Defining and manipulating the epigenetic stability of human embryonic stem cells

This thesis aimed to define and manipulate epigenetic stability of human embryonic stem cells (hESCs). The allele-specific expression of 22 imprinted genes was examined in 22 hESC lines by distinguishing parental single nucleotide polymorphisms in genomic DNA and cDNA. Half of the genes examined (PEG10, PEG1, MESTIT1, IGF2, H19, GTL2, NESP55, PHLDA2 and ATP10C) showed variable allele-specific expression between cell lines, indicating vulnerability to disrupted imprinting. However, 8 genes (KCNQ1OT1, NDN, NDNL1, SNRPN, IPW, PEG3, KCNQ1 and CDKN1C) showed consistent monoallelic expression. Moreover, 4 genes (TP73, IGF2R, WT1 and SLC22A18) known to be monoallelically expressed or to exhibit polymorphic imprinting in human tissues were always biallelically expressed. MEST isoform 1, PEG10 and NESP55 showed an association between the variability observed in interline allele-specific expression status and DNA methylation at their imprinting regulatory regions. These evidences demonstrate gene-specific differences in the stability of imprinted loci in hESC lines and identify disrupted DNA methylation as one potential mechanism. hESOD1 (human embryonic stem cells overexpressing DNMT1) cell lines were established to manipulate epigenetic stability of hESCs. Of ~ 2,200 CpG loci examined by restriction landmark genomic scanning (RLGS), cell lines (cultured over 23 passages) having only endogenous DNMT1 showed in vitro culture induced DNA methylation alterations at 6 loci. However, hESOD1 cell lines showed DNA methylation alterations at only 1 or 2 loci, indicating that overexpression of exogenous DNMT1 resulted in increased epigenetic stability. Of 14 imprinting regulatory regions, 10 tumour-suppressor gene promoters and 3 repetitive sequences examined, 3 loci (DAPK-1, MGMT and TIMP-3) were indentified to be hypermethylated in hESOD1 cell lines, whereas other 21 loci showed normal methylation levels. These evidences demonstrate that overexpression of exogenous DNMT1 can prevent hESCs from accumulating DNA methylation changes upon in vitro culture and cause locus-specific hypermethylation

Defining and manipulating the epigenetic stability of human embryonic stem cells

This thesis aimed to define and manipulate epigenetic stability of human embryonic stem cells (hESCs). The allele-specific expression of 22 imprinted genes was examined in 22 hESC lines by distinguishing parental single nucleotide polymorphisms in genomic DNA and cDNA. Half of the genes examined (PEG10, PEG1, MESTIT1, IGF2, H19, GTL2, NESP55, PHLDA2 and ATP10C) showed variable allele-specific expression between cell lines, indicating vulnerability to disrupted imprinting. However, 8 genes (KCNQ1OT1, NDN, NDNL1, SNRPN, IPW, PEG3, KCNQ1 and CDKN1C) showed consistent monoallelic expression. Moreover, 4 genes (TP73, IGF2R, WT1 and SLC22A18) known to be monoallelically expressed or to exhibit polymorphic imprinting in human tissues were always biallelically expressed. MEST isoform 1, PEG10 and NESP55 showed an association between the variability observed in interline allele-specific expression status and DNA methylation at their imprinting regulatory regions. These evidences demonstrate gene-specific differences in the stability of imprinted loci in hESC lines and identify disrupted DNA methylation as one potential mechanism. hESOD1 (human embryonic stem cells overexpressing DNMT1) cell lines were established to manipulate epigenetic stability of hESCs. Of ~ 2,200 CpG loci examined by restriction landmark genomic scanning (RLGS), cell lines (cultured over 23 passages) having only endogenous DNMT1 showed in vitro culture induced DNA methylation alterations at 6 loci. However, hESOD1 cell lines showed DNA methylation alterations at only 1 or 2 loci, indicating that overexpression of exogenous DNMT1 resulted in increased epigenetic stability. Of 14 imprinting regulatory regions, 10 tumour-suppressor gene promoters and 3 repetitive sequences examined, 3 loci (DAPK-1, MGMT and TIMP-3) were indentified to be hypermethylated in hESOD1 cell lines, whereas other 21 loci showed normal methylation levels. These evidences demonstrate that overexpression of exogenous DNMT1 can prevent hESCs from accumulating DNA methylation changes upon in vitro culture and cause locus-specific hypermethylation

