyzed in both mobilization and ASCT phases of treatment. Overall, there were more MM patients who were treated with G-CSF + CPA combination therapy than G-CSF alone. Length-of-stay was 1.6 times longer in the combination group during the mobilization phase. A reverse trend was observed during the ASCT phase. Direct cost was approximately 1.2 million yen during the mobilization phase and 2.3 million yen during the ASCT phase, with hospitalization basic fee accounting for the highest proportion in both groups and phases. A substantial amount of healthcare resource and cost was consumed in both phases. This study may serve as a basic reference for further health technology assessment of new medicines such as plerixafor. Further investigation of differences between treatment groups is warranted.
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http://dx.doi.org/10.1007/s12185-020-03022-5DOI Listing
February 2021

Potential use of lenvatinib for patients with unresectable hepatocellular carcinoma including after treatment with sorafenib: Real-world evidence and assessment via protein phosphorylation array.

Oncotarget 2020 Jun 30;11(26):2531-2542. Epub 2020 Jun 30.

Department of Gastroenterology and Oncology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.

The efficacy and safety of lenvatinib (LEN) as a second/third-line treatment for unresectable hepatocellular carcinoma (HCC) after sorafenib (SOR) therapy remains unknown. We evaluated the outcomes of second/third-line LEN treatment, investigated the sensitivity of a SOR-resistant HCC cell line (PLC/PRF5-R2) to LEN, and assessed their signal transduction pathways by protein array analysis. We retrospectively enrolled 57 patients with unresectable HCC. Fifty-three radiologically evaluated patients comprised 34 molecular-targeted agent (MTA)-naive (first-line), nine intolerant to SOR (second-line), and 10 resistant to regorafenib (third-line). The objective response rates (ORRs) were 61.8% in first-line, 33.3% in second-line, and 20.0% in third-line groups. The overall survival (OS) in the first-line was significantly longer than that in the third-line group ( < 0.05). Patients with better liver functional reserves (child score, ALBI grade) exhibited higher ORR and longer OS. The IC of LEN against PLC/PRF5-R2 was significantly higher than that against PLC/PRF5. LEN significantly inhibited more LEN-related signal transduction pathways in PLC/PRF5 than in PLC/PRF5-R2 cells. This suggests that LEN is active and safe as a second/third-line treatment for unresectable HCC. LEN seems more effective for patients with HCC with better hepatic reserve functions or before MTA-resistance is acquired because of the partial cross-resistance to SOR.
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http://dx.doi.org/10.18632/oncotarget.27640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335665PMC
June 2020

Erratum: Author Correction: "Dysfunctions" induced by Roux-en-Y gastric bypass surgery are concomitant with metabolic improvement independent of weight loss.

Cell Discov 2020 28;6:25. Epub 2020 Apr 28.

1Key Laboratory of Systems Biology, Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy Sciences, Shanghai, 200031 China.

[This corrects the article DOI: 10.1038/s41421-019-0138-2.].
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http://dx.doi.org/10.1038/s41421-020-0163-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7186218PMC
April 2020

"Dysfunctions" induced by Roux-en-Y gastric bypass surgery are concomitant with metabolic improvement independent of weight loss.

Cell Discov 2020 28;6. Epub 2020 Jan 28.

1Key Laboratory of Systems Biology, Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy Sciences, Shanghai, 200031 China.

Metabolic surgery has been increasingly recommended for obese diabetic patients, but questions remain as to its molecular mechanism that leads to improved metabolic parameters independently of weight loss from a network viewpoint. We evaluated the role of the Roux limb (RL) in Roux-en-Y gastric bypass (RYGB) surgery in nonobese diabetic rat models. Improvements in metabolic parameters were greater in the long-RL RYGB group. Transcriptome profiles reveal that amelioration of diabetes state following RYGB differs remarkably from both normal and diabetic states. According to functional analysis, RYGB surgery significantly affected a major gene group, i.e., the newly changed group, which represented diabetes-irrelevant genes abnormally expressed after RYGB. We hypothesize that novel "dysfunctions" carried by this newly changed gene group induced by RYGB rebalance diabetic states and contribute to amelioration of metabolic parameters. An unusual increase in cholesterol (CHOL) biosynthesis in RL enriched by the newly changed group was concomitant with ameliorated metabolic parameters, as demonstrated by measurements of physiological parameters and biodistribution analysis using [C]-labeled glucose. Our findings demonstrate RYGB-induced "dysfunctions" in the newly changed group as a compensatory role contributes to amelioration of diabetes. Rather than attempting to normalize "abnormal" molecules, we suggest a new disease treatment strategy of turning "normal" molecules "abnormal" in order to achieve a new "normal" physiological balance. It further implies a novel strategy for drug discovery, i.e. targeting also on "normal" molecules, which are traditionally ignored in pharmaceutical development.
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http://dx.doi.org/10.1038/s41421-019-0138-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985254PMC
January 2020

IGF2 Autocrine-Mediated IGF1R Activation Is a Clinically Relevant Mechanism of Osimertinib Resistance in Lung Cancer.

Mol Cancer Res 2020 04 15;18(4):549-559. Epub 2020 Jan 15.

Division of Pulmonary Medicine, Department of Medicine, Keio University, School of Medicine, Tokyo, Japan.

