Yoriko Takahashi

The carbohydrate sequence markup language (CabosML): an XML description of carbohydrate structures

UNLABELLED Bioinformatics resources for glycomics are very poor as compared with those for genomics and proteomics. The complexity of carbohydrate sequences makes it difficult to define a common language to represent them, and the development of bioinformatics tools for glycomics has not progressed. In this study, we developed a carbohydrate sequence markup language (CabosML), an XML description of carbohydrate structures. AVAILABILITY The language definition (XML Schema) and an experimental database of carbohydrate structures using an XML database management system are available at http://www.phoenix.hydra.mki.co.jp/CabosDemo.html CONTACT kikuchi@hydra.mki.co.jp.

Defining Hypo-Methylated Regions of Stem Cell-Specific Promoters in Human iPS Cells Derived from Extra-Embryonic Amnions and Lung Fibroblasts

Background Human induced pluripotent stem (iPS) cells are currently used as powerful resources in regenerative medicine. During very early developmental stages, DNA methylation decreases to an overall low level at the blastocyst stage, from which embryonic stem cells are derived.Therefore, pluripotent stem cells, such as ES and iPS cells, are considered to have hypo-methylated status compared to differentiated cells. However, epigenetic mechanisms of “stemness” remain unknown in iPS cells derived from extra-embryonic and embryonic cells. Methodology/Principal Findings We examined genome-wide DNA methylation (24,949 CpG sites covering 1,3862 genes, mostly selected from promoter regions) with six human iPS cell lines derived from human amniotic cells and fetal lung fibroblasts as well as two human ES cell lines, and eight human differentiated cell lines using Illuminas Infinium HumanMethylation27. A considerable fraction (807 sites) exhibited a distinct difference in the methylation level between the iPS/ES cells and differentiated cells, with 87.6% hyper-methylation seen in iPS/ES cells. However, a limited fraction of CpG sites with hypo-methylation was found in promoters of genes encoding transcription factors. Thus, a group of genes becomes active through a decrease of methylation in their promoters. Twenty-three genes including SOX15, SALL4, TDGF1, PPP1R16B and SOX10 as well as POU5F1 were defined as genes with hypo-methylated SS-DMR (Stem cell-Specific Differentially Methylated Region) and highly expression in iPS/ES cells. Conclusions/Significance We show that DNA methylation profile of human amniotic iPS cells as well as fibroblast iPS cells, and defined the SS-DMRs. Knowledge of epigenetic information across iPS cells derived from different cell types can be used as a signature for “stemness” and may allow us to screen for optimum iPS/ES cells and to validate and monitor iPS/ES cell derivatives for human therapeutic applications.

Gremlin enhances the determined path to cardiomyogenesis.

Background The critical event in heart formation is commitment of mesodermal cells to a cardiomyogenic fate, and cardiac fate determination is regulated by a series of cytokines. Bone morphogenetic proteins (BMPs) and fibroblast growth factors have been shown to be involved in this process, however additional factors needs to be identified for the fate determination, especially at the early stage of cardiomyogenic development. Methodology/Principal Findings Global gene expression analysis using a series of human cells with a cardiomyogenic potential suggested Gremlin (Grem1) is a candidate gene responsible for in vitro cardiomyogenic differentiation. Grem1, a known BMP antagonist, enhanced DMSO-induced cardiomyogenesis of P19CL6 embryonal carcinoma cells (CL6 cells) 10–35 fold in an area of beating differentiated cardiomyocytes. The Grem1 action was most effective at the early differentiation stage when CL6 cells were destined to cardiomyogenesis, and was mediated through inhibition of BMP2. Furthermore, BMP2 inhibited Wnt/β-catenin signaling that promoted CL6 cardiomyogenesis. Conclusions/Significance Grem1 enhances the determined path to cardiomyogenesis in a stage-specific manner, and inhibition of the BMP signaling pathway is involved in initial determination of Grem1-promoted cardiomyogenesis. Our results shed new light on renewal of the cardiovascular system using Grem1 in human.

