Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation☆

Article Outline

• Abstract
• 1. Introduction
• 2. Epigenetic control of gene transcription
  • 2.1. Structural chromatin modifications by histone acetylation/deacetylation
    • 2.1.1. Role of histone deacetylases in the regulation of gene expression
    • 2.1.2. Classification of histone deacetylases and their role in differentiation programs
    • 2.1.3. HDAC inhibitors: types and effects
  • 2.2. DNA methylation
    • 2.2.1. Role of DNA methyltransferases in the regulation of gene expression
    • 2.2.2. Classification of DNA methyltransferases and their role in differentiation programs
    • 2.2.3. DNMT inhibitors: types and effects
  • 2.3. Interplay between DNA methylation and histone acetylation
• 3. Epigenetic modifiers as potent differentiation-promoting compounds in vitro
  • 3.1. Acquisition and stabilisation of a differentiated hepatic phenotype in vitro: an interplay between chromatin remodelling and liver-enriched transcription factors
  • 3.2. Effect of HDAC and DNMT inhibition on liver-specific gene expression
  • 3.3. Epigenetic modifiers: a key factor to (re)program stem cells in vitro?
    • 3.3.1. Stem cell signaling cascades
    • 3.3.2. The epigenetic control of stem cell differentiation
    • 3.3.3. Pluripotent embryonic stem cells and multipotent mesenchymal stem/progenitor cells as source of hepatocytes
      • 3.3.3.1. Pluripotent embryonic stem cells
      • 3.3.3.2. Multipotent mesenchymal stem cells
    • 3.3.4. Stem-cell derived hepatocytes or hepatocyte-like cells?
• 4. Conclusion
• Acknowledgements
• References
• Copyright

Controlling both growth and differentiation of stem cells and their differentiated somatic progeny is a challenge in numerous fields, from preclinical drug development to clinical therapy. Recently, new insights into the underlying molecular mechanisms have unveiled key regulatory roles of epigenetic marks driving cellular pluripotency, differentiation and self-renewal/proliferation. Indeed, the transcription of genes, governing cell-fate decisions during development and maintenance of a cell’s differentiated status in adult life, critically depends on the chromatin accessibility of transcription factors to genomic regulatory and coding regions. In this review, we discuss the epigenetic control of (liver-specific) gene-transcription and the intricate interplay between chromatin modulation, including histone (de)acetylation and DNA (de)methylation, and liver-enriched transcription factors. Special attention is paid to their role in directing hepatic differentiation of primary hepatocytes and stem cells in vitro.

Abbreviations: ADSC, adipose tissue-derived stem cells, ALB, albumin, AFP, alpha-fetoprotein, AhR, aryl hydrocarbon receptor, 5-AzaC, 5-Azacytidine or azacytidine, 5-Aza-dC, 5-Aza-2′-deoxycytidine or decitabine, C/EBP, CCAAT/Enhancer Binding Protein, CYP, cytochrome P450, CBP, CREB-binding protein, CpGs, cytosine-guanine dinucleotides, DHAC, 5-6-Dihydro-5-azacytidine, 4-Me2N-BAVAH, 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide, DNMTs, DNA methyltransferases, DNMTi, DNMT inhibitors, ES, embryonic stem cells, EGF, epidermal growth factor, EGCG, (-)-epigallocatechin-3-gallate, HCC, hepatocellular carcinoma, HNF, hepatocyte nuclear factor, HATs, histone acetyl transferases, HDACs, histone deacetylases, HDACi, hydroxamate-based HDAC inhibitors, iPS, induced pluripotent cells, LETFs, liver-specific transcription factors, MSC, mesenchymal stem/progenitor cells, MBD, methylated DNA-binding protein, P/CAF, p300/CBP-associated factor, PGC-1α, PPAR-gamma coactivator 1alpha, zebularine, 2-Pyrimidone-1-β-D-riboside, TSA, Trichostatin A, VPA, valproic acid

Keywords: Epigenetics, DNA methyltransferase inhibitor, Histone deacetylase inhibitor, Liver-enriched transcription factors, Stem cells, Primary hepatocytes, Differentiation