-mutated lung cancer accounts for a significant proportion of lung cancer cases worldwide. For these cases, osimertinib, a third-generation EGFR tyrosine kinase inhibitor, is extensively used as a first-line or second-line treatment. However, lung cancer cells acquire resistance to osimertinib in 1 to 2 years. Thus, a thorough clarification of resistance mechanisms to osimertinib is highly anticipated. Recent next-generation sequencing (NGS) of lung cancer samples identified several genetically defined resistance mechanisms to osimertinib, such as C797S or amplification. However, nongenetically defined mechanisms are not well evaluated. For a thorough clarification of osimertinib resistance, both genetic and nongenetic mechanisms are essential. By using our comprehensive protein phosphorylation array, we detected IGF1R bypass pathway activation after EGFR abolishment. Both of our established lung cancer cells and patient-derived lung cancer cells demonstrated IGF2 autocrine-mediated IGF1R pathway activation as a mechanism of osimertinib resistance. Notably, this resistance mechanism was not detected by a previously performed NGS, highlighting the essential roles of living cancer cells for a thorough clarification of resistance mechanisms. Interestingly, the immunohistochemical analysis confirmed the increased IGF2 expression in lung cancer patients who were treated with osimertinib and met the established clinical definition of acquired resistance. The findings highlight the crucial roles of cell-autonomous ligand expression in osimertinib resistance. Here, we report for the first time the IGF2 autocrine-mediated IGF1R activation as a nongenetic mechanism of osimertinib resistance in lung cancer at a clinically relevant level. IMPLICATIONS: Using comprehensive protein phosphorylation array and patient-derived lung cancer cells, we found that IGF2 autocrine-mediated IGF1R pathway activation is a clinically relevant and common mechanism of acquired resistance to osimertinib.
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http://dx.doi.org/10.1158/1541-7786.MCR-19-0956DOI Listing
April 2020

CD4 T-cell Immunity in the Peripheral Blood Correlates with Response to Anti-PD-1 Therapy.

Cancer Immunol Res 2020 03 23;8(3):334-344. Epub 2019 Dec 23.

Division of Respiratory Medicine, Saitama Medical University International Medical Center, Hidaka, Saitama, Japan.

Accumulating evidence indicates that CD8 T cells in the tumor microenvironment and systemic CD4 T-cell immunity play an important role in mediating durable antitumor responses. We longitudinally examined T-cell immunity in the peripheral blood of patients with non-small lung cancer and found that responders had significantly ( < 0.0001) higher percentages of effector, CD62L CD4 T cells prior to PD-1 blockade. Conversely, the percentage of CD25FOXP3 CD4 T cells was significantly ( = 0.034) higher in nonresponders. We developed a formula, which demonstrated 85.7% sensitivity and 100% specificity, based on the percentages of CD62L CD4 T cells and CD25FOXP3 cells to predict nonresponders. Mass cytometry analysis revealed that the CD62L CD4 T-cell subset expressed T-bet, CD27, FOXP3, and CXCR3, indicative of a Th1 subpopulation. CD62L CD4 T cells significantly correlated with effector CD8 T cells ( = 0.0091) and with PD-1 expression on effector CD8 T cells ( = 0.0015). Gene expression analysis revealed that , and were preferentially expressed in CD62L CD4 T cells derived from responders. Notably, long-term responders, who had >500-day progression-free survival, showed significantly higher numbers of CD62L CD4 T cells prior to PD-1 blockade therapy. Decreased CD62L CD4 T-cell percentages after therapy resulted in acquired resistance, with long-term survivors maintaining high CD62L CD4 T-cell percentages. These results pave the way for new treatment strategies for patients by monitoring CD4 T-cell immune statuses in their peripheral blood.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0574DOI Listing
March 2020

Promotion of the Warburg effect is associated with poor benefit from adjuvant chemotherapy in colorectal cancer.

Cancer Sci 2020 Feb 9;111(2):658-666. Epub 2020 Jan 9.

Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan.

Metabolic reprogramming, including the Warburg effect, is a hallmark of cancer. Indeed, the diversity of cancer metabolism leads to cancer heterogeneity, but accurate assessment of metabolic properties in tumors has not yet been undertaken. Here, we performed absolute quantification of the expression levels of 113 proteins related to carbohydrate metabolism and antioxidant pathways, in stage III colorectal cancer surgical specimens from 70 patients. The Warburg effect appeared in absolute protein levels between tumor and normal mucosa specimens demonstrated. Notably, the levels of proteins associated with the tricarboxylic citric acid cycle were remarkably reduced in the malignant tumors which had relapsed after surgery and treatment with 5-fluorouracil-based adjuvant therapy. In addition, the efficacy of 5-fluorouracil also decreased in the cultured cancer cell lines with promotion of the Warburg effect. We further identified nine and eight important proteins, which are closely related to the Warburg effect, for relapse risk and 5-fluorouracil benefit, respectively, using a biomarker exploration procedure. These results provide us a clue for bridging between metabolic protein expression profiles and benefit from 5-fluorouracil adjuvant chemotherapy.
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http://dx.doi.org/10.1111/cas.14275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7004516PMC
February 2020

ToGo-WF: prediction of RNA tertiary structures and RNA-RNA/protein interactions using the KNIME workflow.

J Comput Aided Mol Des 2019 05 6;33(5):497-507. Epub 2019 Mar 6.

Molecular Profiling for Drug Discovery Research Center (molprof), National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo, 135-0064, Japan.