Epigenetic clustering of lung adenocarcinomas based on DNA methylation profiles in adjacent lung tissue: Its correlation with smoking history and chronic obstructive pulmonary disease

The aim of this study was to clarify the significance of DNA methylation alterations during lung carcinogenesis. Infinium assay was performed using 139 paired samples of non‐cancerous lung tissue (N) and tumorous tissue (T) from a learning cohort of patients with lung adenocarcinomas (LADCs). Fifty paired N and T samples from a validation cohort were also analyzed. DNA methylation alterations on 1,928 probes occurred in N samples relative to normal lung tissue from patients without primary lung tumors, and were inherited by, or strengthened in, T samples. Unsupervised hierarchical clustering using DNA methylation levels in N samples on all 26,447 probes subclustered patients into Cluster I (n = 32), Cluster II (n = 35) and Cluster III (n = 72). LADCs in Cluster I developed from the inflammatory background in chronic obstructive pulmonary disease (COPD) in heavy smokers and were locally invasive. Most patients in Cluster II were non‐smokers and had a favorable outcome. LADCs in Cluster III developed in light smokers were most aggressive (frequently showing lymphatic and blood vessel invasion, lymph node metastasis and an advanced pathological stage), and had a poor outcome. DNA methylation levels of hallmark genes for each cluster, such as IRX2, HOXD8, SPARCL1, RGS5 and EI24, were again correlated with clinicopathological characteristics in the validation cohort. DNA methylation profiles reflecting carcinogenetic factors such as smoking and COPD appear to be established in non‐cancerous lung tissue from patients with LADCs and may determine the aggressiveness of tumors developing in individual patients, and thus patient outcome.

Human sclera maintains common characteristics with cartilage throughout evolution.

Background The sclera maintains and protects the eye ball, which receives visual inputs. Although the sclera does not contribute significantly to visual perception, scleral diseases such as refractory scleritis, scleral perforation and pathological myopia are considered incurable or difficult to cure. The aim of this study is to identify characteristics of the human sclera as one of the connective tissues derived from the neural crest and mesoderm. Methodology/Principal Findings We have demonstrated microarray data of cultured human infant scleral cells. Hierarchical clustering was performed to group scleral cells and other mesenchymal cells into subcategories. Hierarchical clustering analysis showed similarity between scleral cells and auricular cartilage-derived cells. Cultured micromasses of scleral cells exposed to TGF-βs and BMP2 produced an abundant matrix. The expression of cartilage-associated genes, such as Indian hedge hog, type X collagen, and MMP13, was up-regulated within 3 weeks in vitro. These results suggest that human ‘sclera’-derived cells can be considered chondrocytes when cultured ex vivo. Conclusions/Significance Our present study shows a chondrogenic potential of human sclera. Interestingly, the sclera of certain vertebrates, such as birds and fish, is composed of hyaline cartilage. Although the human sclera is not a cartilaginous tissue, the human sclera maintains chondrogenic potential throughout evolution. In addition, our findings directly explain an enigma that the sclera and the joint cartilage are common targets of inflammatory cells in rheumatic arthritis. The present global gene expression database will contribute to the clarification of the pathogenesis of developmental diseases such as high myopia.

Cells of Extraembryonic Mesodermal Origin Confer Human Dystrophin in the Mdx Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is an X‐linked recessive genetic disease characterized by severe skeletal muscular degeneration. The placenta is considered to be a promising candidate cell source for cellular therapeutics because it contains a large number of cells and heterogenous cell populations with myogenic potentials. We analyzed the myogenic potential of cells obtained from six parts of the placenta, that is, umbilical cord, amniotic epithelium, amniotic mesoderm, chorionic plate, villous chorion, and decidua basalis. In vitro cells derived from amniotic mesoderm, chorionic plate, and villous chorion efficiently transdifferentiate into myotubes. In addition, in vivo implantation of placenta‐derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of human dystrophin. Differential contribution to myogenesis in this study may be attributed to placental portion‐dependent default cell state. Molecular taxonomic characterization of placenta‐derived maternal and fetal cells in vitro will help determine the feasibility of cell‐based therapy. J. Cell. Physiol. 223:695–702, 2010.

Hyaline cartilage formation and enchondral ossification modeled with KUM5 and OP9 chondroblasts

What is it that defines a bone marrow‐derived chondrocyte? We attempted to identify marrow‐derived cells with chondrogenic nature and immortality without transformation, defining “immortality” simply as indefinite cell division. KUM5 mesenchymal cells, a marrow stromal cell line, generated hyaline cartilage in vivo and exhibited enchondral ossification at a later stage after implantation. Selection of KUM5 chondroblasts based on the activity of the chondrocyte‐specific cis‐regulatory element of the collagen α2(XI) gene resulted in enhancement of their chondrogenic nature. Gene chip analysis revealed that OP9 cells, another marrow stromal cell line, derived from macrophage colony‐stimulating factor‐deficient osteopetrotic mice and also known to be niche‐constituting cells for hematopoietic stem cells expressed chondrocyte‐specific or ‐associated genes such as type II collagen α1, Sox9, and cartilage oligomeric matrix protein at an extremely high level, as did KUM5 cells. After cultured OP9 micromasses exposed to TGF‐β3 and BMP2 were implanted in mice, they produced abundant metachromatic matrix with the toluidine blue stain and formed type II collagen‐positive hyaline cartilage within 2 weeks in vivo. Hierarchical clustering and principal component analysis based on microarray data of the expression of cell surface markers and cell‐type‐specific genes resulted in grouping of KUM5 and OP9 cells into the same subcategory of “chondroblast,” that is, a distinct cell type group. We here show that these two cell lines exhibit the unique characteristics of hyaline cartilage formation and enchondral ossification in vitro and in vivo. J. Cell. Biochem. 100: 1240–1254, 2007.

