Title: Epigenetic aberrations in human pluripotent stem cells
Abstract: Review14 May 2019free access Epigenetic aberrations in human pluripotent stem cells Shiran Bar Department of Genetics, The Azrieli Center for Stem Cells and Genetic Research, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel Search for more papers by this author Nissim Benvenisty Corresponding Author [email protected] orcid.org/0000-0001-8234-2685 Department of Genetics, The Azrieli Center for Stem Cells and Genetic Research, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel Search for more papers by this author Shiran Bar Department of Genetics, The Azrieli Center for Stem Cells and Genetic Research, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel Search for more papers by this author Nissim Benvenisty Corresponding Author [email protected] orcid.org/0000-0001-8234-2685 Department of Genetics, The Azrieli Center for Stem Cells and Genetic Research, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel Search for more papers by this author Author Information Shiran Bar1 and Nissim Benvenisty *,1 1Department of Genetics, The Azrieli Center for Stem Cells and Genetic Research, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel *Corresponding author. Tel: +972 2 6586774; E-mail: [email protected] EMBO J (2019)38:e101033https://doi.org/10.15252/embj.2018101033 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Human pluripotent stem cells (hPSCs) are being increasingly utilized worldwide in investigating human development, and modeling and discovering therapies for a wide range of diseases as well as a source for cellular therapy. Yet, since the first isolation of human embryonic stem cells (hESCs) 20 years ago, followed by the successful reprogramming of human-induced pluripotent stem cells (hiPSCs) 10 years later, various studies shed light on abnormalities that sometimes accumulate in these cells in vitro. Whereas genetic aberrations are well documented, epigenetic alterations are not as thoroughly discussed. In this review, we highlight frequent epigenetic aberrations found in hPSCs, including alterations in DNA methylation patterns, parental imprinting, and X chromosome inactivation. We discuss the potential origins of these abnormalities in hESCs and hiPSCs, survey the different methods for detecting them, and elaborate on their potential consequences for the different utilities of hPSCs. Introduction Human pluripotent stem cells (hPSCs) represent the in vitro counterparts of embryonic cells in human pre-implantation development. Their ability to self-renew and differentiate into all three germ layers in culture establishes them as highly valuable in research of early human embryogenesis and disease modeling as well as a promising source for regenerative therapy (Ben-David et al, 2012). Apart from their abundant potential, the unique properties of hPSCs also contribute to their tumorigenicity and genomic instability. Injecting hPSCs under the skin of immunodeficient mice leads to the formation of benign teratomas, which are comprised of differentiated cells of all three germ layers. Yet, following extended growth in culture, these cells may undergo selection for altered karyotypes, which can subsequently give rise to aggressive tumors. The prevalence and consequences of genetic aberrations in hPSCs are the subject of extensive discussions (Ben-David & Benvenisty, 2011). These modifications include chromosomal abnormalities, copy number variations, and point mutations which are repeatedly selected during hPSC propagation. Whereas variations in TP53 were recently found to significantly dominate the landscape of point mutations in hPSCs (Merkle et al, 2017), the most common chromosomal abnormalities in hPSCs include full or partial trisomies of chromosomes 1, 8, 12, 17, and X, and duplications of 20q11.21 (Lund et al, 2012; Weissbein et al, 2014). As similarly observed in a variety of tumors, these increased copy number variations are thought to confer selective advantage of cultured cells by upregulating growth-promoting genes. Notwithstanding, in addition to such genetic changes, epigenetic aberrations are also inherently capable of affecting the dosage of gene expression. Markedly, epigenetic changes affect cellular phenotypes and emerge extensively in culture, but although they are inherited through mitosis, they are not associated with changes in genomic sequences. Epigenetic abnormalities include deviations in DNA methylation, histone modifications, and other inherited chromatin marks, which drive changes in gene expression and cellular integrity (Flavahan et al, 2017). In this review, we focus on three epigenetic abnormalities, which were most studied in hPSCs and include DNA methylation alterations, loss of parental imprinting, and variation in X chromosome inactivation. These aberrations significantly influence transcription of individual genes, up