Recent progress in molecular biology has revealed that many non-coding RNAs regulate gene expression or catalyze biochemical reactions in tumors, viruses and several other diseases. The tertiary structure of RNA molecules and RNA-RNA/protein interaction sites are of increasing importance as potential targets for new medicines that treat a broad array of human diseases. Current RNA drugs are split into two groups: antisense RNA molecules and aptamers. In this report, we present a novel workflow to predict RNA tertiary structures and RNA-RNA/protein interactions using the KNIME environment, which enabled us to assemble a combination of RNA-related analytical tools and databases. In this study, three analytical workflows for comprehensive structural analysis of RNA are introduced: (1) prediction of the tertiary structure of RNA; (2) prediction of the structure of RNA-RNA complexes and analysis of their interactions; and (3) prediction of the structure of RNA-protein complexes and analysis of their interactions. In an RNA-protein case study, we modeled the tertiary structure of pegaptanib, an aptamer drug, and performed docking calculations of the pegaptanib-vascular endothelial growth factor complex using a fragment of the interaction site of the aptamer. We also present molecular dynamics simulations of the RNA-protein complex to evaluate the affinity of the complex by mutating bases at the interaction interface. The results provide valuable information for designing novel features of aptamer-protein complexes.
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http://dx.doi.org/10.1007/s10822-019-00195-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088279PMC
May 2019

Drug repositioning for dengue haemorrhagic fever by integrating multiple omics analyses.

Sci Rep 2019 01 24;9(1):523. Epub 2019 Jan 24.

Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, 135-0064, Japan.

To detect drug candidates for dengue haemorrhagic fever (DHF), we employed a computational drug repositioning method to perform an integrated multiple omics analysis based on transcriptomic, proteomic, and interactomic data. We identified 3,892 significant genes, 389 proteins, and 221 human proteins by transcriptomic analysis, proteomic analysis, and human-dengue virus protein-protein interactions, respectively. The drug candidates were selected using gene expression profiles for inverse drug-disease relationships compared with DHF patients and healthy controls as well as interactomic relationships between the signature proteins and chemical compounds. Integrating the results of the multiple omics analysis, we identified eight candidates for drug repositioning to treat DHF that targeted five proteins (ACTG1, CALR, ERC1, HSPA5, SYNE2) involved in human-dengue virus protein-protein interactions, and the signature proteins in the proteomic analysis mapped to significant pathways. Interestingly, five of these drug candidates, valparoic acid, sirolimus, resveratrol, vorinostat, and Y-27632, have been reported previously as effective treatments for flavivirus-induced diseases. The computational approach using multiple omics data for drug repositioning described in this study can be used effectively to identify novel drug candidates.
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http://dx.doi.org/10.1038/s41598-018-36636-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346040PMC
January 2019

Prospective exosome-focused translational research for afatinib study of non-small cell lung cancer patients expressing EGFR (EXTRA study).

Thorac Cancer 2019 02 8;10(2):395-400. Epub 2018 Dec 8.

Division of Medical Oncology, Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan.

Patients with EGFR-mutated non-small cell lung cancer (NSCLC) exhibit resistance to EGFR-tyrosine kinase inhibitors (TKIs) within 9-14 months of therapy. Recently, EGFR-mutated NSCLC has demonstrated the potential for heterogeneity; therefore, the manner of clonal heterogeneity may impact the duration of progression-free and overall survival and other parameters affecting EGFR-TKI treatment efficacy. However no predictive biomarker of these favorable treatment efficacies has been identified to date. The exosome-focused translational research for afatinib (EXTRA) study aims to identify a novel predictive biomarker and a resistance marker for afatinib by analyzing data from association studies of the clinical efficacy of afatinib and four "OMICs" (genomics, proteomics, epigenomics, and metabolomics) using peripheral blood from patients treated with afatinib. This study aims to: (i) conduct comprehensive multi-OMIC analyses in a prospective clinical trial, and (ii) focus on both sera/plasma and exosome as a source for OMIC analyses to identify a novel predictor of the efficacy of a specific drug. To eliminate the carryover bias of prior treatment, systemic treatment-naïve patients were enrolled. The candidates to be screened for biomarkers comprise a discovery cohort of 60 patients and an independent validation cohort of 40 patients. The EXTRA study is the first trial to screen novel biomarkers of longer treatment efficacy of EGFR-TKIs using four-OMICs analyses, focusing on both "naked or free" molecules and "capsulated" exosomal components in serially collected peripheral blood.
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http://dx.doi.org/10.1111/1759-7714.12923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360199PMC
February 2019

Plasma amino acid profile associated with fatty liver disease and co-occurrence of metabolic risk factors.

Sci Rep 2017 11 3;7(1):14485. Epub 2017 Nov 3.

Center for Multiphasic Health Testing and Services, Mitsui Memorial Hospital, 1 Kanda, Izumicho, Chiyoda-ku, Tokyo, 101-8643, Japan.

Fatty liver disease (FLD) increases the risk of diabetes, cardiovascular disease, and steatohepatitis, which leads to fibrosis, cirrhosis, and hepatocellular carcinoma. Thus, the early detection of FLD is necessary. We aimed to find a quantitative and feasible model for discriminating the FLD, based on plasma free amino acid (PFAA) profiles. We constructed models of the relationship between PFAA levels in 2,000 generally healthy Japanese subjects and the diagnosis of FLD by abdominal ultrasound scan by multiple logistic regression analysis with variable selection. The performance of these models for FLD discrimination was validated using an independent data set of 2,160 subjects. The generated PFAA-based model was able to identify FLD patients. The area under the receiver operating characteristic curve for the model was 0.83, which was higher than those of other existing liver function-associated markers ranging from 0.53 to 0.80. The value of the linear discriminant in the model yielded the adjusted odds ratio (with 95% confidence intervals) for a 1 standard deviation increase of 2.63 (2.14-3.25) in the multiple logistic regression analysis with known liver function-associated covariates. Interestingly, the linear discriminant values were significantly associated with the progression of FLD, and patients with nonalcoholic steatohepatitis also exhibited higher values.
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http://dx.doi.org/10.1038/s41598-017-14974-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5670226PMC
November 2017

Relationship between regulatory pattern of gene expression level and gene function.