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1. Introduction 

Hepatocytes contain a rich source of xenobiotic biotransformation enzymes and consequently, the liver represents a primary target for xenobiotic-induced acute and systemic toxicity. Hence, hepatocytes are the ultimate source for toxicological screening/profiling of potential drug candidates. The drawback, however, is that upon isolation and subsequent culture of hepatocytes, a rapid and substantial decline of hepatic functionality occurs. In particular, the xenobiotic biotransformation capacity undergoes phenotypic changes [1]. This progressive loss of a differentiated hepatic phenotype in vitro plausibly results from a proliferative response, elicited during hepatocyte isolation from the liver. In fact, the cell cycle entrance triggers the activation of several signal transduction pathways, ultimately leading to profound alterations in gene expression [2]. The acquisition and stabilisation of a differentiated hepatic geno- and consequently phenotype, i.e. liver-specific gene/protein expression, very often rely on the concerted binding of liver-enriched transcription factors (LETFs) and other trans-acting DNA-binding proteins to well-defined regulatory and coding regions of target genes [3], [4], [5]. As DNA is tightly condensed into chromatin fibers by histones and other proteins, modulation of chromatin compaction is a prerequisite to facilitate binding of transcription factors and consequential transcriptional activation [6], [7]. Epigenetic events, including covalent histone modifications and DNA methylation, are therefore broadly acknowledged to play a fundamental role in the organisation of chromatin architecture and hence in the strict control of gene transcription [8], [9]. For example, in proliferating hepatocellular carcinoma (HCC) and HCC-derived hepatoma cell lines, inhibition of histone deacetylation and DNA methylation is found to drastically down- and up-regulate genes involved in cellular proliferation and xenobiotic metabolism, respectively [10], [11]. Consequently, it was thought that epigenetic events may display a predominant role in the acquisition and maintenance of the hepatocyte’s differentiated phenotype of dedifferentiating primary hepatocytes in vitro. Alternatively, stem cells have been proposed to produce functional hepatocytes as well. New insights into the molecular mechanisms governing the balance between self-renewal/proliferation and lineage-directed differentiation of embryonic stem cells have unveiled the presence of epigenetic marks as being key regulatory players [9], [12]. In fact, progression from unsoiled stem cells towards their differentiated progeny is characterized by alterations in the epigenetic landscapes of gene regulatory and coding regions. [9], [12], [13], [14], [15]. More specifically, locus-specific modifications on histones and DNA, progressively silence the transcription of pluripotent genes (euchromatic→heterochromatic state), whilst typical differentiated, lineage-specific genes become activated (heterochromatic→euchromatic state) [9], [12], [13], [14], [15]. Anticipation with nuclear chromatin might thus involve a key strategy for cell fate re-programmation.

In this review, we will discuss the key regulatory role of epigenetic modification in gene transcription, with particular focus on the maintenance and the acquisition of a differentiated geno/phenotype of primary hepatocytes and stem cells, i.e. pluripotent embryonic stem cells (ESCs) and multipotent mesenchymal stem/progenitor cells (MSC), respectively.

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2. Epigenetic control of gene transcription 

2.1. Structural chromatin modifications by histone acetylation/deacetylation 

The chromatin higher order structure can be subjected to a number of reversible posttranslational modifications [16]. Although the functional relevance of the individual reactions is often unclear, it is generally believed that the global repertoire of histone tail modifications constitutes a (epigenetic) code, which affects chromatin structure and/or gene expression [17]. To date, histone acetylation, methylation, phosphorylation/ubiquination/sumoylation, ADP-ribosylation and glycosylation of histones were reported [6], [16]. Histone acetylation, the best-understood posttranslational histone modification, is discussed in the following paragraphs.

Two opposing enzyme activities, i.e. histone acetyl transferases (HATs) (recently also referred to as lysine (K)-acetyltransfereases or briefly KATs [18]) and histone deacetylases (HDACs), determine the acetylation status of the lysine residues at the N-terminal histone tails extending out of the nucleosome [6]. Upon acetylation (Fig. 1), the positive charges on the side chains of these lysine residues are partially neutralised, thereby weakening the interaction with the negatively charged phosphate groups in the DNA backbone and affecting the nucleosome stability. The degree of acetylation of core histones can thus modulate DNA accessibility and chromatin activity in transcription, replication, recombination and repair [19]. Whereas actively transcribed genes are characterized by highly acetylated core histones, hypoacetylated histones are preferentially found in transcriptionally silenced chromatin regions [20]. Consequently, the long-standing paradigm existed that HDAC inhibition, leading to histone hyperacetylation, was exclusively associated with transcriptional activation. Yet, evidence is accumulating of HDACs functioning as both transcriptional activators or repressors. Indeed, by removal of acetyl groups from histone tails, HDACs do not only modulate the physical interaction between histones and DNA in the nucleosomal units, but also the message encrypted in the histones’ posttranscriptional modifications, and thus the epigenetic/histone code [21]. Consequently, specific effector proteins, e.g. transcription factors, are recruited resulting in further transcriptional stimulation or silencing, depending on the message comprised. Additionally, HDACs have targets other than histones, including the transcription factors p53, c-myc, NF-κB, YY-1, E2F and GATA family [22]. HDAC-mediated deacetylation of these non-histone proteins may affect their stability, localization, DNA-binding activity or ability to interact with other proteins [23]. As a result, the activity of the target protein might be augmented/reduced (Fig. 1).

