Link for full list of publications
(2023) Cell. 186, 4, p. 683-685 Abstract
Transgenerational epigenetic inheritance in mammals has long been debatable. In this issue of Cell, Takahashi et al. induce DNA methylation at promoter-associated CpG islands (CGIs) of two metabolism-related genes and show that the acquired epigenetic changes and associated metabolic phenotypes are stably propagated across several generations in transgenic mice.
(2023) Nature reviews. Molecular cell biology. Abstract
Understanding how the same DNA sequence instructs an abundance of transcriptional programmes associated with different cell states cannot be complete without considering epigenetics. The ability of cells to commit and memorize specific epigenetic programmes is essential for the construction of complex tissues and organs during development. However, because transcriptional and epigenetic changes go hand in hand, determining the roles of epigenetic mechanisms in cell state acquisition remains a notoriously difficult task. During my undergraduate studies, I stumbled upon a landmark paper from the lab of Howard Cedar, which sparked my long-term interest in this problem.
(2022) Stem Cell Reports. 17, 11, p. 2484-2500 Abstract[All authors]
The recent derivation of human trophoblast stem cells (TSCs) from placental cytotrophoblasts and blastocysts opened opportunities for studying the development and function of the human placenta. Recent reports have suggested that human naïve, but not primed, pluripotent stem cells (PSCs) retain an exclusive potential to generate TSCs. Here we report that, in the absence of WNT stimulation, transforming growth factor β (TGF-β) pathway inhibition leads to direct and robust conversion of primed human PSCs into TSCs. The resulting primed PSC-derived TSC lines exhibit self-renewal, can differentiate into the main trophoblast lineages, and present RNA and epigenetic profiles that are indistinguishable from recently established TSC lines derived from human placenta, blastocysts, or isogenic human naïve PSCs expanded under human enhanced naïve stem cell medium (HENSM) conditions. Activation of nuclear Yes-associated protein (YAP) signaling is sufficient for this conversion and necessary for human TSC maintenance. Our findings underscore a residual plasticity in primed human PSCs that allows their in vitro conversion into extra-embryonic trophoblast lineages.
Histone exchange sensors reveal variant specific dynamics in mouse embryonic stem cells(2022) BioRxiv. Abstract
Eviction of histones from nucleosomes and their exchange with newly synthesized or alternative variants is a central epigenetic determinant. Here, we define the genome-wide incorporation and exchange pattern of canonical and non-canonical histone variants in mouse embryonic stem cells by implementing a recently established, genetically encoded exchange sensor. While exchange of all measured variants scales with transcription, we describe variant-specific associations with transcription elongation and Polycomb binding. We found considerable exchange of H3.1 and H2B variants in heterochromatin and repeat elements, contrasting the stable incorporation and little exchange of H3.3 in these regions. This unexpected association between H3.3 incorporation and exchange of canonical variants is also evident in active promoters and enhancers, and further validated by reduced H3.1 dynamics following depletion of the HIRA H3.3-specific chaperone. The sensor system provides a powerful tool for studying regulation of histone dynamics toward understanding its role in shaping the epigenetic landscape in vivo.
Time-Aligned Hourglass Gastrulation Models in Rabbit and Mouse(2022) BioRxiv. Abstract[All authors]
The hourglass model describes the convergence of species within the same phylum to a similar body plan during development, yet the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0-8.5, and compare the species using a new framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell state compositions at E7.5, underlied by quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages, and divergence of primordial germ cells programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a new basis for studying the evolution of gastrulation dynamics across mammals.
Mouse embryo model derived exclusively from embryonic stem cells undergoes neurulation and heart development(2022) Cell Stem Cell. 29, p. 1-14 Abstract
Several in vitro models have been developed to recapitulate mouse embryogenesis solely from embryonic stem cells (ESCs). Despite mimicking many aspects of early development, they fail to capture the interactions between embryonic and extraembryonic tissues. To overcome this difficulty, we have developed a mouse ESC-based in vitro model that reconstitutes the pluripotent ESC lineage and the two extra-embryonic lineages of the post-implantation embryo by transcription factor-mediated induction. This unified model recapitulates developmental events from embryonic day 5.5 to 8.5, including gastrulation, and formation of the anterior-posterior axis, brain, a beating heart structure, and the development of extraembryonic tissues, including yolk sac and chorion. Comparing single-cell RNA sequencing from individual structures with time-matched natural embryos identified remarkably similar transcriptional programs across lineages, but also showed when and where the model diverges from the natural program. Our findings demonstrate an extraordinary plasticity of ESCs to self-organise and generate a whole embryo-like structure.