PLoS One 2017 11;12(5):e0177430. Epub 2017 May 11.

Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.

Regulation of gene expression levels is essential for all living systems and transcription factors (TFs) are the main regulators of gene expression through their ability to repress or induce transcription. A balance between synthesis and degradation rates controls gene expression levels. To determine which rate is dominant, we analyzed the correlation between expression levels of a TF and its regulated gene based on a mathematical model. We selected about 280,000 expression patterns of 355 TFs and 647 regulated genes using DNA microarray data from the Gene Expression Omnibus (GEO) data repository. Based on our model, correlation between the expressions of TF-regulated gene pairs corresponds to tuning of the synthesis rate, whereas no correlation indicates excessive synthesis and requires tuning of the degradation rate. The gene expression relationships between TF-regulated gene pairs were classified into four types that correspond to different gene regulatory mechanisms. It was surprising that fewer than 20% of these genes were governed by the familiar regulatory mechanism, i.e., through the synthesis rate. Moreover, we performed pathway analysis and found that each classification type corresponded to distinct gene functions: cellular regulation pathways were dominant in the type with synthesis rate regulation and terms associated with diseases such as cancer, Parkinson's disease, and Alzheimer's disease were dominant in the type with degradation rate regulation. Interestingly, these diseases are caused by the accumulation of proteins. These results indicated that gene expression is regulated structurally, not arbitrarily, according to the gene function. This funding is indicative of a systematic control of transcription processes at the whole-cell level.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177430PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426767PMC
September 2017

A large-scale targeted proteomics assay resource based on an in vitro human proteome.

Nat Methods 2017 03 26;14(3):251-258. Epub 2016 Dec 26.

Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

Targeted proteomics approaches are of value for deep and accurate quantification of protein abundance. Extending such methods to quantify large numbers of proteins requires the construction of predefined targeted assays. We developed a targeted proteomics platform-in vitro proteome-assisted multiple reaction monitoring (MRM) for protein absolute quantification (iMPAQT)-by using >18,000 human recombinant proteins, thus enabling protein absolute quantification on a genome-wide scale. Our platform comprises experimentally confirmed MRM assays of mass tag (mTRAQ)-labeled peptides to allow for rapid and straightforward measurement of the absolute abundance of predefined sets of proteins by mass spectrometry. We applied iMPAQT to delineate the quantitative metabolic landscape of normal and transformed human fibroblasts. Oncogenic transformation gave rise to relatively small but global changes in metabolic pathways resulting in aerobic glycolysis (Warburg effect) and increased rates of macromolecule synthesis. iMPAQT should facilitate quantitative biology studies based on protein abundance measurements.
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http://dx.doi.org/10.1038/nmeth.4116DOI Listing
March 2017

Plasma Free Amino Acid Profiles Predict Four-Year Risk of Developing Diabetes, Metabolic Syndrome, Dyslipidemia, and Hypertension in Japanese Population.

Sci Rep 2015 Jul 9;5:11918. Epub 2015 Jul 9.

Center for Multiphasic Health Testing and Services, Mitsui Memorial Hospital, 1 Kanda, Izumicho, Chiyoda-ku, Tokyo 101-8643, Japan.

Plasma free amino acid (PFAA) profile is highlighted in its association with visceral obesity and hyperinsulinemia, and future diabetes. Indeed PFAA profiling potentially can evaluate individuals' future risks of developing lifestyle-related diseases, in addition to diabetes. However, few studies have been performed especially in Asian populations, about the optimal combination of PFAAs for evaluating health risks. We quantified PFAA levels in 3,701 Japanese subjects, and determined visceral fat area (VFA) and two-hour post-challenge insulin (Ins120 min) values in 865 and 1,160 subjects, respectively. Then, models between PFAA levels and the VFA or Ins120 min values were constructed by multiple linear regression analysis with variable selection. Finally, a cohort study of 2,984 subjects to examine capabilities of the obtained models for predicting four-year risk of developing new-onset lifestyle-related diseases was conducted. The correlation coefficients of the obtained PFAA models against VFA or Ins120 min were higher than single PFAA level. Our models work well for future risk prediction. Even after adjusting for commonly accepted multiple risk factors, these models can predict future development of diabetes, metabolic syndrome, and dyslipidemia. PFAA profiles confer independent and differing contributions to increasing the lifestyle-related disease risks in addition to the currently known factors in a general Japanese population.
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http://dx.doi.org/10.1038/srep11918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496670PMC
July 2015

Cytokinetic Failure-induced Tetraploidy Develops into Aneuploidy, Triggering Skin Aging in Phosphovimentin-deficient Mice.

J Biol Chem 2015 May 6;290(21):12984-98. Epub 2015 Apr 6.