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• Fig. 1. 

  HAT/HDAC-mediated transcriptional control. (A), Histone-related pathway: HATs/HDACs acetylate/deacetylate histones resulting in reduced/augmented chromatin compaction and alternations of the histone code, respectively. (B) Non-histone related pathway: HDACs directly interfere with non-histone protein targets, including transcription factors, nuclear hormone receptors, nuclear import factors, structural proteins and adhesion proteins. Deacetylation of latter non-histone proteins might affect diverse aspects of their protein physiology, resulting in either decreased or increased activity of the target protein. Both pathways interconnect with each other. The transcriptional outcome thus relies on the sum of all – transcription-stimulating/inhibiting – actions.

In light of these data, it is clear that HDACs operate at more than one level in the transcriptional regulation scheme. We refer to Smith for a scrupulous review regarding the transcriptional machinery underlying HDACs-mediated transcriptional (in)activation [24].

Today, 18 HDACs have been characterized. Based on sequence similarity, they can be classified into four distinct classes. Class I (HDAC-1,-2,-3,-8) and class II HDACs (HDAC-4,-5,-6,-7,-9,-10) closely resemble the yeast RPD3 and HDA1 proteins, respectively. Class III HDACs or ‘sirtuins’ are structurally unrelated to the other HDACs and apparently insensitive to hydroxamate-based HDAC inhibitors (HDACi) such as Trichostatin A (TSA) [22]. HDAC-11 is the single representative of mammalian class IV HDACs. It displays conserved residues in the catalytic core that share some, yet insufficient, homology to both class I and II enzymes [25], [26], [27]. Most HDACs lack intrinsic DNA-binding activities and are therefore capable of homo- and hetero-dimerisation. The HDAC catalytic domain is formed by a stretch of ca. 390 amino acids consisting of a set of conserved amino acids, which differ between class I and class II HDACs [6], [28]. The active site consists of a gently curved tubular pocket, a zinc-binding site and two Asp-His charge relay systems [29].

