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Published in final edited form as: J Cell Biochem. 2021 Oct 19;123(2):147–154. doi: 10.1002/jcb.30164

Role of Epigenetics in Cellular Reprogramming; from iPSCs to Disease Modeling and Cell Therapy

Yong Li 1, Radbod Darabi 2
PMCID: PMC8860854  NIHMSID: NIHMS1747974  PMID: 34668236

Abstract

Epigenetics play a fundamental role in iPSC technology due to their effect on iPSC’s reprogramming efficiency and their subsequent role in iPSC differentiation toward a specific lineage. Epigenetics can skew the differentiation course of iPSCs toward a specific lineage based on the epigenetic memory of the source cells, or even lead to acquisition of new cell phenotypes, due to its aberrations during reprogramming. This viewpoint discusses key features of the epigenetic process during iPSC reprogramming/differentiation and outlines important epigenetic factors that need to be considered for successful generation and differentiation of iPSCs for downstream applications.

Introduction

Epigenetics can be defined as any mechanism involved in alteration of gene expression without modification of the gene sequence (Berger, Kouzarides, Shiekhattar, & Shilatifard, 2009). There are about 25,000 genes in mammals which direct the development of almost 200 different cell types in the body. This specification is closely orchestrated at the level of gene expression through epigenetics. Epigenetics often function through chromatin modification to allow for inheritance of altered gene expression, without changing the gene sequence (Kornberg, 1974). During the early stages of embryo development, different tissue progenitors are initially formed through activation of the lineage-specific transcription factors and genes. Meanwhile, this process is simultaneously modified through complex epigenetic mechanisms that alter gene expressions at the level of chromatin and DNA. These modifications stabilize of the gene expression program and guide the differentiation of specific cell subsets forming different tissues (Xu, Li, Liu, & Gao, 2021). Interestingly, epigenetic modifications are rather stable and can be inherited though subsequent cell divisions, leading to the generation of daughter cells that are committed to their “differentiation fate”. This process is the landmark of the “epigenetic memory” in the cells, which is quite stable and inheritable during cell division in somatic cells (Zovkic, 2021).

At the cytological level, active or repressed chromatin can be identified through staining. Using chromatin dyes, active regions represent as decondensed, light stained chromatin, called “euchromatin”. Repressed chromatin stain as compacted regions with dense staining called “heterochromatin” (Shahid, Simpson, Miao, & Singh, 2021). This state “on versus off” situation of the chromatin in each cell’s nucleus is tightly regulated by several mechanisms which modify the genes at the level of DNA. Among these, DNA methylation is one of the well-known epigenetic mechanisms modifying chemical structure of the DNA and thus altering its expression (Holliday & Pugh, 1975). In this process, catalyzed by DNA methyltransferases (DNMTs) (Gruenbaum, Cedar, & Razin, 1982), a methyl group is added predominantly at cytosine bases at CpG dinucleotide sites in noncoding regions and leads to formation of 5‑methylcytosine (5mC) (Bird, Taggart, Frommer, Miller, & Macleod, 1985). The presence of the methyl group interferes with binding of the transcription factors and thus represses the gene expression (Razin & Riggs, 1980). In addition, recruitment of other proteins with repressing activity to the methylation sites is another mechanism of gene silencing in this case. The importance of DNA methylation in gene expression is also well documented through study of cancerous cell lines, in which global DNA hypomethylation (activation) along with hypermethylation (suppression) of tumor suppressor genes have been demonstrated (Feinberg & Tycko, 2004). Thus DNA methylation is a major epigenetic mechanism in suppression of transcription in human genome.

Another important epigenetic mechanism is histone modification through acetylation, phosphorylation, or methylation (Allfrey, Faulkner, & Mirsky, 1964). Histones are protein octamers responsible for DNA wrapping and packaging by forming nucleosomes, the basic chromatin units. In case of histone acetylation, by addition of a negatively charged acetyl group to lysine residues of the histone proteins through histone acetyl transferases (HATs), electrostatic affinity between DNA and histone is reduced leading to a more euchromatin state and promoting gene transcription. Conversely, histone deacetylation occurs through the actions of histone deacetylase (HDAC) and leads to repression of transcription (Hassig & Schreiber, 1997). Phosphorylation is also another histone modification which has important roles in chromosome segregation during cell division as well as activation of DNA repair mechanisms (Mahadevan, Willis, & Barratt, 1991). Finally, methylation can also happen at the level of histone, which is one of the most complex epigenetic mechanisms leading to transcriptional activation or repression, based on methylation of different sites. For example, methylation at H3K4, H3K36, and H3K79 can lead to activation of transcription, while at H3K9, H3K27, H3K64, and H4K20, it has the reverse effect leading to repression (Verdin & Ott, 2015). Several histone demethylases have also been identified so far, which play important and different roles in epigenetic regulation of gene expression.