(2022) Cell. 185, p. 1-17 Abstract[All authors]
Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, Tet-mutant cells retain near-complete differentiation potential, whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors (e.g., Dppa4 and Gdf3) and failure to activate multiple signaling from nascent mesoderm (Lefty, FGF, and Notch) as likely cell-intrinsic drivers of TET loss phenotypes. We further suggest loss of enhancer demethylation as the underlying mechanism. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications.
(2022) Nature Communications. 13, 4391. Abstract
Mammalian parental imprinting represents an exquisite form of epigenetic control regulating the parent-specific monoallelic expression of genes in clusters. While imprinting perturbations are widely associated with developmental abnormalities, the intricate regional interplay between imprinted genes makes interpreting the contribution of gene dosage effects to phenotypes a challenging task. Using mouse models with distinct deletions in an intergenic region controlling imprinting across the Dlk1-Dio3 domain, we link changes in genetic and epigenetic states to allelic-expression and phenotypic outcome in vivo. This determined how hierarchical interactions between regulatory elements orchestrate robust parent-specific expression, with implications for non-imprinted gene regulation. Strikingly, flipping imprinting on the parental chromosomes by crossing genotypes of complete and partial intergenic element deletions rescues the lethality of each deletion on its own. Our work indicates that parental origin of an epigenetic state is irrelevant as long as appropriate balanced gene expression is established and maintained at imprinted loci.
(2021) Nature Methods. 18, 9, p. 1060-1067 Abstract[All authors]
N6-methyladenosine (m6A) is the most prevalent modification of messenger RNA in mammals. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels: site, gene and sample. Current approaches address these needs in a limited manner. Here we develop m6A-seq2, relying on multiplexed m6A-immunoprecipitation of barcoded and pooled samples. m6A-seq2 allows a big increase in throughput while reducing technical variability, requirements of input material and cost. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an orthogonal alternative to mass spectrometry-based approaches. Finally, we develop a computational approach for gene-level quantitation of m6A. We demonstrate that using this metric, roughly 30% of the variability in RNA half life in mouse embryonic stem cells can be explained, establishing m6A as a main driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-mediated regulation at three different levels.
(2021) Molecular Cell. 81, 11, p. 2374-2387 Abstract
Adenosine-to-inosine editing is catalyzed by ADAR1 at thousands of sites transcriptome-wide. Despite intense interest in ADAR1 from physiological, bioengineering, and therapeutic perspectives, the rules of ADAR1 substrate selection are poorly understood. Here, we used large-scale systematic probing of ∼2,000 synthetic constructs to explore the structure and sequence context determining editability. We uncover two structural layers determining the formation and propagation of A-to-I editing, independent of sequence. First, editing is robustly induced at fixed intervals of 35 bp upstream and 30 bp downstream of structural disruptions. Second, editing is symmetrically introduced on opposite sites on a double-stranded structure. Our findings suggest a recursive model for RNA editing, whereby the structural alteration induced by the editing at one site iteratively gives rise to the formation of an additional editing site at a fixed periodicity, serving as a basis for the propagation of editing along and across both strands of double-stranded RNA structures.
(2021) Cell. 184, p. 1-18 Abstract[All authors]
Mouse embryonic development is a canonical model system for studying mammalian cell fate acquisition. Recently, single-cell atlases comprehensively charted embryonic transcriptional landscapes, yet inference of the coordinated dynamics of cells over such atlases remains challenging. Here, we introduce a temporal model for mouse gastrulation, consisting of data from 153 individually sampled embryos spanning 36 h of molecular diversification. Using algorithms and precise timing, we infer differentiation flows and lineage specification dynamics over the embryonic transcriptional manifold. Rapid transcriptional bifurcations characterize the commitment of early specialized node and blood cells. However, for most lineages, we observe combinatorial multi-furcation dynamics rather than hierarchical transcriptional transitions. In the mesoderm, dozens of transcription factors combinatorially regulate multifurcations, as we exemplify using time-matched chimeric embryos of Foxc1/Foxc2 mutants. Our study rejects the notion of differentiation being governed by a series of binary choices, providing an alternative quantitative model for cell fate acquisition.