From the Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya 464-8681, the Departments of Cellular Oncology and

Tetraploidy, a state in which cells have doubled chromosomal sets, is observed in ∼20% of solid tumors and is considered to frequently precede aneuploidy in carcinogenesis. Tetraploidy is also detected during terminal differentiation and represents a hallmark of aging. Most tetraploid cultured cells are arrested by p53 stabilization. However, the fate of tetraploid cells in vivo remains largely unknown. Here, we analyze the ability to repair wounds in the skin of phosphovimentin-deficient (VIM(SA/SA)) mice. Early into wound healing, subcutaneous fibroblasts failed to undergo cytokinesis, resulting in binucleate tetraploidy. Accordingly, the mRNA level of p21 (a p53-responsive gene) was elevated in a VIM(SA/SA)-specific manner. Disappearance of tetraploidy coincided with an increase in aneuploidy. Thereafter, senescence-related markers were significantly elevated in VIM(SA/SA) mice. Because our tetraploidy-prone mouse model also exhibited subcutaneous fat loss at the age of 14 months, another premature aging phenotype, our data suggest that following cytokinetic failure, a subset of tetraploid cells enters a new cell cycle and develops into aneuploid cells in vivo, which promote premature aging.
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http://dx.doi.org/10.1074/jbc.M114.633891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505553PMC
May 2015

Telomerase reverse transcriptase has an extratelomeric function in somatic cell reprogramming.

J Biol Chem 2014 May 14;289(22):15776-87. Epub 2014 Apr 14.

From the Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo, 160-8582.

Reactivation of the endogenous telomerase reverse transcriptase (TERT) catalytic subunit and telomere elongation occur during the reprogramming of somatic cells to induced pluripotent stem (iPS) cells. However, the role of TERT in the reprogramming process is unclear. To clarify its function, the reprogramming process was examined in TERT-KO somatic cells. To exclude the effect of telomere elongation, tail-tip fibroblasts (TTFs) from first generation TERT-KO mice were used. Although iPS cells were successfully generated from TERT-KO TTFs, the efficiency of reprogramming these cells was markedly lower than that of WT TTFs. The gene expression profiles of iPS cells induced from TERT-KO TTFs were similar to those of WT iPS cells and ES cells, and TERT-KO iPS cells formed teratomas that differentiated into all three germ layers. These data indicate that TERT plays an extratelomeric role in the reprogramming process, but its function is dispensable. However, TERT-KO iPS cells showed transient defects in growth and teratoma formation during continuous growth. In addition, TERT-KO iPS cells developed chromosome fusions that accumulated with increasing passage numbers, consistent with the fact that TERT is essential for the maintenance of genome structure and stability in iPS cells. In a rescue experiment, an enzymatically inactive mutant of TERT (D702A) had a positive effect on somatic cell reprogramming of TERT-KO TTFs, which confirmed the extratelomeric role of TERT in this process.
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http://dx.doi.org/10.1074/jbc.M113.536037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140932PMC
May 2014

Identification of master regulator candidates in conjunction with network screening and inference.

Int J Data Min Bioinform 2013 ;8(3):366-80

Computational Biology Research Centre, National Institute of Advanced Industrial Science and Technology, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.

We developed a procedure for identifying transcriptional Master Regulators (MRs) related to special biological phenomena, such as diseases, in conjunction with network screening and inference. Network screening is a system for detecting activated transcriptional regulatory networks under particular conditions, based on the estimation of graph structure consistency with the measured data. Since network screening utilises the known Transcriptional Factor (TF)-gene relationships as the experimental evidence for molecular relationships, its performance depends on the ensemble of known TF networks used for its analysis. To compensate for its restrictions, a network inference method, the path consistency algorithm, is concomitantly utilised to identify MRs. The performance is illustrated by means of the known MRs in brain tumours that were computationally inferred and experimentally verified. As a result, the present procedure worked well for identifying MRs, in comparison to the previous computational selection for experimental verification.
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http://dx.doi.org/10.1504/ijdmb.2013.056077DOI Listing
February 2014

MIDDAS-M: motif-independent de novo detection of secondary metabolite gene clusters through the integration of genome sequencing and transcriptome data.

PLoS One 2013 31;8(12):e84028. Epub 2013 Dec 31.

Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan.

Many bioactive natural products are produced as "secondary metabolites" by plants, bacteria, and fungi. During the middle of the 20th century, several secondary metabolites from fungi revolutionized the pharmaceutical industry, for example, penicillin, lovastatin, and cyclosporine. They are generally biosynthesized by enzymes encoded by clusters of coordinately regulated genes, and several motif-based methods have been developed to detect secondary metabolite biosynthetic (SMB) gene clusters using the sequence information of typical SMB core genes such as polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). However, no detection method exists for SMB gene clusters that are functional and do not include core SMB genes at present. To advance the exploration of SMB gene clusters, especially those without known core genes, we developed MIDDAS-M, a motif-independent de novodetection algorithm for SMB gene clusters. We integrated virtual gene cluster generation in an annotated genome sequence with highly sensitive scoring of the cooperative transcriptional regulation of cluster member genes. MIDDAS-M accurately predicted 38 SMB gene clusters that have been experimentally confirmed and/or predicted by other motif-based methods in 3 fungal strains. MIDDAS-M further identified a new SMB gene cluster for ustiloxin B, which was experimentally validated. Sequence analysis of the cluster genes indicated a novel mechanism for peptide biosynthesis independent of NRPS. Because it is fully computational and independent of empirical knowledge about SMB core genes, MIDDAS-M allows a large-scale, comprehensive analysis of SMB gene clusters, including those with novel biosynthetic mechanisms that do not contain any functionally characterized genes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084028PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3877130PMC
September 2014

Gaussian graphical model for identifying significantly responsive regulatory networks from time course high-throughput data.