Aside these histone modifications, ATP-dependent chromatin-remodeling complexes are another layer of epigenetic regulation of the gene expression at the histone level (Tsukiyama & Wu, 1995). These protein complexes can change the structural properties of the chromatin and nucleosome thus affecting transcription of the genes. In addition, recent data also support the interdependence and cross talk between histone modification and DNA methylation mechanisms (Cheung, Allis, & Sassone-Corsi, 2000; Kondo, 2009), which might play an important role in the control of the epigenetic changes.

Noncoding RNAs (ncRNAs) are another important mechanisms which can modify gene expression or chromatin structure through transcriptional silencing or recruitment of protein complexes responsible for chromatin modification (Fire et al., 1998; Volpe et al., 2002). These include RNA interference (RNAi), microRNAs (miRNA) and long non-coding RNAs (lncRNAs), which are responsible for transcriptional or post-transcriptional gene silencing. In the case of lncRNAs, they can directly interact with DNA or histone methylation machinery to recruit modifying enzymes at particular genomic sites (Di Ruscio et al., 2013). lncRNAs can also play the role of scaffolds for recruitment of different protein complexes at a genomic locus for gene activation or silencing(Sun et al., 2016). Thus RNA molecules add another layer of control over the gene transcription and play crucial role in epigenetic regulation of transcription.

Epigenetic inheritance and its role in cell fate determination

During early embryogenesis, tissue-specific transcription factors along with epigenetic modifiers harmonize together to determine the identity of different cellular progenitors. These allow the maintenance of precise configuration of gene expression and stable chromatin patterns in specific cell types which allows for the formation of different organs in a developing embryo (Elsherbiny & Dobreva, 2021). After initial specification of the progenitors’ identities, stability of the chromatin patterns and the epigenetic profile of each cell type is essential for propagation of identical cells during multiple rounds of division. This is also referred as “epigenetic memory” (Bird, 2002). Thus, epigenetic memory allows for maintaining similar cell identity during multiple divisions without modification of DNA sequence or any inductive signaling.

Fine tuning of these processes are essential for the formation of a multicellular organism with a diversified portfolio of tissue progenitors as well as their subsequent differentiation toward mature cells. In addition, epigenetic inheritance in cells requires a stable epigenetic signature to remain unchanged during DNA replication, and with immediate functionality in the newly synthesized DNA of the daughter cells (Lacal & Ventura, 2018). This property is well reflected during cell division and mitosis of different tissue stem cells. In “symmetric cell division”, parental histone H3/H4 tetramers are equally distributed between the two newly synthesized DNAs during DNA replication (Petryk et al., 2018). This equal distribution of parental histones allows for formation of nascent chromatin containing similar ratios of parental versus new chromatin tetramers in new daughter cells and keeping a rather stable epigenetic memory. Consequently, in the symmetric cell division, the progenies will be identical to the parent stem cell. On the other hand, to promote cell differentiation, many stem cells go through “asymmetric cell divisions” as well. In this scenario, H3—H4 tetramers are asymmetrically segregated toward daughter cells and one cell receives majority of the parental histones, leading to maintenance of parental stem cell properties, while the other cell will contain mostly new synthesized histone with a remodeled epigenetic pattern, which derives the cell toward a different differentiation path (Wooten, Ranjan, & Chen, 2020). It is important to mention that the pattern of chromatin inheritance is more conserved in condensed heterochromatin/repressed regions, while is less efficient and more complex in open chromatin and active genes.

