Showing papers by "In-Hyun Park published in 2009"

Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts.

Abstract: Induced pluripotent stem (iPS) cells are derived by epigenetic reprogramming, but their DNA methylation patterns have not yet been analyzed on a genome-wide scale. Here, we find substantial hypermethylation and hypomethylation of cytosine-phosphate-guanine (CpG) island shores in nine human iPS cell lines as compared to their parental fibroblasts. The differentially methylated regions (DMRs) in the reprogrammed cells (denoted R-DMRs) were significantly enriched in tissue-specific (T-DMRs; 2.6-fold, P < 10(-4)) and cancer-specific DMRs (C-DMRs; 3.6-fold, P < 10(-4)). Notably, even though the iPS cells are derived from fibroblasts, their R-DMRs can distinguish between normal brain, liver and spleen cells and between colon cancer and normal colon cells. Thus, many DMRs are broadly involved in tissue differentiation, epigenetic reprogramming and cancer. We observed colocalization of hypomethylated R-DMRs with hypermethylated C-DMRs and bivalent chromatin marks, and colocalization of hypermethylated R-DMRs with hypomethylated C-DMRs and the absence of bivalent marks, suggesting two mechanisms for epigenetic reprogramming in iPS cells and cancer.

Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells.

Abstract: Studies of epigenetic modifications would benefit from improved methods for high-throughput methylation profiling. We introduce two complementary approaches that use next-generation sequencing technology to detect cytosine methylation. In the first method, we designed approximately 10,000 bisulfite padlock probes to profile approximately 7,000 CpG locations distributed over the ENCODE pilot project regions and applied them to human B-lymphocytes, fibroblasts and induced pluripotent stem cells. This unbiased choice of targets takes advantage of existing expression and chromatin immunoprecipitation data and enabled us to observe a pattern of low promoter methylation and high gene-body methylation in highly expressed genes. The second method, methyl-sensitive cut counting, generated nontargeted genome-scale data for approximately 1.4 million HpaII sites in the DNA of B-lymphocytes and confirmed that gene-body methylation in highly expressed genes is a consistent phenomenon throughout the human genome. Our observations highlight the usefulness of techniques that are not inherently or intentionally biased towards particular subsets like CpG islands or promoter regions.

Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells

Abstract: Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by enforced expression of transcription factors. Using serial live imaging of human fibroblasts undergoing reprogramming, we identified distinct colony types that morphologically resemble embryonic stem (ES) cells yet differ in molecular phenotype and differentiation potential. By analyzing expression of pluripotency markers, methylation at the OCT4 and NANOG promoters and differentiation into teratomas, we determined that only one colony type represents true iPS cells, whereas the others represent reprogramming intermediates. Proviral silencing and expression of TRA-1-60, DNMT3B and REX1 can be used to distinguish the fully reprogrammed state, whereas alkaline phosphatase, SSEA-4, GDF3, hTERT and NANOG are insufficient as markers. We also show that reprogramming using chemically defined medium favors formation of fully reprogrammed over partially reprogrammed colonies. Our data define molecular markers of the fully reprogrammed state and highlight the need for rigorous characterization and standardization of putative iPS cells.

Corrigendum: Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells

Abstract: Corrigendum: Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells

Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming

Abstract: Current DNA methylation assays are limited in the flexibility and efficiency of characterizing a large number of genomic targets. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. A set of ~30,000 padlock probes was designed to assess methylation of ~66,000 CpG sites within 2,020 CpG islands on human chromosome 12, chromosome 20, and 34 selected regions. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts. Induced pluripotent stem (iPS) cells appeared to display more methylation than embryonic stem cells. We found 288 regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes.

Cardiomyocyte differentiation of human induced pluripotent stem cells.

Abstract: Background— The ability to derive human induced pluripotent stem (hiPS) cell lines by reprogramming of adult fibroblasts with a set of transcription factors offers unique opportunities for basic and translational cardiovascular research. In the present study, we aimed to characterize the cardiomyocyte differentiation potential of hiPS cells and to study the molecular, structural, and functional properties of the generated hiPS-derived cardiomyocytes. Methods and Results— Cardiomyocyte differentiation of the hiPS cells was induced with the embryoid body differentiation system. Gene expression studies demonstrated that the cardiomyocyte differentiation process of the hiPS cells was characterized by an initial increase in mesoderm and cardiomesoderm markers, followed by expression of cardiac-specific transcription factors and finally by cardiac-specific structural genes. Cells in the contracting embryoid bodies were stained positively for cardiac troponin-I, sarcomeric α-actinin, and connexin-43. Reverse-tra...