IET Syst Biol 2013 Oct;7(5):143-52

With rapid accumulation of functional relationships between biological molecules, knowledge-based networks have been constructed and stocked in many databases. These networks provide curated and comprehensive information for functional linkages among genes and proteins, whereas their activities are highly related with specific phenotypes and conditions. To evaluate a knowledge-based network in a specific condition, the consistency between its structure and conditionally specific gene expression profiling data are an important criterion. In this study, the authors propose a Gaussian graphical model to evaluate the documented regulatory networks by the consistency between network architectures and time course gene expression profiles. They derive a dynamic Bayesian network model to evaluate gene regulatory networks in both simulated and true time course microarray data. The regulatory networks are evaluated by matching network structure with gene expression to achieve consistency measurement. To demonstrate the effectiveness of the authors method, they identify significant regulatory networks in response to the time course of circadian rhythm. The knowledge-based networks are screened and ranked by their structural consistencies with dynamic gene expression profiling.
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http://dx.doi.org/10.1049/iet-syb.2012.0062DOI Listing
October 2013

Identification of upstream regulators for prognostic expression signature genes in colorectal cancer.

BMC Syst Biol 2013 Sep 4;7:86. Epub 2013 Sep 4.

Medicinal Bioconvergence Research Center, Advanced Institutes of Convergence Technology, Suwon 443-270, South Korea.

Background: Gene expression signatures have been commonly used as diagnostic and prognostic markers for cancer subtyping. However, expression signatures frequently include many passengers, which are not directly related to cancer progression. Their upstream regulators such as transcription factors (TFs) may take a more critical role as drivers or master regulators to provide better clues on the underlying regulatory mechanisms and therapeutic applications.

Results: In order to identify prognostic master regulators, we took the known 85 prognostic signature genes for colorectal cancer and inferred their upstream TFs. To this end, a global transcriptional regulatory network was constructed with total >200,000 TF-target links using the ARACNE algorithm. We selected the top 10 TFs as candidate master regulators to show the highest coverage of the signature genes among the total 846 TF-target sub-networks or regulons. The selected TFs showed a comparable or slightly better prognostic performance than the original 85 signature genes in spite of greatly reduced number of marker genes from 85 to 10. Notably, these TFs were selected solely from inferred regulatory links using gene expression profiles and included many TFs regulating tumorigenic processes such as proliferation, metastasis, and differentiation.

Conclusions: Our network approach leads to the identification of the upstream transcription factors for prognostic signature genes to provide leads to their regulatory mechanisms. We demonstrate that our approach could identify upstream biomarkers for a given set of signature genes with markedly smaller size and comparable performances. The utility of our method may be expandable to other types of signatures such as diagnosis and drug response.
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http://dx.doi.org/10.1186/1752-0509-7-86DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3847874PMC
September 2013

Association of Rev-erbα in adipose tissues with Type 2 diabetes mellitus amelioration after gastric bypass surgery in Goto-Kakizaki rats.

Am J Physiol Regul Integr Comp Physiol 2013 Jul 1;305(2):R134-46. Epub 2013 May 1.

Clinical Medical College of Yangzhou University, Yangzhou, Jiangsu, China.

We estimated the key molecules related to Type 2 diabetes mellitus (T2DM) in adipose, liver, and muscle tissues, from nonobese diabetic Goto-Kakizaki (GK) rats and their Wistar controls, by computationally analyzing the expression profiles in open source data. With the aid of information from previous reports, Rev-erbα in adipose tissue emerged as one of the most plausible candidates. Here, in animal models, including GK rats surgically treated to ameliorate T2DM, we examined the association of Rev-erbα in adipose tissue with T2DM progression. After analyses of the Rev-erbα mRNA expression in the adipose tissue of our animal models, we compared the Rev-erbα protein expression levels in the adipose, liver, and muscle tissues of GK and Wistar controls at the ages of 1 mo (M), 3M, and 6M. The Rev-erbα protein levels in adipose tissue showed a distinctive pattern, with the negative correlation of an increasing trend in GK rats, and a decreasing trend in Wistar rats during aging, from those in liver and muscle tissues. Moreover, dysregulation of the circadian Rev-erbα expression in the adipose tissue of 6-mo-old GK rats was also observed. In particular, we ameliorated T2DM in GK rats by gastric bypass surgery, and revealed that T2DM amelioration in diabetic GK rats was associated with improved circadian Rev-erbα expression, in a comparison between the surgically treated and untreated GK rats. The roles of Rev-erbα in adipose tissue were further investigated by observations of Rev-erbα-related molecules, with reference to previous reports.
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http://dx.doi.org/10.1152/ajpregu.00520.2012DOI Listing
July 2013

Identification of drug candidate against prostate cancer from the aspect of somatic cell reprogramming.

Cancer Sci 2013 Aug 26;104(8):1017-26. Epub 2013 May 26.

Department of Urology, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo, Japan.