to an entire chromosome. We discuss the forces driving these aberrations (including when and how they might arise), outline different methods to detect them, and examine their consequences for applications of hPSCs. Human pluripotent stem cell types hPSCs can be classified by the method used to derive them. Human embryonic stem cells (hESCs) were the first hPSCs to be successfully propagated in vitro and they are isolated from the inner cell mass (ICM) of in vitro fertilized embryos. Yet, this requirement for human blastocysts bears ethical concerns and limits the capacity to generate profuse and diverse hESC lines. Therefore, the generation of human-induced pluripotent stem cells (hiPSCs) by direct reprogramming from somatic cells was a promising advancement and enabled more laboratories to produce new cell lines and join the field (Hochedlinger & Jaenisch, 2015). Moreover, isolating hiPSCs from disease patients potentially adds a significant progress toward transplantations of autologous differentiated cells. More recently, reprogramming was also achieved by introducing a somatic nucleus into an enucleated oocyte to generate human somatic cell nuclear transfer PSCs (SCNT-PSCs; Tachibana et al, 2013; Yamada et al, 2014). Although hPSCs that are grown in standard culture conditions mimic most functional properties of ICM in differentiation, they significantly diverge in their epigenetic and transcriptional landscapes. These dissimilarities prompted the search for culture conditions that will reset and maintain hPSCs in a “naïve” state, which presumably better resemble pluripotent cells in vivo. Consequently, there are currently several different protocols for isolating and resetting hPSCs to become naive (Sagi & Benvenisty, 2016; Bates & Silva, 2017; Collier & Rugg-Gunn, 2018). In this review, we will discuss and compare epigenetic aberrations in the different types of hPSCs, including hESCs, hiPSCs, SCNT-PSCs, and naïve hPSCs. Types of epigenetic aberrations in hPSCs, their origins, and detection methods DNA methylation patterns The most extensively studied epigenetic modification is DNA methylation, in which methyl groups are added to the fifth carbon of cytosine residues, thus forming 5-methylcytosine (5-mC), mainly in the context of CpG dinucleotides. In mammals, 5-mC is prevalent in diverse genomic regions, including transposable elements, imprinted regions, and gene bodies, as well as in some inactive promoters, whereas CpG-rich regions are mostly devoid of methylation (Suzuki & Bird, 2008). This modification is maintained during replication and is considered relatively stable in somatic cells, yet various reports also emphasize its dynamic regulation during development (Hackett & Surani, 2013; Messerschmidt et al, 2014), its sensitivity to aging and environmental forces (Jung & Pfeifer, 2015; Mitchell et al, 2016), and high frequency of alterations in many cancers (Flavahan et al, 2017). Although DNA methylation is mostly associated with gene repression, its regulatory function is emerging as more complex and dependent on CpG density. Thus, in many instances, 5-mC is apparently secondary to other repressive marks and contributes to sustaining heterochromatic memory and long-term gene silencing. Accordingly, the absence of methylation at a promoter does not necessarily prompt the activation of its cognate gene, as additional factors are required for transcription initiation (Hackett & Surani, 2013). Nevertheless, the function of 5-mC is critical in silencing transposons and also in disrupting their sequence over time by favoring mutations through deamination. These roles were suggested to be the drivers for 5-mC selection during evolution (Yoder et al, 1997). After fertilization, DNA methylation is globally erased, reaches a minimum in the blastocyst stage, and is then re-established during differentiation (Iurlaro et al, 2017). Although human ICM cells exhibit very low levels of genome-wide DNA methylation, hPSCs are globally hypermethylated even when compared with somatic cells (Nishino & Umezawa, 2016). Moreover, the global persistence of DNA methylation is essential for hPSC survival, as knockout of DNA methyltransferase 1 (DNMT1), which is responsible for catalyzing the addition of 5-mC on the newly replicated strand during S phase, results in rapid cell death (Liao et al, 2015). Similar consequences are observed following treatment with the chemical demethylating agent 5-aza-2′-deoxycytidine (5-azadc; Bar-Nur et al, 2012). The necessity for DNMT1 in hPSCs is in complete contrast to mouse PSCs, which are resistant to simultaneous knockout of Dnmt1, Dnmt3a, and Dnmt3b (Tsumura et al, 2006; Liao et al, 2015). The significant differences in DNA methylation between human blastocysts and hPSCs, along with discrepancies in gene expression, are a significant driver in the research of naive hPSCs. Therefore, naive hPSCs should exhibit similar