Another example of epigenetic inheritance in heterochromatin is histone methylation. Among histone modifications, histone methylation has an undeniable role in epigenetic memory of the cells due to its relatively long half-life and high stability. For example, histone lysine trimethylation at lysine 27 (H3K27me3) is unique marker of heterochromatin/gene repression and its inheritance during cell division plays an important role in controlling cell fate. H3K27me3 is deposited by Polycomb repressive complexes (PRC) and subsequently leads to chromatin compaction (Di Croce & Helin, 2013). Recent data indicates the inheritance pattern of H3K27me3 during cell division plays important roles in repression of critical developmental genes and cell fate control during early phases of embryo development (Jadhav et al., 2020). Similar roles have also been discovered for inheritance of H3K9me3 heterochromatin domains and its modifiers in silencing inappropriate genes during early embryogenesis (Nicetto & Zaret, 2019).

Inheritance of active euchromatin such as histone acetylation is however more complex and less predictable than heterochromatin regions. As parental histone patterns from euchromatin regions are not generally inherited over cell division, continued expression of active genes in these regions of chromatin is mainly induced by the presence of other regulatory factors and signals in the cells. One of the major mechanisms governing inheritance of active gene program during cell division is through “mitotic bookmarking” of the cells (Festuccia, Gonzalez, Owens, & Navarro, 2017). In this process, during formation of mitotic chromatin, lineage-specific transcription factors (TFs) can bind to their specific binding sites in mitotic chromatin and “bookmark” lineage-specific programs that are needed to be activated in daughter cells, thus directly facilitate the inheritance of active gene programs in daughter cells.

Finally, other variables in cellular microenvironment can also change the epigenetic landscape of the dividing cells and thus affect the cell fate. One example is the changes in the oxygen level and the presence of hypoxia. Hypoxia-inducible factors (HIFs) are major players of this scenario which can modulate cellular epigenetics(Nakamura, Shi, Darabi, & Li, 2021). They can modify DNA methylation by changing the level of universal methyl donor, S-adenosylmethionine (SAM) (Hermes, von Hippel, Osswald, & Kloor, 2005), affecting expression of DNMTs (Skowronski, Dubey, Rodenhiser, & Coomber, 2010), or even cause histone modifications by altering the expression of histone modifiers (Krieg et al., 2010).

Role of epigenetics in stem cell differentiation and generation of lineage progenitors

As pluripotent stem cells gradually differentiate into various lineage progenitors, there is a global gain of methylation to warrant the silencing of pluripotency or non-specific genes (Meissner et al., 2008). Meanwhile, some specific loci also gradually lose methylation. This demethylation and subsequently gene activation happens in lineage-specific genes to allow appropriate cell fate determination (Nagae et al., 2011). For example, in case of skeletal muscle development, fine tuning and activation of myogenic genes and transcription factors through demethylation is tightly regulated by epigenetics. The role of DNA demethylation in activation of muscle-specific genes was initially confirmed during the study of the effect of 5-azacytidine treatment (a potent DNA methylation/DNMT inhibitor) on mouse fibroblasts and their cell fate change into myogenic cells due to MyoD activation (Constantinides, Jones, & Gevers, 1977; Davis, Weintraub, & Lassar, 1987). Indeed demethylation of promoter and enhancer regions of myogenic regulatory factors (MRFs) such as Myf5 and MyoD is the key driver of activation of myogenic program during early development (Brunk, Goldhamer, & Emerson, 1996; Carrio et al., 2015). Study of the CpG islands in human skeletal muscle samples also indicated that while major non-muscle lineage-specific markers (such as genes associated with osteogenic, adipogenic and neurogenic lineages) are hypermethylated in muscle progenitor cells leading to their silencing, genes related to muscle contractile proteins are specifically hypomethylated (Calvanese et al., 2012; Sorensen, Jacobsen, Reiner, Andersen, & Collas, 2010). As muscle cell progenitors shift from initial activation and proliferation phases to later stages of differentiation into mature myofibers, temporal shift in myogenic gene activation is carefully orchestrated by specific DNMT and TET isoforms (ten eleven translocation enzymes, responsible for oxidation of 5mCs and locus –specific reversal of DNA methylation) modifying methylation status at specific loci (Aguirre-Arteta, Grunewald, Cardoso, & Leonhardt, 2000; Tsumagari et al., 2013). This pattern can be observed by differentiation-dependent demethylation of promoter or enhancer regions of the late myogenic genes such as Myogenin and Desmin at terminal stage of myotube differentiation in myogenic cells (Lindahl Allen, Koch, Clelland, Dunham, & Antoniou, 2009; Lucarelli, Fuso, Strom, & Scarpa, 2001). Also hypomethylation and/or hydroxymethylation of the Notch signaling pathway ligands and receptor genes (an important governing pathway for myogenesis) have been demonstrated in skeletal muscle tissue (Terragni et al., 2014), which is another indication of the important role of epigenetics in controlling gene expression and signaling pathways in a tissue-specific manner and throughout differentiation process.