A Mixed-effects Height-Diameter Model for Pinus densiflora Trees in Gangwon Province, Korea

A new mixed-effects model was developed that predicts individual-tree total height for Pinus densiflora trees in Gangwon province as a function of individual-tree diameter (cm). The mixed-effects model contains two random-effects parameters. Maximum likelihood estimation was used to fit the model to 560 height-diameter observations of individual trees measured throughout Gwangwon province in 2007 as part of the National Forest Inventory Program in Korea. The new model is an improvement over fixed effects models because it can be calibrated to a local area, such as an inventory plot or individual stand. The new model also appears to be an improvement over the Forest Resources Evaluation and Prediction Program for the ten calibration trees used in this study. An example is provided that describes how to estimate the random-effects parameters using ten calibration trees

Development of Korean Academy of Medical Sciences Guideline on the Skin and Related System: Impairment Evaluation of Disfigurement in Skin and Appearance

The purpose of this study is to develop new standards for the disability evaluation with reference to existing laws and other study reports regarding disabilities for the rational evaluation of the diverse kinds of disfigurement in appearance and skin. Three plastic surgery specialists and 3 dermatology specialists developed a new standard for the disability evaluation which is appropriate for circumstances in Korea. Disability rate does not take into account the social occupation, gender or age of the patient, but instead, evaluate the Activity of Daily Living and the social adaptability of the appearance and skin disfigurement regardless of the balance between different disabilities. We tried to include most cutaneous disorders and categorized them into 3 types; congenital (Type 1), acquired (Type 2) as well as any permanent skin impairment sequelae of disease, trauma or treatment process (Type 3). For type 3 disorders, we tried to rate the score according to the size of involved skin lesion. The disability rate is determined by dividing the disability class into 8 steps based on the seriousness of each type of disability

Direct Conversion of Mouse Fibroblasts into Cholangiocyte Progenitor Cells

Disorders of the biliary epithelium, known as cholangiopathies, cause severe and irreversible liver diseases. The limited accessibility of bile duct precludes modeling of several cholangiocyte-mediated diseases. Therefore, novel approaches for obtaining functional cholangiocytes with high purity are needed. Previous work has shown that the combination of Hnf1β and Foxa3 could directly convert mouse fibroblasts into bipotential hepatic stem cell-like cells, termed iHepSCs. However, the efficiency of converting fibroblasts into iHepSCs is low, and these iHepSCs exhibit extremely low differentiation potential into cholangiocytes, thus hindering the translation of iHepSCs to the clinic. Here, we describe that the expression of Hnf1α and Foxa3 dramatically facilitates the robust generation of iHepSCs. Notably, prolonged in vitro culture of Hnf1α- and Foxa3-derived iHepSCs induces a Notch signaling-mediated secondary conversion into cholangiocyte progenitor-like cells that display dramatically enhanced differentiation capacity into mature cholangiocytes. Our study provides a robust two-step approach for obtaining cholangiocyte progenitor-like cells using defined factors

Integrin-linked kinase controls retinal angiogenesis and is linked to wnt signaling and exudative vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR

Reversible Pulmonary Hypertension in Adolescent with Left Atrial Myxoma

We report a patient of left atrial huge myxoma presenting with severe pulmonary hypertension in adolescents. A patient was a 14-year-old boy presented with sudden onset dyspnea. Transthoracic echocardiographic study revealed the presence of a nodular, 4.34 × 8.11 cm sized, mobile, hyperechoic mass in the left atrium and severe pulmonary hypertension with tricuspid insufficiency. After surgical therapy, tricuspid regurgitation and pulmonary hypertension was decreased and the patient was stabilized and had an uneventful clinical course

Induced neural stem cells from distinct genetic backgrounds exhibit different reprogramming status

Somatic cells could be directly converted into induced neural stem cells (iNSCs) by ectopic expression of defined transcription factors. However, the underlying mechanism of direct lineage transition into iNSCs is largely unknown. In this study, we examined the effect of genetic background on the direct conversion process into an iNSC state. The iNSCs from two different mouse strains exhibited the distinct efficiency of lineage conversion as well as clonal expansion. Furthermore, the expression levels of endogenous NSC markers, silencing of transgenes, and in vitro differentiation potential were also different between iNSC lines from different strains. Therefore, our data suggest that the genetic background of starting cells influences the conversion efficiency as well as reprogramming status of directly converted iNSCs.ope