epigenetic features to that of ICM, including global hypomethylation (Theunissen et al, 2016 ) and resistance to DNMT1 downregulation. DNA methylation aberrations have been observed in various hPSC lines (Lund et al, 2012). These include 5-mC variations in gene promoters (Fig 1) and non-coding regions, as well as residual DNA methylation signatures from somatic cells in iPSCs. Accumulating evidence illustrates similarities between gene-specific DNA methylation aberrations in hPSCs and those prevalent in malignancies (Planello et al, 2014; Konki et al, 2016; Weissbein et al, 2017). The generation of more accurate and comprehensive maps of whole-genome methylation should promote additional analyses on gene-specific 5-mC aberrations in hPSCs, which may enhance our understanding of their implications and elaborate on the epigenetic dynamics in these cells. Figure 1. Types of epigenetic aberrations in hPSCsTop: DNA methylation aberrations in hPSCs are mainly caused by promoter hypermethylation and lead to gene silencing. Center: In normal hPSCs, imprinted genes are expressed from either the paternal or maternal allele. Loss of imprinting involves hypomethylation of imprinted DMRs, driving aberrant biallelic expression of imprinted genes. Bottom: Female cells in early pre-implantation development have two active X chromosomes. Later in development, they initiate XCI, which is achieved by XIST coating and results in silencing one X chromosome randomly. Many hPSCs exhibit aberrant erosion of XCI, which is characterized by XIST repression and partial reactivation of genes from the silent X chromosome. Download figure Download PowerPoint Origins of DNA methylation aberrations Analyzing DNA methylation in multiple hPSC lines identified characteristic patterns that distinguish them from somatic cells (Bibikova et al, 2006; Deng et al, 2009), including abnormal hypermethylation of tumor suppressor genes (Calvanese et al, 2008). During growth in culture, it has been shown that these cells acquire variable DNA methylation aberrations in multiple sites (Bock et al, 2011; Nazor et al, 2012), many of which also appear in tumors (International Stem Cell Initiative et al, 2011). These culture-induced aberrations are mostly stable and persist throughout differentiation (Allegrucci et al, 2007; Nazor et al, 2012). Using genome-wide methylation maps of hPSCs as a resource, a separate study concentrated on recurrent gene-specific alterations and detected several genes that repeatedly gain methylation in hPSCs of higher culture passage, and whose expression was silenced. Subsequently, this study focused on the gene TSPYL5, which was also implicated in several tumors, demonstrating that its deletion in cells in which it was still active, resulted in overexpression of pluripotency and growth-related genes as well as downregulation of tumor suppressors and genes associated with differentiation (Weissbein et al, 2017). Additional studies identified frequent hypermethylation and reduced expression of the antioxidant gene catalase (CAT; Bock et al, 2011), occurring specifically in hPSCs featuring an abnormal karyotype (Konki et al, 2016). Particularly, hPSC lines, which are prone to chromosomal instability, undergo a gradual methylation increase near the start site of this gene in normal and abnormal cells, suggesting that DNA methylation aberrations in a gene involved in reducing oxidative stress and DNA damage may predispose these cells toward the accumulation of genetic aberrations. Correspondingly, the authors also found the same aberrations in embryonal carcinoma cell lines and downregulation of CAT in several other cancers (Konki et al, 2016). Interestingly, the majority of reported aberrations in hPSCs involve gaining of methylation accompanied by gene silencing (Fig 1). Such hypermethylation requires the alteration of both alleles, whereas gene activation is potentially feasible following hypomethylation of a single allele. A possible explanation for this potential discrepancy may be that loss of methylation of a growth-promoting gene might not be sufficient for its activation. Alternatively, this could also be attributed to the global tendency of hPSCs toward hypermethylation, or to a technical limitation of studies utilizing methylation arrays, which are mostly enriched in non-methylated CpG islands. Overall, these studies signify that gene-specific methylation aberrations, which are also present in cancers, are strongly selected during hPSC growth in vitro (Fig 2). As these selection forces are apparently similar to those driving genetic aberrations, previous recommendations for improving cell-culture practices aimed at minimizing cellular stress (Weissbein et al, 2014) could also be beneficial for reducing the selection toward DNA methylation abnormalities in hPSC. Still, since epigenetic modifications are also directly affected by the environment, further research aimed at