A role for Lin28 in primordial germ-cell development and germ-cell malignancy

Abstract: An RNA interference screen of 30 gene candidates has identified Lin28, a negative regulator of let-7 microRNA processing, as a potentially key regulator of primordial germ cell development, the process in the developing embryo that selects the cells destined to produce sperm and eggs. In addition, Lin28 levels are elevated in primary human germ cell tumours, suggesting that it may also be implicated in germ cell malignancy. In order to investigate the earliest molecular mechanisms of germ cell specification, mouse embryonic stem cells were differentiated into putative primordial germ cells (PGCs) in vitro. The use of inhibitory RNAs to then screen candidate genes for effects on the development of these cells demonstrates a genetic pathway for PGC specification involving Lin28, a negative regulator of let-7 microRNA processing. The rarity and inaccessibility of the earliest primordial germ cells (PGCs) in the mouse embryo thwart efforts to investigate molecular mechanisms of germ-cell specification. stella (also called Dppa3) marks the rare founder population of the germ lineage1,2. Here we differentiate mouse embryonic stem cells carrying a stella transgenic reporter into putative PGCs in vitro. The Stella+ cells possess a transcriptional profile similar to embryo-derived PGCs, and like their counterparts in vivo, lose imprints in a time-dependent manner. Using inhibitory RNAs to screen candidate genes for effects on the development of Stella+ cells in vitro, we discovered that Lin28, a negative regulator of let-7 microRNA processing3,4,5,6, is essential for proper PGC development. Furthermore, we show that Blimp1 (also called Prdm1), a let-7 target and a master regulator of PGC specification7,8,9, can rescue the effect of Lin28 deficiency during PGC development, thereby establishing a mechanism of action for Lin28 during PGC specification. Overexpression of Lin28 promotes formation of Stella+ cells in vitro and PGCs in chimaeric embryos, and is associated with human germ-cell tumours. The differentiation of putative PGCs from embryonic stem cells in vitro recapitulates the early stages of gamete development in vivo, and provides an accessible system for discovering novel genes involved in germ-cell development and malignancy.

Down's syndrome suppression of tumour growth and the role of the calcineurin inhibitor DSCR1

Abstract: Individuals with Down's syndrome are known to have a lower rate of certain solid cancers. New work from Baek et al. shows that a mouse model with an extra copy of the chromosome 21 gene Dscr1 (encoding Down syndrome critical region protein 1) exhibits decreased tumour growth due to reduced angiogenesis. They provide evidence that together with another chromosome 21 gene, Dyrk1a, a modest increase in Dscr1 expression limits angiogenesis by decreasing the activity of the calcineurin pathway. These data provide a mechanism for the reduced cancer incidence in Down's syndrome and identify the calcineurin signalling pathway, and its regulators DSCR1 and DYRK1A, as potential therapeutic targets in cancers arising in all individuals. Individuals with Down's syndrome are known to have a lower rate of certain solid cancers. Now, a mouse model with one extra copy of Dscr1, a gene located on chromosome 21, is shown to display decreased tumour growth; this is thought to be via suppression of angiogenesis mediated by decreasing the activity of the calcineurin pathway. The incidence of many cancer types is significantly reduced in individuals with Down’s syndrome1,2,3,4, and it is thought that this broad cancer protection is conferred by the increased expression of one or more of the 231 supernumerary genes on the extra copy of chromosome 21. One such gene is Down’s syndrome candidate region-1 (DSCR1, also known as RCAN1), which encodes a protein that suppresses vascular endothelial growth factor (VEGF)-mediated angiogenic signalling by the calcineurin pathway5,6,7,8,9,10. Here we show that DSCR1 is increased in Down’s syndrome tissues and in a mouse model of Down’s syndrome. Furthermore, we show that the modest increase in expression afforded by a single extra transgenic copy of Dscr1 is sufficient to confer significant suppression of tumour growth in mice, and that such resistance is a consequence of a deficit in tumour angiogenesis arising from suppression of the calcineurin pathway. We also provide evidence that attenuation of calcineurin activity by DSCR1, together with another chromosome 21 gene Dyrk1a, may be sufficient to markedly diminish angiogenesis. These data provide a mechanism for the reduced cancer incidence in Down’s syndrome and identify the calcineurin signalling pathway, and its regulators DSCR1 and DYRK1A, as potential therapeutic targets in cancers arising in all individuals.