(2021) Nature (London). Abstract[All authors]
Establishment of the mammalian body plan occurs shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. While methods for in vitro culture of pre- and peri-implantation mouse embryos are routinely utilized, approaches for robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we develop highly conducive ex utero post-implantation mouse embryo culture platforms, that enable appropriate development of embryos before gastrulation (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in 3D rotating bottles settings, while extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of novel static and rotating bottle culture platforms. Histological, molecular, and single-cell RNA-seq analyses validate that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to introducing a variety of embryonic perturbations and micro-manipulations that can be followed ex utero for up to six days. Establishment of a system to robustly grow normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool to investigate embryogenesis, eliminating the uterine barrier to mechanistically interrogate post-implantation morphogenesis and tissue specification in mammals.
(2019) Molecular Cell. 75, 5, p. 905-+ Abstract[All authors]
Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo.
Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules(2019) Cell Stem Cell. 24, 2, p. 328-341.e9 Abstract[All authors]
The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.
(2016) Cell reports (Cambridge). 16, 12, p. 3167-3180 Abstract
Parent-specific differentially methylated regions (DMRs) are established during gametogenesis and regulate parent-specific expression of imprinted genes. Monoallelic expression of imprinted genes is essential for development, suggesting that imprints are faithfully maintained in embryos and adults. To test this hypothesis, we targeted a reporter for genomic methylation to the imprinted Dlk1-Dio3 intergenic DMR (IG-DMR) to assess the methylation of both parental alleles at single-cell resolution. Biallelic gain or loss of IG-DMR methylation occurred in a small fraction of mouse embryonic stem cells, significantly affecting developmental potency. Mice carrying the reporter in either parental allele showed striking parent-specific changes in IG-DMR methylation, causing substantial and consistent tissue- and cell-type-dependent signatures in embryos and postnatal animals. Furthermore, dynamics in DNA methylation persisted during adult neurogenesis, resulting in inter-individual diversity. This substantial cell-cell DNA methylation heterogeneity implies that dynamic DNA methylation variations in the adult may be of functional importance.
(2016) Cell (Cambridge). 167, 1, p. 233-247.e17 Abstract[All authors]
Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.
Parkinson-associated risk variant in distal enhancer of α-synuclein modulates target gene expression(2016) Nature (London). 533, 7601, p. 95-99 Abstract[All authors]
Genome-wide association studies (GWAS) have identified numerous genetic variants associated with complex diseases, but mechanistic insights are impeded by a lack of understanding of how specific risk variants functionally contribute to the underlying pathogenesis. It has been proposed that cis-acting effects of non-coding risk variants on gene expression are a major factor for phenotypic variation of complex traits and disease susceptibility. Recent genome-scale epigenetic studies have highlighted the enrichment of GWAS-identified variants in regulatory DNA elements of disease-relevant cell types. Furthermore, single nucleotide polymorphism (SNP)-specific changes in transcription factor binding are correlated with heritable alterations in chromatin state and considered a major mediator of sequence-dependent regulation of gene expression. Here we describe a novel strategy to functionally dissect the cis-acting effect of genetic risk variants in regulatory elements on gene expression by combining genome-wide epigenetic information with clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 genome editing in human pluripotent stem cells. By generating a genetically precisely controlled experimental system, we identify a common Parkinson's disease associated risk variant in a non-coding distal enhancer element that regulates the expression of α-synuclein (SNCA), a key gene implicated in the pathogenesis of Parkinson's disease. Our data suggest that the transcriptional deregulation of SNCA is associated with sequence-dependent binding of the brain-specific transcription factors EMX2 and NKX6-1. This work establishes an experimental paradigm to functionally connect genetic variation with disease-relevant phenotypes.