Considering the similarities between the transcriptional programming involved in cancer progression and somatic cell reprogramming, we tried to identify drugs that would be effective against malignant cancers. We used the early transposon Oct4 and Sox2 enhancer (EOS) system to select human prostate cancer (PCA) cells expressing high levels of OCT4. Patients with metastatic castration-resistant PCA that does not respond to treatment with docetaxel have few therapeutic options. The OCT4-expressing PCA cells selected using the EOS system showed increased tumorigenicity and high resistance to docetaxel, both in vitro and in vivo. By using their gene expression data, expression signature-based prediction for compound candidates identified an antiviral drug, ribavirin, as a conversion modulator from drug resistance to sensitivity. Treatment of PCA cells with ribavirin decreased their resistance against treatment with docetaxel. This indicated that ribavirin reversed the gene expression, including that of humoral factors, in the OCT4-expressing PCA cells selected using the EOS system. Thereby, ribavirin increased the efficacy of docetaxel for cancer cells. We propose a novel cell reprogramming approach, named drug efficacy reprogramming, as a new model for identifying candidate antitumor drugs.
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http://dx.doi.org/10.1111/cas.12183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657195PMC
August 2013

Gene expression profiling of hepatitis B- and hepatitis C-related hepatocellular carcinoma using graphical Gaussian modeling.

Genomics 2013 Apr 26;101(4):238-48. Epub 2013 Feb 26.

Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, Kanazawa, Takara-Machi 13-1, Kanazawa 920-8641, Japan.

Background & Aims: Gene expression profiling of hepatocellular carcinoma (HCC) and background liver has been studied extensively; however, the relationship between the gene expression profiles of different lesions has not been assessed.

Methods: We examined the expression profiles of 34 HCC specimens (17 hepatitis B virus [HBV]-related and 17 hepatitis C virus [HCV]-related) and 71 non-tumor liver specimens (36 chronic hepatitis B [CH-B] and 35 chronic hepatitis C [CH-C]) using an in-house cDNA microarray consisting of liver-predominant genes. Graphical Gaussian modeling (GGM) was applied to elucidate the interactions of gene clusters among the HCC and non-tumor lesions.

Results: In CH-B-related HCC, the expression of vascular endothelial growth factor-family signaling and regulation of T cell differentiation, apoptosis, and survival, as well as development-related genes was up-regulated. In CH-C-related HCC, the expression of ectodermal development and cell proliferation, wnt receptor signaling, cell adhesion, and defense response genes was also up-regulated. Many of the metabolism-related genes were down-regulated in both CH-B- and CH-C-related HCC. GGM analysis of the HCC and non-tumor lesions revealed that DNA damage response genes were associated with AP1 signaling in non-tumor lesions, which mediates the expression of many genes in CH-B-related HCC. In contrast, signal transducer and activator of transcription 1 and phosphatase and tensin homolog were associated with early growth response protein 1 signaling in non-tumor lesions, which potentially promotes angiogenesis, fibrogenesis, and tumorigenesis in CH-C-related HCC.

Conclusions: Gene expression profiling of HCC and non-tumor lesions revealed the predisposing changes of gene expression in HCC. This approach has potential for the early diagnosis and possible prevention of HCC.
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http://dx.doi.org/10.1016/j.ygeno.2013.02.007DOI Listing
April 2013

Induction of pluripotent stem cells from primordial germ cells by single reprogramming factors.

Stem Cells 2013 Mar;31(3):479-87

Department of Cell Differentiation, The Sakaguchi Laboratory and , Keio University, Tokyo, Japan.

Germ cells are similar to pluripotent stem cells in terms of gene expression patterns and the capacity to convert to pluripotent stem cells in culture. The factors involved in germ cell development are also able to reprogram somatic cells. This suggests that germ cells are useful tools for investigating the mechanisms responsible for somatic cell reprograming. In this study, the expression of reprograming factors in primordial germ cells (PGCs) was analyzed. PGCs expressed Oct3/4, Sox2, and c-Myc but not Klf4. However, Klf2, Klf5, Essrb, or Essrg, which were expressed in PGCs, could compensate for Klf4 during somatic cell reprograming. Furthermore, PGCs could be converted to a pluripotent state by infection with any of the known reprogramming factors (Oct3/4, Sox2, Klf4, and c-Myc). These cells were designated as multipotent PGCs (mPGCs). Contrary to differences in the origins of somatic cells in somatic cell reprogramming, we hypothesized that the gene expression levels of the reprogramming factors would vary in mPGCs. Candidate genes involved in the regulation of tumorigenicity and/or reprogramming efficiency were identified by comparing the gene expression profiles of mPGCs generated by the exogenous expression of c-Myc or L-Myc.
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http://dx.doi.org/10.1002/stem.1303DOI Listing
March 2013

Network completion using dynamic programming and least-squares fitting.

ScientificWorldJournal 2012 1;2012:957620. Epub 2012 Nov 1.

Bioinformatics Center, Institute for Chemical Research, Kyoto University Gokasho, Uji, Kyoto 611-0011, Japan.

We consider the problem of network completion, which is to make the minimum amount of modifications to a given network so that the resulting network is most consistent with the observed data. We employ here a certain type of differential equations as gene regulation rules in a genetic network, gene expression time series data as observed data, and deletions and additions of edges as basic modification operations. In addition, we assume that the numbers of deleted and added edges are specified. For this problem, we present a novel method using dynamic programming and least-squares fitting and show that it outputs a network with the minimum sum squared error in polynomial time if the maximum indegree of the network is bounded by a constant. We also perform computational experiments using both artificially generated and real gene expression time series data.
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http://dx.doi.org/10.1100/2012/957620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504398PMC
June 2013

A computational procedure for identifying master regulator candidates: a case study on diabetes progression in Goto-Kakizaki rats.