Hematopoietic Development from Human Induced Pluripotent Stem Cells

Abstract: A decade of research on human embryonic stem cells (ESC) has paved the way for the discovery of alternative approaches to generating pluripotent stem cells. Combinatorial overexpression of a limited number of proteins linked to pluripotency in ESC was recently found to reprogram differentiated somatic cells back to a pluripotent state, enabling the derivation of isogenic (patient-specific) pluripotent stem cell lines. Current research is focusing on improving reprogramming protocols (e.g., circumventing the use of retroviral technology and oncoproteins), and on methods for differentiation into transplantable tissues of interest. In mouse ESC, we have previously shown that the embryonic morphogens BMP4 and Wnt3a direct blood formation via activation of Cdx and Hox genes. Ectopic expression of Cdx4 and HoxB4 enables the generation of mouse ESC-derived hematopoietic stem cells (HSC) capable of multilineage reconstitution of lethally irradiated adult mice. Here, we explore hematopoietic development from human induced pluripotent stem (iPS) cells generated in our laboratory. Our data show robust differentiation of iPS cells to mesoderm and to blood lineages, as shown by generation of CD34 + CD45 + cells, hematopoietic colony activity, and gene expression data, and suggest conservation of blood patterning pathways between mouse and human hematopoietic development.

A Robust Approach to Identifying Tissue-Specific Gene Expression Regulatory Variants Using Personalized Human Induced Pluripotent Stem Cells

Abstract: Normal variation in gene expression due to regulatory polymorphisms is often masked by biological and experimental noise. In addition, some regulatory polymorphisms may become apparent only in specific tissues. We derived human induced pluripotent stem (iPS) cells from adult skin primary fibroblasts and attempted to detect tissue-specific cis-regulatory variants using in vitro cell differentiation. We used padlock probes and high-throughput sequencing for digital RNA allelotyping and measured allele-specific gene expression in primary fibroblasts, lymphoblastoid cells, iPS cells, and their differentiated derivatives. We show that allele-specific expression is both cell type and genotype-dependent, but the majority of detectable allele-specific expression loci remains consistent despite large changes in the cell type or the experimental condition following iPS reprogramming, except on the X-chromosome. We show that our approach to mapping cis-regulatory variants reduces in vitro experimental noise and reveals additional tissue-specific variants using skin-derived human iPS cells.

Human iPS cell derivation/reprogramming.

Abstract: This unit describes a protocol for deriving induced pluripotent stem (iPS) cells from human fibroblast cells. Human fibroblast cells are infected with retroviral vectors expressing four transcription factors (Oct4, Sox2, Klf4, and Myc) and selected for 3 to 4 weeks under human embryonic stem (hES) cell culture conditions. iPS cell colonies are mechanically isolated using a dissection microscope and handled like hES cells thereafter. Human iPS cells share similarities with hES cells including the expression of pluripotency genes, and differentiation as embryoid bodies in vitro into three germ layers (EB) and in vivo as teratomas.

Detection of human somatic cell reprogramming

Abstract: The methods and kits described herein are based, in part, to the discovery of a phenotype representing a fully-reprogrammed iPS cell and several reprogramming intermediates. The methods and kits described herein permit identification of fully-reprogrammed iPS cells and further permits one of skill in the art to monitor the emergence of iPS cells during the reprogramming process. The methods/kits can also be performed using real time using live cell imaging. Also described herein are methods for screening candidate reprogramming agents by monitoring the emergence of fully-reprogrammed iPS cells in the presence and absence of such an agent.