finding methods to amend culture conditions to support the stability of DNA methylation is necessary. Figure 2. Origins of epigenetic aberrations in hESCs and hiPSCs5-methylcytosine (5-mC) aberrations accumulate following continuous growth in culture. Newly reprogrammed hiPSCs preserve somatic DNA methylation memory. Loss of imprinting (LOI) emerges mainly during reprogramming and transition to naïve pluripotency, but can also spread in culture of hPSCs and somatic cells. Erosion of X chromosome inactivation (XCI) in hESCs appears during their derivation and becomes widespread in very early passages. hiPSCs maintain XCI in early passages, but erosion can occur over time. Dark color represents in vitro processes that are more frequently associated with the epigenetic aberration. Download figure Download PowerPoint In addition to their emergence over time in culture, DNA methylation aberrations were also observed to be highly induced upon reprogramming of human somatic cells to iPSCs (Fig 2). This process encompasses substantial epigenetic changes, transforming the chromatin landscape of a differentiated cell to that of an undifferentiated one by inducing the expression of key pluripotency genes, thus leading to broad changes in the overall transcription pattern. Accordingly, the efficiency of reprogramming was found to be inversely correlated with the extent of differences in CpG methylation between the somatic cell-of-origin and hPSCs (Ruiz et al, 2012). Correspondingly, many reported hiPSC lines were insufficient in completely erasing their somatic identity, thus carrying residual methylation of their source cells in various regions. This resulted in an epigenetic memory at distinct regions which bear differential methylation between hESCs and hiPSCs (Kim et al, 2010, 2011; Bar-Nur et al, 2011; Lister et al, 2011; Ohi et al, 2011; Roost et al, 2017). hiPSCs were also shown to maintain somatic non-CG methylation, especially near centromeres and telomeres in regions marked by H3K9me3 (Lister et al, 2011). Nevertheless, reprogramming mouse and human somatic cells by nuclear transfer facilitated a less aberrant methylation pattern which is similar to that of ESCs (Kim et al, 2010; Ma et al, 2014). Additional methylation alterations, which are not found in either the normal somatic source cells or hESCs, are acquired during reprogramming and vary between different hiPSC lines (Doi et al, 2009; Bock et al, 2011; Lister et al, 2011; Nishino et al, 2011; Koyanagi-Aoi et al, 2013; Planello et al, 2014; Nishino & Umezawa, 2016; Tesarova et al, 2016). It was shown that employing different combinations of reprogramming factors result in distinct patterns of 5-mC aberrations. Reprogramming with Yamanaka factors (OCT4, SOX2, KLF4, and cMYC) is mostly associated with increased methylation in specific regions, while Thomson factors (OCT4, SOX2, NANOG, and LIN28) induce reduced methylation in different locations (Planello et al, 2014). This suggests that induction of such abnormalities is possibly due to the massive epigenetic perturbation which is provoked in this process (Liang & Zhang, 2013; Fig 2). Importantly, some of these methylation aberrations are also implicated in different tumors (Ohm et al, 2010) and are transmitted throughout differentiation (Lister et al, 2011; Ruiz et al, 2012). Nevertheless, while somatic memory and aberrations in DNA methylation exist in early-passage iPSCs, some studies found that many of them were diminished over time, at which point iPSCs become highly similar to ESCs in their methylation pattern (Nishino et al, 2011; Nishino & Umezawa, 2016; Tesarova et al, 2016), whereas others demonstrated a preservation of epigenetic memory over time (Kim et al, 2011). Even though reprogramming is often considered the cause for acquiring methylation aberrations, it was shown that it can also enable the reversal of 5-mC alterations in some tumor suppressors and cancer-related genes, which were abnormally methylated and silenced in somatic cells (Ron-Bigger et al, 2010). Overall, various studies illustrate the dynamic regulation of DNA methylation in iPSCs, yet these variations are mostly stabilized during prolonged growth in culture. However, while reprogramming-related methylation aberrations are resolved at high passages, growth-related changes could also be selected at the same time, raising a conflict regarding the recommended culture practice for human iPSCs. Analysis of DNA methylation DNA methylation can be inspected at various resolutions for unraveling the distribution, specificity, stability, and effects of this modification. In the past, treating extracted DNA with methylation-sensitive enzymes (Cedar et al, 1979) followed by PCR amplification facilitated the regional analysis of 5-mC changes at specific genomic locations. In order to extend this resolution and obtain single nucleotide information, additional methods were developed