Epigenetic clustering of gastric carcinomas based on DNA methylation profiles at the precancerous stage: its correlation with tumor aggressiveness and patient outcome

Summary Single-CpG resolution genome-wide DNA methylation analysis indicated that distinct DNA methylation profiles are established during field cancerization in gastric mucosae, and such profiles at the precancerous stage are inherited by gastric cancers, thus determining tumor aggressiveness and patient outcome.

PDGFR-β Plays a Key Role in the Ectopic Migration of Neuroblasts in Cerebral Stroke

The neuroprotective agents and induction of endogenous neurogenesis remain to be the urgent issues to be established for the care of cerebral stroke. Platelet‐derived growth factor receptor beta (PDGFR‐β) is mainly expressed in neural stem/progenitor cells (NSPCs), neurons and vascular pericytes of the brain; however, the role in pathological neurogenesis remains elusive. To this end, we examined the role of PDGFR‐β in the migration and proliferation of NSPCs after stroke. A transient middle cerebral‐arterial occlusion (MCAO) was introduced into the mice with conditional Pdgfrb‐gene inactivation, including N‐PRβ‐KO mice where the Pdgfrb‐gene was mostly inactivated in the brain except that in vascular pericytes, and E‐PRβ‐KO mice with tamoxifen‐induced systemic Pdgfrb‐gene inactivation. The migration of the DCX+ neuroblasts from the subventricular zone toward the ischemic core was highly increased in N‐PRβ‐KO, but not in E‐PRβ‐KO as compared to Pdgfrb‐gene preserving control mice. We showed that CXCL12, a potent chemoattractant for CXCR4‐expressing NSPCs, was upregulated in the ischemic lesion of N‐PRβ‐KO mice. Furthermore, integrin α3 intrinsically expressed in NSPCs that critically mediates extracellular matrix‐dependent migration, was upregulated in N‐PRβ‐KO after MCAO. NSPCs isolated from N‐PRβ‐KO rapidly migrated on the surface coated with collagen type IV or fibronectin that are abundant in vascular niche and ischemic core. PDGFR‐β was suggested to be critically involved in pathological neurogenesis through the regulation of lesion‐derived chemoattractant as well as intrinsic signal of NSPCs, and we believe that a coordinated regulation of these molecular events may be able to improve neurogenesis in injured brain for further functional recovery. Stem Cells 2016;34:685–698

Alterations of the spindle checkpoint pathway in clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas

CpG‐island methylator phenotype (CIMP)‐positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poor patient outcome. The aim of this study was to clarify the molecular pathways participating in CIMP‐positive renal carcinogenesis. Genome (whole‐exome and copy number), transcriptome and proteome (two‐dimensional image converted analysis of liquid chromatography‐mass spectrometry) analyses were performed using tissue specimens of 87 CIMP‐negative and 14 CIMP‐positive clear cell RCCs and corresponding specimens of non‐cancerous renal cortex. Genes encoding microtubule‐associated proteins, such as DNAH2, DNAH5, DNAH10, RP1 and HAUS8, showed a 10% or higher incidence of genetic aberrations (non‐synonymous single‐nucleotide mutations and insertions/deletions) in CIMP‐positive RCCs, whereas CIMP‐negative RCCs lacked distinct genetic characteristics. MetaCore pathway analysis of CIMP‐positive RCCs revealed that alterations of mRNA or protein expression were significantly accumulated in six pathways, all participating in the spindle checkpoint, including the “The metaphase checkpoint (p = 1.427 × 10−6),” “Role of Anaphase Promoting Complex in cell cycle regulation (p = 7.444 × 10−6)” and “Spindle assembly and chromosome separation (p = 9.260 × 10−6)” pathways. Quantitative RT‐PCR analysis revealed that mRNA expression levels for genes included in such pathways, i.e., AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25, were significantly higher in CIMP‐positive than in CIMP‐negative RCCs. All CIMP‐positive RCCs showed overexpression of Aurora kinases, AURKA and AURKB, and this overexpression was mainly attributable to increased copy number. These data suggest that abnormalities of the spindle checkpoint pathway participate in CIMP‐positive renal carcinogenesis, and that AURKA and AURKB may be potential therapeutic targets in more aggressive CIMP‐positive RCCs.