scholarly journals Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells

2016 ◽  
Vol 113 (34) ◽  
pp. 9545-9550 ◽  
Author(s):  
Norikatsu Miyoshi ◽  
Jente M. Stel ◽  
Keiko Shioda ◽  
Na Qu ◽  
Junko Odajima ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cell Model ◽  
Methylation Status ◽  
Primordial Germ Cell ◽  
Dna Demethylation ◽  
Imprinted Genes ◽  
Sequencing Data ◽  
Genome Wide

The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.

scholarly journals TDG regulates cell cycle progression in human neural progenitors

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 497
Author(s):  
Igal Germanguz ◽  
Jenny C. Park ◽  
Jessica Cinkornpumin ◽  
Aryeh Solomon ◽  
Minori Ohashi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Methylation ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Methylation Status ◽  
Human Pluripotent Stem Cells ◽  
Dna Demethylation ◽  
Neural Progenitors ◽  
Specific Cell ◽  
Active Demethylation

Background: As cells divide, they must both replicate their DNA and generate a new set of histone proteins.  The newly synthesized daughter strands and histones are unmodified, and must therefore be covalently modified to allow for transmission of important epigenetic marks to daughter cells.  Human pluripotent stem cells (hPSCs) display a unique cell cycle profile, and control of the cell cycle is known to be critical for their proper differentiation and survival.  A major unresolved question is how hPSCs regulate their DNA methylation status through the cell cycle, namely how passive and active demethylation work to maintain a stable genome. Thymine-DNA glycosylase (TDG), an embryonic essential gene, has been recently implicated as a major enzyme involved in demethylation. Methods: We use human pluripotent stem cells and their derivatives to investigate the role of TDG in differentiation and proliferation.  To perform loss of function of TDG, RNA Interference was used.  To study the cell cyle, we engineered human pluripotent stem cells to express the FUCCI tool which marks cells at various stages of the cell cycle with distinct patterns of fluorescent proteins.  We also used cell cycle profiling by FACS, and DNA methylation analysis to probe a connection between DNA demethylation and cell cycle. Results: Here we present data showing that TDG regulates cell cycle dynamics in human neural progenitors (NPCs) derived from hPSCs, leading to changes in  cell cycle related gene expression and neural differentiation capacity.  These data show that loss of TDG function can block differentiation by driving proliferation of neural progenitors.  We also identify specific cell cycle related genes whose expression changes upon loss of TDG expression. Conclusions: These observations suggest that TDG and active demethylation play an important role in hPSC cell cycle regulation and differentiation.

scholarly journals Generation of Mouse Parthenogenetic Epiblast Stem Cells and Their Imprinting Patterns

2019 ◽  
Vol 20 (21) ◽  
pp. 5428
Author(s):  
Bong Jong Seo ◽  
Hyun Sik Jang ◽  
Hyuk Song ◽  
Chankyu Park ◽  
Kwonho Hong ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Expression Patterns ◽  
Methylation Status ◽  
Embryonic Stem ◽  
Methylation Pattern ◽  
Imprinted Genes ◽  
Differentiation Potential ◽  
Epiblast Stem Cells

Pluripotent stem cells can be established from parthenogenetic embryos, which only possess maternal alleles with maternal-specific imprinting patterns. Previously, we and others showed that parthenogenetic embryonic stem cells (pESCs) and parthenogenetic induced pluripotent stem cells (piPSCs) progressively lose the bimaternal imprinting patterns. As ESCs and iPSCs are naïve pluripotent stem cells, parthenogenetic primed pluripotent stem cells have not yet been established, and thus, their imprinting patterns have not been studied. Here, we first established parthenogenetic epiblast stem cells (pEpiSCs) from 7.5 dpc parthenogenetic implantation embryos and compared the expression patterns and DNA methylation status of the representative imprinted genes with biparental EpiSCs. We found that there were no striking differences between pEpiSCs and biparental EpiSCs with respect to morphology, pluripotency gene expression, and differentiation potential, but there were differences in the expression and DNA methylation status of imprinted genes (H19, Igf2, Peg1, and Peg3). Moreover, pEpiSCs displayed a different DNA methylation pattern compared with that of parthenogenetic neural stem cells (pNSCs), which showed a typical bimaternal imprinting pattern. These results suggest that both naïve pluripotent stem cells and primed pluripotent stem cells have an unstable imprinting status.