(2015) Cell (Cambridge). 163, 1, p. 218-229 Abstract
Mammalian DNA methylation plays an essential role in development. To date, only snapshots of different mouse and human cell types have been generated, providing a static view on DNA methylation. To enable monitoring of methylation status as it changes over time, we establish a reporter of genomic methylation (RGM) that relies on a minimal imprinted gene promoter driving a fluorescent protein. We show that insertion of RGM proximal to promoter-associated CpG islands reports the gain or loss of DNA methylation. We further utilized RGM to report endogenous methylation dynamics of non-coding regulatory elements, such as the pluripotency-specific super enhancers of Sox2 and miR290. Loci-specific DNA methylation changes and their correlation with transcription were visualized during cell-state transition following differentiation of mouse embryonic stem cells and during reprogramming of somatic cells to pluripotency. RGM will allow the investigation of dynamic methylation changes during development and disease at single-cell resolution.
Differentiation of human parthenogenetic pluripotent stem cells reveals multiple tissue- and isoform-specific imprinted transcripts(2015) Cell reports (Cambridge). 11, 2, p. 308-320 Abstract
Parental imprinting results in monoallelic parent-of-origin-dependent gene expression. However, many imprinted genes identified by differential methylation do not exhibit complete monoallelic expression. Previous studies demonstrated complex tissue-dependent expression patterns for some imprinted genes. Still, the complete magnitude of this phenomenon remains largely unknown. By differentiating human parthenogenetic induced pluripotent stem cells into different cell types and combining DNA methylation with a 5' RNA sequencing methodology, we were able to identify tissue- and isoform-dependent imprinted genes in a genome-wide manner. We demonstrate that nearly half of all imprinted genes express both biallelic and monoallelic isoforms that are controlled by tissue-specific alternative promoters. This study provides a global analysis of tissue-specific imprinting in humans and suggests that alternative promoters are central in the regulation of imprinted genes.
(2015) Cold Spring Harbor Symposia on Quantitative Biology. 80, p. 199-206 Abstract
DNA methylation is a broadly studied epigenetic modification that is essential for normal mammalian development. Over the years, numerous methodologies were developed trying to cope with the intrinsic challenge of reading the "second dimension" epigenetic code. The recent rapid expansion of sequencing technologies has made it possible to fully chart the methylation landscape of different cell types at single-base resolution. Surprisingly, accumulating data suggest that, in addition to the massive epigenome remodeling during early development, cell type and tissue specification is associated with high levels of DNA methylation dynamics at distal regulatory elements. However, current methods provide only a static "snapshot" of DNA methylation, thus precluding the study of real-time methylation dynamics during cell fate changes. Here we review the principles of a new approach that enables monitoring loci-specific DNA methylation dynamics at single-cell resolution. We also discuss potential applications and promises for implementing this methodology to study DNA methylation changes during development and disease.
Systematic Identification of Culture Conditions for Induction and Maintenance of Naive Human Pluripotency(2014) Cell Stem Cell. 15, 4, p. 471-487 Abstract[All authors]
Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, “naive” state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells.
The noncoding RNA IPW regulates the imprinted DLK1-DIO3 locus in an induced pluripotent stem cell model of Prader-Willi syndrome(2014) Nature Genetics. 46, 6, p. 551-557 Abstract
Parental imprinting is a form of epigenetic regulation that results in parent-of-origin differential gene expression. To study Prader-Willi syndrome (PWS), a developmental imprinting disorder, we generated case-derived induced pluripotent stem cells (iPSCs) harboring distinct aberrations in the affected region on chromosome 15. In studying PWS-iPSCs and human parthenogenetic iPSCs, we unexpectedly found substantial upregulation of virtually all maternally expressed genes (MEGs) in the imprinted DLK1-DIO3 locus on chromosome 14. Subsequently, we determined that IPW, a long noncoding RNA in the critical region of the PWS locus, is a regulator of the DLK1-DIO3 region, as its overexpression in PWS and parthenogenetic iPSCs resulted in downregulation of MEGs in this locus. We further show that gene expression changes in the DLK1-DIO3 region coincide with chromatin modifications rather than DNA methylation levels. Our results suggest that a subset of PWS phenotypes may arise from dysregulation of an imprinted locus distinct from the PWS region.