BMC Syst Biol 2012 16;6 Suppl 1:S2. Epub 2012 Jul 16.

School of Life Sciences, University of Science and Technology of China, Hefei, China.

Background: We have recently identified a number of active regulatory networks involved in diabetes progression in Goto-Kakizaki (GK) rats by network screening. The networks were quite consistent with the previous knowledge of the regulatory relationships between transcription factors (TFs) and their regulated genes. To study the underlying molecular mechanisms directly related to phenotype changes, such as diseases, we also previously developed a computational procedure for identifying transcriptional master regulators (MRs) in conjunction with network screening and network inference, by effectively perturbing the phenotype states.

Results: In this work, we further improved our previous method for identifying MR candidates, by listing them in a more reliable manner, and applied the method to reveal the MR candidates for diabetes progression in GK rats from the active networks. Specifically, the active TF-gene pairs for different time periods in GK rats were first extracted from the networks by network screening. Another set of active TF-gene pairs was selected by network inference, by considering the gene expression signatures for those periods between GK and Wistar-Kyoto (WKY) rats. The TF-gene pairs extracted by the two methods were then further selected, from the viewpoints of the emergence specificity of TF in GK rats and the regulated-gene coverage of TF in the expression signature. Finally, we narrowed all of the genes down to only 5 TFs (Etv4, Fus, Nr2f1, Sp2, and Tcfap2b) as the candidates of MRs, with 54 regulated genes, by merging the selected TF-gene pairs.

Conclusions: The present method has successfully identified biologically plausible MR candidates, including the TFs related to diabetes in previous reports. Although the experimental verifications of the candidates and the present procedure are beyond the scope of this study, we narrowed down the candidates to 5 TFs, which can be used to perform the verification experiments relatively easily. The numerical results showed that our computational method is an efficient way to detect the key molecules responsible for biological phenomena.
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http://dx.doi.org/10.1186/1752-0509-6-S1-S2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3403593PMC
April 2013

Optimal ratio of transcription factors for somatic cell reprogramming.

J Biol Chem 2012 Oct 6;287(43):36273-82. Epub 2012 Sep 6.

Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582, Japan.

Somatic cell reprogramming is achieved by four reprogramming transcription factors (RTFs), Oct3/4, Sox2, Klf4, and c-Myc. However, in addition to the induction of pluripotent cells, these RTFs also generate pseudo-pluripotent cells, which do not show Nanog promoter activity. Therefore, it should be possible to fine-tune the RTFs to produce only fully pluripotent cells. For this study, a tagging system was developed to sort induced pluripotent stem (iPS) cells according to the expression levels of each of the four RTFs. Using this system, the most effective ratio (Oct3/4-high, Sox2-low, Klf4-high, c-Myc-high) of the RTFs was 88 times more efficient at producing iPS cells than the worst effective ratio (Oct3/4-low, Sox2-high, Klf4-low, c-Myc-low). Among the various RTF combinations, Oct3/4-high and Sox2-low produced the most efficient results. To investigate the molecular basis, microarray analysis was performed on iPS cells generated under high (Oct3/4-high and Sox2-low) and low (Oct3/4-low and Sox2-high) efficiency reprogramming conditions. Pathway analysis revealed that the G protein-coupled receptor (GPCR) pathway was up-regulated significantly under the high efficiency condition and treatment with the chemokine, C-C motif ligand 2, a member of the GPCR family, enhanced somatic cell reprogramming 12.3 times. Furthermore, data from the analysis of the signature gene expression profiles of mouse embryonic fibroblasts at 2 days after RTF infection revealed that the genetic modifier, Whsc1l1 (variant 1), also improved the efficiency of somatic cell reprogramming. Finally, comparison of the overall gene expression profiles between the high and low efficiency conditions will provide novel insights into mechanisms underlying somatic cell reprogramming.
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http://dx.doi.org/10.1074/jbc.M112.380683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3476294PMC
October 2012

Tracing the conversion process from primordial germ cells to pluripotent stem cells in mice.

Biol Reprod 2012 Jun 14;86(6):182. Epub 2012 Jun 14.

Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo, Japan.

To understand mechanisms underlying acquisition of pluripotency, it is critical to identify cells that can be converted to pluripotent stem cells. For this purpose, we focused on unipotent primordial germ cells (PGCs), which can be reprogrammed into pluripotent embryonic germ (EG) cells under defined conditions. Treatment of PGCs with combinations of signaling inhibitors, including inhibitors of MAP2K (MEK), GSK3B (GSK-3beta), and TGFB (TGFbeta) type 1 receptors, induced cells to enter a pluripotent state at a high frequency (12.1%) by Day 10 of culture. When we employed fluorescence-activated cell sorting to monitor conversion of candidate cells to a pluripotent state, we observed a cell cycle shift to S phase, indicating enrichment of pluripotent cells, during the early phase of EG formation. Transcriptome analysis revealed that PGCs retained expression of some pluripotent stem cell-associated genes, such as Pou5f1 and Sox2, during EG cell formation. On the other hand, PGCs lost their germ lineage characteristics and acquired expression of pluripotent stem cell markers, such as Klf4 and Eras. The overall gene expression profiles revealed by this system provide novel insight into how pluripotency is acquired in germ-committed cells.
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http://dx.doi.org/10.1095/biolreprod.111.096792DOI Listing
June 2012