It is also noteworthy to mention that expression and activity of DNMT and TET isoforms is also regulated by metabolism through the availability of methyl donors or precursors generated by different metabolic pathways and within the microenvironment (Teperino, Schoonjans, & Auwerx, 2010). Consequently any significant change in epigenetic landscape or microenvironment of the iPSCs during differentiation process might have the potential to induce a major shift in differentiation stage of the target cells. This is a critical point if the iPSCs are used for developmental studies, disease modeling or regenerative purposes, as for each application a specific cell type in a particular stage of differentiation (such as progenitors, committed or fully differentiated cells) might be needed. Therefore, optimization of the cell culture media composition (microenvironment) and growth incubation parameters (such as O2 and CO2 percent) is essential to achieve a sustainable differentiation with appropriate cell type for each application.

Role of epigenetics in Cellular Reprogramming

Initial reprograming experiments on somatic cell nuclear transfer (SCNT) and successful generation of cloned frogs from differentiated amphibian cell nuclei, revealed that cellular differentiation is due to reversible epigenetic alterations (Briggs & King, 1952). Thus, the nuclei of differentiated cells contain all genomic information necessary to reactivate pluripotency and can reverse cellular differentiation. In later studies, generation of induced pluripotent stem cells (iPSCs) from somatic cells by ectopic expression of Yamanka factors (OCT4, SOX2, KLF4, and cMYC—OSKM) further confirmed the reversibility of the differentiation into pluripotency by overexpression of key transcription factors (Takahashi & Yamanaka, 2006). The pattern of iPSC reprogramming is a stepwise and well organized process involving gradual silencing of the somatic genes, followed by upregulation of pluripotency factors. In an ideal condition, reprogramming is managed by remodeling of the epigenetic landscape of the somatic cells into the pluripotent state. However, some evidence points to the low efficiency of this process during reprogramming into iPSCs. The first indication is overall low efficiency of iPSC reprogramming (typically less than 1%) among identical cells receiving reprogramming factors. This reprogramming barrier is mainly due to the transcriptional and epigenetic status of the source cells (Bhutani et al., 2010). Since the binding of Yamanaka factors to enhancement and promoter regions is a necessary step for recruitment and activation of transcription machinery to initiate pluripotency program in somatic cells during reprogramming, reported low efficiency might be partly due to condensed chromatin in somatic cells, which can be bypassed using chromatin modifiers(Singhal et al., 2010). In addition, genome-wide epigenetic evaluations also have confirmed correct reprogramming of epigenetic landscape (i.e. histone modifications and DNA methylation pattern in somatic vs. pluripotency genes) in validated iPSC lines, while in partially reprogrammed iPSCs this pattern is incomplete (Mikkelsen et al., 2008).

Another piece of evidence which supports the important role of epigenetics in reprogramming is the considerable variability of the reprogramming efficiency among different somatic cell types, based on their tissue source, age or their differentiation status (Eminli et al., 2009; Nazor et al., 2012). Comparative epigenetic studies on iPSCs have demonstrated the presence of significant variations in DNA methylation patterns among different iPSC lines. These variable patterns in DNA methylation of iPSCs can be due to the partial persistence of the source cell memory or generation of the aberrant methylation patterns during reprogramming (Kim et al., 2010). Indeed, the presence of residual repressive hypermethylation from source cells (due to insufficient demethylation during reprogramming), leads to resistance and weak differentiation potential of the derived iPSCs into other lineages, with skewed tendency toward their parental lineage (due to insufficient methylation/repression of their source cell somatic-specific gene loci). This “remaining epigenetic memory” from source cell can be weakened or erased by serial passaging of iPSCs or using chromatin modifying chemicals (Polo et al., 2010). Epigenetic memory of the iPSCs can also be rearranged to the desired pattern in case of regenerative applications. This can be done using chromatin modifying agents or by their differentiation into a target lineage, followed by subsequent reprogramming into iPSCs, which significantly improves differentiation of newly generated iPSCs into target lineage (Kim et al., 2010).