(2013) Nature Communications. 4, 1, p. 2724-2724 Abstract
The monoallelic nature of imprinted genes renders them highly susceptible to genetic and epigenetic perturbations, potentially resulting in transformation and disease. Here we show, using parthenogenetic induced pluripotent stem cells, an imprinted transcript that serves as an antisense regulator of onco-miR-372-3 (named anti-miR-371-3). As miR-372-3 have been shown to have an oncogenic role in testicular germ cell tumours, we study the involvement of their antisense transcript in these cells. Our results suggest that hypermethylation, leading to loss-of-expression of the imprinted antisense transcript, contributes to tumorigenic transformation by affecting the downstream target LATS2. Finally, we provide evidence for a tumour suppressive role of anti-miR-371-3, as its overexpression in tumour cells results in cell growth arrest and apoptosis, and prevents tumour formation on injection into immunodeficient mice.
Identification of novel imprinted differentially methylated regions by global analysis of human-parthenogenetic-induced pluripotent stem cells(2013) Stem Cell Reports. 1, 1, p. 79-89 Abstract
Parental imprinting is an epigenetic phenomenon by which genes are expressed in a monoallelic fashion, according to their parent of origin. DNA methylation is considered the hallmark mechanism regulating parental imprinting. To identify imprinted differentially methylated regions (DMRs), we compared the DNA methylation status between multiple normal and parthenogenetic human pluripotent stem cells (PSCs) by performing reduced representation bisulfite sequencing. Our analysis identified over 20 previously unknown imprinted DMRs in addition to the known DMRs. These include DMRs in loci associated with human disorders, and a class of intergenic DMRs that do not seem to be related to gene expression. Furthermore, the study showed some DMRs to be unstable, liable to differentiation or reprogramming. A comprehensive comparison between mouse and human DMRs identified almost half of the imprinted DMRs to be species specific. Taken together, our data map novel DMRs in the human genome, their evolutionary conservation, and relation to gene expression.
(2011) Nature Structural & Molecular Biology. 18, 6, p. 735-741 Abstract
To study the role of parental imprinting in human embryogenesis, we generated parthenogenetic human induced pluripotent stem cells (iPSCs) with a homozygote diploid karyotype. Global gene expression and DNA methylation analyses of the parthenogenetic cells enabled the identification of the entire repertoire of paternally expressed genes. We thus demonstrated that the gene U5D, encoding a variant of the U5 small RNA component of the spliceosome, is an imprinted gene. Introduction of the U5D gene into parthenogenetic cells partially corrected their molecular phenotype. Our analysis also uncovered multiple miRNAs existing as imprinted clustered transcripts, whose putative targets we then studied further. Examination of the consequences of parthenogenesis on human development identified marked effects on the differentiation of extraembryonic trophectoderm and embryonic liver and muscle tissues. This analysis suggests that distinct regulatory imprinted small RNAs and their targets have substantial roles in human development.
(2009) Stem cells (Dayton, Ohio). 27, 11, p. 2686-2690 Abstract
Genomic imprinting is an epigenetic phenomenon whereby genes are expressed in a monoallelic manner, which is inherited either maternally or paternally. Expression of imprinted genes has been examined in human embryonic stem (ES) cells, and the cells show a substantial degree of genomic imprinting stability. Recently, human somatic cells were reprogrammed to a pluripotent state using various defined factors. These induced pluripotent stem (iPS) cells are thought to have a great potential for studying genetic diseases and to be a source of patient-specific stem cells. Thus, studying the expression of imprinted genes in these cells is important. We examined the allelic expression of various imprinted genes in several iPS cell lines and found polymorphisms in four genes. After analyzing parent-specific expression of these genes, we observed overall normal monoallelic expression in the iPS cell lines. However, we found biallelic expression of the H19 gene in one iPS cell line and biallelic expression of the KCNQ10T1 gene in another iPS cell line. We further analyzed the DNA methylation levels of the promoter region of the H19 gene and found that the cell line that showed biallelic expression had undergone extensive DNA demethylation. Additionally we studied the imprinting gene expression pattern of multiple human iPS cell lines via DNA microarray analyses and divided the pattern of expression into three groups: (a) genes that showed significantly stable levels of expression in iPS cells, (b) genes that showed a substantial degree of variability in expression in both human ES and iPS cells, and (c) genes that showed aberrant expression levels in some human iPS cell lines, as compared with human ES cells. In general, iPS cells have a rather stable expression of their imprinted genes. However, we found a significant number of cell lines with abnormal expression of imprinted genes, and thus we believe that imprinted genes should be examined for each cell line if it is to be used for studying genetic diseases or for the purpose of regenerative medicine.