based on bisulfite treatment, which specifically converts unmethylated cytosine to uracil, allowing the distinction between methylated and unmethylated CpGs. Bisulfite sequencing PCR or pyrosequencing implements this distinction to obtain single CpG data for similar loci-specific analysis (Frommer et al, 1992; Tost & Gut, 2007; Table 1). However, these techniques require prior knowledge of putative genes which are susceptible to aberrations. Thus, an unbiased screen of DNA methylation aberrations calls for a genome-wide evaluation of 5-mC at single nucleotide resolution. Formerly, this was mostly established by employing DNA methylation arrays, in which multiple probes enable the analysis of methylation at various genomic regions (Gitan et al, 2002; Weber et al, 2005), or by reduced representation bisulfite sequencing (RRBS), which combines bisulfite treatment with specific restriction enzymes to quantify methylation at regions with high CpG content (Meissner et al, 2005). Lately, the significant cost reduction of whole-genome sequencing is driving more laboratories to apply analysis of DNA methylation across the entire genome, by executing whole-genome bisulfite sequencing (WGBS; Lister et al, 2009; Table 1). More recently, WGBS was also performed in single cells (Smallwood et al, 2014), enhancing the sensitivity of such analyses. A newly established DNA methylation reporter offers investigation of 5-mC in single live cells, indicating dynamic methylation changes at specific loci (Stelzer et al, 2015). Table 1. Methodologies for detecting epigenetic aberrations While the abovementioned methods are useful for analyzing 5-mC, it does not readily indicate transcriptional alterations. Therefore, combining these methods with RNA quantification, as recently performed in parallel single-cell sequencing (Angermueller et al, 2016), is highly valuable for associating changes in methylation and gene expression, to ultimately achieve a comprehensive picture of DNA methylation aberrations in hPSCs. Parental imprinting Parental imprinting is a phenomenon that evokes monoallelic epigenetic silencing of certain genes in a parent-of-origin mode by incorporating 5-mC to discriminate between the maternal and paternal alleles in mammalian cells. This is enabled by the establishment of differentially methylated regions (DMRs) at specific loci of the oocyte and sperm genomes, which are subsequently maintained in the zygote and throughout development. These DMRs directly regulate the expression of nearby genes, leading to monoallelic silencing of ~ 100 imprinted genes (Bartolomei & Ferguson-Smith, 2011). Elegant nuclear transfer experiments that enforced activation of oocytes containing same-sex genomes demonstrated that imprinting poses the main barrier for uniparental reproduction in mammals (McGrath & Solter, 1984; Surani et al, 1984). Only extensive artificial editing and removal of several imprinted regions could overcome this impediment and successfully generate all-maternal (parthenogenetic), and recently also all-paternal (androgenetic) mice (Kono et al, 2004; Kawahara et al, 2007; Li et al, 2018). In humans, spontaneous parthenogenetic and androgenetic development may commence, but result in tumors. Moreover, loss of imprinting (LOI) at specific loci leads to developmental disorders such as Prader–Willi syndrome and Beckwith–Wiedemann syndrome. Nevertheless, imprinting is considered highly stable throughout life and across tissues, with only a few examples of tissue-specific imprinting mostly observed in the placenta (Frost & Moore, 2010). However, various studies identified considerable incidence of LOI in hPSCs, resulting in the expression of imprinted genes from both alleles instead of one, along with DNA methylation changes at imprinting DMRs (Rugg-Gunn et al, 2005; International Stem Cell Initiative et al, 2007; Pick et al, 2009; Nazor et al, 2012; Ma et al, 2014; Johannesson et al, 2014; Bar et al, 2017; Fig 1). Many imprinted genes reside in clusters and are conversely regulated by the same germline DMR. Thus, alteration in a single region can drive both biallelic expression and silencing of multiple genes (Bartolomei & Ferguson-Smith, 2011). Notably, imprinting aberrations persist following differentiation toward various cell types, thereby aggravating the consequences of LOI on hPSCs growth and integrity. The sources for loss of parental imprinting hPSCs acquire distinct DNA methylation signatures as compared to their cell type of origin (as detailed above). These epigenetic dynamics are a potential cause for the multiple reports on LOI in hPSCs. While studies examining the status of imprinted genes in ESCs concluded that they are for the most part stable and have relatively rare instances of LOI (International Stem Cell Initiative et al, 2007; Rugg-Gunn et al, 2007), inspection of iPSCs or SCNT-PSCs identified higher degrees of imprinting aberrations in