Role of epigenetics in disease modeling and tissue regeneration using iPSCs

With the significant advancement of iPSC technology for generation of integration-free iPSCs, the application for in vitro disease modeling as well as development of patient-specific stem cell therapy is more in reach. For either of these applications, generation of tissue-specific progenitors or differentiated cells from iPSCs is essential. However, due to the presence of potential genetic modifiers (such as chromosomal abnormality or copy number variations) and epigenetic variations (such as aberrant DNA methylation, source cell memory or erosion of dosage compensation due to variability in X chromosome inactivation) in iPSCs, their potential to differentiate into a specific tissue lineage might be significantly varied among different iPSC lines (Bar & Benvenisty, 2019; Liang & Zhang, 2013). In addition, the presence of these clonal variations in iPSCs might lead to unexpected alterations or addition of new cellular phenotypes in differentiated cells (Mekhoubad et al., 2012). This is particularly important in case of in vitro disease modeling, as it might lead to wrong interpretation of the new phenotype as a disease related one. On the other hand, for cell therapy using iPSCs, these variables play different roles. For example, source cell memory and biased differentiation of iPSCs to their source lineage might be beneficial if the iPSC cell source was collected from affected tissue in patient. If not, as mentioned above, source cell memory can be suppressed by serial passaging of iPSCs or be reshaped toward desired lineage by iPSC differentiation into desired lineage, followed by repeated reprogramming into new iPSCs, which will have higher tendency to differentiate into target lineage (Kim et al., 2010).

Another consequence of genetic or epigenetic clonal variations in iPSCs is the risk of development of abnormal cell phenotypes during differentiation. This can lead to generation of cell populations with limited expansion or differentiation, early senescence or reduced self-renewal potential (Feng et al., 2010). In addition, these epigenetic variations might affect oncogenic genes and increase the risk of tumorigenesis (Anguera et al., 2012; Ben-David & Benvenisty, 2011). These risks can be reduced by genomic and epigenetic screening of iPSCs and in vivo transplantation studies in animal models to study their long-term regeneration potential, identify any side-effects and eventually exclude affected iPSCs lines.

Closing remarks

Although iPSCs provide a great opportunity for personalized medicine, there are still major safety concerns such as the risk of tumorigenicity due to genetic or epigenetic alterations during reprogramming, reactivation of pluripotency genes after differentiation into target cells or the presence of poorly differentiated and potentially tumorigenic cells due to incomplete characterization of differentiated cells. Since epigenetics plays a key role in all steps of iPSC technology, from its initial generation steps to its final differentiation, it is important to pay attention to these variables during the entire process. Based on the application purpose of the iPSCs, the risk of uncontrollable genetic or epigenetic variations can be significantly reduced using few strategies (as demonstrated in Figure-1). These include proper and wise selection of source cells to obtain a homogenous cell population with correct epigenetic profile (based on downstream application), selection of chemically defined media and cytokines during reprogramming and differentiation to provide appropriate external signaling and minimize epigenetic aberrations, using non-integrating methods for iPSC generation, proper culture and passaging of iPSCs to reduce epigenetic memory of the source cells and stabilize iPSCs, comprehensive genetic and epigenetic screening of the iPSC lines to ensure lack abnormalities, paying extra attention to the possible role of epigenetic abnormalities as a potential cause of abnormal cell phenotype in disease modeling studies using iPSCs, and full characterization using in vitro differentiation as well as in vivo teratoma/tumorigenesis studies to ensure safe cell prep for regenerative applications. These precautionary guidelines mark the way toward successful derivation of quality iPSC lines with proper genetic and epigenetic profile useful for disease modeling or regenerative applications.

Figure 1. Important factors affecting the epigenetic landscape of the iPSCs.

Figure 1

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As demonstrated, important factors affecting the epigenetic landscape of the iPSCs have been listed for each stage of the reprogramming process. Careful consideration of these factors are essential for generation of ideal iPSCs for downstream applications.

Acknowledgements

This work was supported by the funding from the National Institute of Arthritis, and Musculoskeletal and Skin Diseases (NIAMS) of the NIH under award numbers 1R01AR068293 and 1R01AR076770 to R.D. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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