scholarly journals Generation of human oogonia from induced pluripotent stem cells in vitro

Science ◽  
2018 ◽  
Vol 362 (6412) ◽  
pp. 356-360 ◽  
Author(s):  
Chika Yamashiro ◽  
Kotaro Sasaki ◽  
Yukihiro Yabuta ◽  
Yoji Kojima ◽  
Tomonori Nakamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cell ◽  
Dna Demethylation ◽  
Inactive X Chromosome ◽  
Genome Wide ◽  
Aberrant Dna Methylation ◽  
Induced Pluripotent

Human in vitro gametogenesis may transform reproductive medicine. Human pluripotent stem cells (hPSCs) have been induced into primordial germ cell–like cells (hPGCLCs); however, further differentiation to a mature germ cell has not been achieved. Here, we show that hPGCLCs differentiate progressively into oogonia-like cells during a long-term in vitro culture (approximately 4 months) in xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. The hPGCLC-derived oogonia display hallmarks of epigenetic reprogramming—genome-wide DNA demethylation, imprint erasure, and extinguishment of aberrant DNA methylation in hPSCs—and acquire an immediate precursory state for meiotic recombination. Furthermore, the inactive X chromosome shows a progressive demethylation and reactivation, albeit partially. These findings establish the germline competence of hPSCs and provide a critical step toward human in vitro gametogenesis.

scholarly journals Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 876
Author(s):  
Raquel Bernad ◽  
Cian J. Lynch ◽  
Rocio G. Urdinguio ◽  
Camille Stephan-Otto Attolini ◽  
Mario F. Fraga ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cell ◽  
Normal Karyotype ◽  
Cell Types ◽  
Dna Demethylation ◽  
Starting State ◽  
Teratoma Formation

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.

scholarly journals Genome-Wide DNA Methylation Pattern of Cancer Stem Cells in Esophageal Cancer

2020 ◽  
Vol 19 ◽  
pp. 153303382098379
Author(s):  
Xiying Yu ◽  
Ying Teng ◽  
Xingran Jiang ◽  
Hui Yuan ◽  
Wei Jiang
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Esophageal Cancer ◽  
Cancer Stem Cells ◽  
Side Population ◽  
Methylation Status ◽  
Methylation Profile ◽  
Cpg Dinucleotides ◽  
Genome Wide ◽  
Differentially Methylated Genes

Background: Cancer stem cells (CSCs) are considered the main cause of cancer recurrence and metastasis, and DNA methylation is involved in the maintenance of CSCs. However, the methylation profile of esophageal CSCs remains unknown. Methods: Side population (SP) cells were isolated from esophageal squamous cell carcinoma (ESCC) cell lines KYSE150 and EC109. Sphere-forming cells were collected from human primary esophageal cancer cells. SP cells and sphere-forming cells were used as substitutes for cancer stem-like cells. We investigated the genome-wide DNA methylation profile in esophageal cancer stem-like cells using reduced representation bisulfite sequencing (RRBS). Results: Methylated cytosine (mC) was found mostly in CpG dinucleotides, located mostly in the intronic, intergenic, and exonic regions. Forty intersected differentially methylated regions (DMRs) were identified in these 3 groups of samples. Thirteen differentially methylated genes with the same alteration trend were detected; these included OTX1, SPACA1, CD163L1, ST8SIA2, TECR, CADM3, GRM1, LRRK1, CHSY1, PROKR2, LINC00658, LOC100506688, and NKD2. DMRs covering ST8SIA2 and GRM1 were located in exons. These differentially methylated genes were involved in 10 categories of biological processes and 3 cell signaling pathways. Conclusions: When compared to non-CSCs, cancer stem-like cells have a differential methylation status, which provides an important biological base for understanding esophageal CSCs and developing therapeutic targets for esophageal cancer.