these cells (Pick et al, 2009; Johannesson et al, 2014; Ma et al, 2014). Recently, a large-scale analysis of LOI in hundreds of hPSC samples was performed. The results corroborated significantly higher levels of LOI in iPSCs compared with ESCs (Bar et al, 2017). This analysis also revealed that some imprinted genes are already expressed biallelically in a proportion of parental fibroblast cells used for reprogramming, which is maintained and even expanded in some of the hiPSC lines, since they are derived from single-cell clones. Overall, these results suggest that imprinting is lost primarily during the reprogramming process, but also upon culturing fibroblasts and hPSCs (Fig 2). Nevertheless, while DNA methylation aberrations in most genes were shown to be relieved in iPSCs upon culture adaptation (Nishino et al, 2011; Nishino & Umezawa, 2016; Tesarova et al, 2016), the differential methylation at imprinted regions cannot be restored since restoration requires the distinction between maternal and paternal alleles, which is feasible only in the gametes. Furthermore, recent studies in mouse and human PSCs suggest that globally, DNA methylation in these cells is highly dynamic, cycling between de novo methylation and its erasure (Shipony et al, 2014; Rulands et al, 2018). However, this turnover is not observed in imprinted regions (Shipony et al, 2014), suggesting a special regulation for maintaining imprinting in PSCs. Collectively, these findings indicate that DMRs are more susceptible to aberrations during somatic cell reprogramming toward pluripotency, while being more tightly preserved in standard culture. In naive hPSCs, DNA methylation is substantially reduced, reaching similarly low levels as in pre-implantation development. However, contrary to the tight conservation of imprinting in the early embryo, methylation at most imprinted regions is lost in naive hPSCs of all current protocols (Pastor et al, 2016; Theunissen et al, 2016). Recent investigations also consistently discovered global erasure of DMRs in mouse naive PSCs following prolonged culture in 2i medium (Choi et al, 2017), propounding that further effort is required to improve these culture conditions as to prevent LOI. In addition to differences in LOI rates between various hPSC types, the tendency of different imprinted genes to exhibit biallelic expression also poses an intriguing comparison. Several lines of evidence establish that while some genes are mostly resistant to imprinting aberrations (SNRPN, PEG3), other genes feature frequent LOI across many hPSC lines (H19, IGF2, MEG3, ZDBF2; Kim et al, 2007; Nazor et al, 2012). The main difference between genes with distinct LOI frequencies was linked to the parent-of-origin methylation regulating their monoallelic expression: Imprinted genes governed by a paternally methylated DMR are much more prone to LOI as compared to those controlled by a maternally methylated DMR (Rugg-Gunn et al, 2007; Bar et al, 2017). Particularly, this difference is prominent in iPSCs, signifying that imprinted paternally methylated regions are more sensitive to aberrations during reprogramming. This implies that maternal and paternal alleles might incorporate different mechanisms for protecting imprinted regions from demethylation. A supportive indication for this suggestion is observed in the zygote, where maternal and paternal pronuclei are both extensively demethylated after fertilization, but at distinct rates, and thus they may recruit different pathways to regulate this process and maintain parental imprinting. Yet, another stimulating possibility for resolving the varying sensitivity toward aberrations of different imprinted genes was suggested to involve correlation with the exact developmental timing of gene activation, whereby prominent transcription during early pre-implantation stages is associated with increased safeguarding of imprinting in hPSCs (Rugg-Gunn et al, 2007). Interrogation of the exact expression time point of additional imprinted genes, which were implicated in more recent studies of LOI, is required to better assess this hypothesis. While LOI which emerges during reprogramming could take over the culture by means of clonal expansion, its spontaneous appearance during hPSCs growth is thought to occur rarely, only in a small subset of cells. Therefore, in order for the biallelic expression to spread, it should probably offer these cells a selective advantage of some sort. Such beneficial effect may include faster proliferation, reduced apoptosis, decreased differentiation, and increased colony formation. Biallelic activation of an imprinted gene is expected to cause a twofold increase in its expression, but while some genes follow this simple linear model, others are transcribed at higher (RTL1, IGF2) or lower (PEG10, SGCE, NDN) levels than anticipated, indicative of a more complex regulatory network (Bar et al, 2017). Correspon