scholarly journals Putative Epimutagens in Maternal Peripheral and Cord Blood Samples Identified Using Human Induced Pluripotent Stem Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Yoshikazu Arai ◽  
Koji Hayakawa ◽  
Daisuke Arai ◽  
Rie Ito ◽  
Yusuke Iwasaki ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Cord Blood ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Methylation Status ◽  
Combined Exposure ◽  
Blood Samples ◽  
Low Concentrations ◽  
Induced Pluripotent

The regulation of transcription and genome stability by epigenetic systems are crucial for the proper development of mammalian embryos. Chemicals that disturb epigenetic systems are termed epimutagens. We previously performed chemical screening that focused on heterochromatin formation and DNA methylation status in mouse embryonic stem cells and identified five epimutagens: diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se), and octachlorodipropyl ether (S-421). Here, we used human induced pluripotent stem cells (hiPSCs) to confirm the effects of 20 chemicals, including the five epimutagens, detected at low concentrations in maternal peripheral and cord blood samples. Of note, these individual chemicals did not exhibit epimutagenic activity in hiPSCs. However, because the fetal environment contains various chemicals, we evaluated the effects of combined exposure to chemicals (DEP, Hg, cotinine, Se, and S-421) on hiPSCs. The combined exposure caused a decrease in the number of heterochromatin signals and aberrant DNA methylation status at multiple gene loci in hiPSCs. The combined exposure also affected embryoid body formation and neural differentiation from hiPSCs. Therefore, DEP, Hg, cotinine, Se, and S-421 were defined as an “epimutagen combination” that is effective at low concentrations as detected in maternal peripheral and cord blood.

scholarly journals Identifying regulators of parental imprinting by CRISPR/Cas9 screening in haploid human embryonic stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shiran Bar ◽  
Dan Vershkov ◽  
Gal Keshet ◽  
Elyad Lezmi ◽  
Naama Meller ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Human Embryos ◽  
Monoallelic Expression ◽  
Imprinted Genes ◽  
Genome Wide ◽  
A Genome ◽  
Parental Imprinting

AbstractIn mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.

scholarly journals Dynamics of DNA Methylation Reprogramming Influenced by X Chromosome Dosage in Induced Pluripotent Stem Cells

2018 ◽  
Vol 11 ◽  
pp. 251686571880293 ◽  
Author(s):  
Adrian Janiszewski ◽  
Juan Song ◽  
Lotte Vanheer ◽  
Natalie De Geest ◽  
Vincent Pasque
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
X Chromosome ◽  
Pluripotent Stem Cells ◽  
Somatic Cells ◽  
Global Dna Methylation ◽  
X Chromosomes ◽  
Genome Wide ◽  
Induced Pluripotent

How the epigenome of one cell type is remodeled during reprogramming into another unrelated type of cell remains unclear. Overexpression of transcription factors in somatic cells enables the induction of induced pluripotent stem cells (iPSCs). This process entails genome-wide remodeling of DNA methylation, chromatin, and transcription. Recent work suggests that the number of active X chromosomes present in a cell influences remodeling of DNA methylation during somatic cell reprogramming to mouse iPSCs. Female iPSCs with 2 active X chromosomes display global DNA hypomethylation, whereas male XY iPSCs show DNA methylation levels similar to the somatic cells they are derived from. Global DNA methylation erasure in female iPSCs takes place genome-wide and involves repression of DNA methyltransferases. However, on loss of one X chromosome, female iPSCs acquire a DNA methylation landscape resembling that of XY iPSCs. Therefore, it is the X chromosome dosage that dictates global DNA methylation levels in iPSCs. Here, we discuss the evidence that links X chromosome dosage with the regulation of DNA methylation in pluripotent stem cells. We focus on iPSCs reprogramming studies, where X chromosome status is a novel factor impacting our understanding of epigenetic remodeling.

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