Class I HDACs (HDACs-1,-2,-3,-8) are generally small nuclear enzymes [27] that participate in cell cycle progression [25] and the regulation of housekeeping genes [28]. Class II HDACs (HDACs-4,-5,-6,-7,-9,-10) share some domain similarity with yeast HDA1 [25], [27]. They can be subdivided into class IIa HDACs (HDACs-4,-5,-7,-9) and class IIb HDACs (HDAC-6 and -10) [30]. With the exception of HDAC-10, class II HDACs show a restricted tissue-specific expression pattern with the highest expression found in heart, brain and skeletal muscle [28]. They are larger than class I HDACs and shuttle between cytoplasm and nucleus. In the nucleus, they mediate cellular proliferation and transcriptional repression of differentiation-related genes, leading to loss of the differentiated phenotype [25]. This was particularly shown for class IIa HDACs-4,-5,-7,-9 isoforms in muscle cell differentiation [31]. Class IIa HDACs, and principally HDAC-4, are also implemented in stress signaling processes, such as cardiac/chondrocyte hypertrophy and neuronal cell death. [32], [33], [34]. Most of these functions are mediated by interactions with the MEF2 transcription factor family [32]. In contrast to other HDACs, Class IIb HDAC-6 can accomplish cytoplasm-located functions as well. More specifically, HDAC-6 functions at the crossroads between two cellular signaling systems, i.e. protein lysine acetylation and ubiquitination [35]. This unique feature expounds its protective role against the accumulation of cytotoxic misfolded protein aggregates within cells [35]. In addition, HDAC-6 and class I HDAC-3 regulate osteoblast differentiation and bone formation via interaction with transcriptional regulators such as Runx2 [35], [36]. Likewise, class I HDAC-1, in complex with myoD, mediates repression of muscle-specific gene expression in undifferentiated myoblasts [37]. In general, class I HDACs overexpression coincides with increased cell proliferation and a concomitant shift towards dedifferentiation, while levels drop during differentiation processes. For example, in normal small intestine, the HDAC-3 expression is maximal in proliferating cells at the crypt base and is markedly decreased at the villus tip, harbouring more differentiated cells [38]. In comparison, 90% of cells residing in adenomas of small intestine are HDAC-3⁺. Basically, overexpression of distinct HDACs appears in various tumor specimens, e.g. HDAC-1 in prostate, gastric and colon tumors, HDAC-2 in colorectal, cervical and gastric cancer [26], and HDAC-3 in colon cancer [38]. Specific inhibition of HDAC-3 by RNA interference could inhibit proliferation of colon cancer cell lines and increase both expression and activity of the differentiation marker alkaline phosphatase [38]. As for liver-specific functions of HDACs, recently a crucial role was credited to HDAC-3 in liver homeostasis and development. In this respect, HDAC-3 absence in zebrafish leads to abnormalities in liver development, [39] whilst conditioned deletion of HDAC-3 in mice induces severe disruption of carbohydrate and lipid metabolism, resulting in organ hypertrophy and hepatocellular damage [40]. HDAC-1 overexpression in transgenic mice, on the other hand, results in a high incidence of hepatic steatosis and nuclear pleomorphism concomitant with altered expression of genes involved in cell cycle, apoptosis, and lipid metabolism such as p53, PPARγ, Bak and p21 [41], [42]. Also, a number of studies provide evidence for the involvement of HDACs and HATs in the transcriptional regulation of liver-specific genes by LETFs. This issue will be thoroughly discussed in a later section.

Briefly, these data indicate that modulating the expression of specific HDACs might involve a strategy to (re)activate differentiation programs.

Currently, several structurally diverse compounds both natural and synthetic, are known as HDACi. These include short-chain fatty acids, (non)-cyclic hydroxamates, (non)-epoxyketone-containing cyclic tetrapeptides, benzamides and miscellaneous structures [43]. Hydroxamate-based inhibitors of classes I and II are promising since they were repeatedly shown to selectively inhibit tumor growth in animals at low (micromolar) and apparently non-toxic doses [32]. Basically, in recent years, HDACi have emerged as promising therapeutics for the treatment of several malignancies, including leukaemia, solid tumors and non-solid cancers such as multiple myeloma [27]. In that respect, Vorinostat^® has recently been approved by the FDA for the treatment of advanced primary cutaneous T-cell lymphoma, whilst several other hydroxamate-containing HDACi are being tested in phases I and II clinical trials for their therapeutic potential [44], [45]. Having seen the growth-inhibiting and differentiation-promoting features of hydroxamate-based HDACi in tumor cells, including hepatoma cells [10], [11], [46], [47], [48], our group successfully applied these compounds to stabilize the differentiated phenotype of normal primary hepatocytes in vitro [1], [2], [49], [50], [51], [52]. This will be discussed later in this review.

2.2. DNA methylation 

Reversible DNA methylation occurs at the cytosine–guanine dinucleotides (CpGs) in the DNA and includes addition of a methyl group to the carbon-5 position of cytosine [53]. DNA methylation patterns are established by DNA methyltransferases (DNMTs), catalyzing the addition of a methyl group derived from the methyl donor S-adenosyl methionine [17], [54]. The catalytic activity of these enzymes is accomplished by a highly conserved C-terminal domain, present in all DNMTs.

In the mammalian genome, CpGs are not uniformly distributed. CpG islands, comprising >1 CpG per 80 base pairs [55], are particulary present in/near gene promotor regions. They are usually unmethylated, thereby allowing gene expression [53], [54], [55]. The distribution of (un)methylated CpGs differs, however, within distinct cell types due to the interplay between DNA methylation/demethylation, giving a cell-type specific DNA methylation pattern [17]. Passive DNA demethylation occurs during DNA replication by chemically blocking DNMTs [17]. The exact mechanism of active DNA replication-independent demethylation still remains elusive. Recent data suggest the involvement of identical enzymes in both the establishment of DNA methylation and demethylation patterns [56]. For example, the methylated DNA-binding protein MBD2 also displays demethylase activity [57]. Additional information is needed to unravel this tangled web.

Based on structural differences in their regulatory N-terminal domain, three distinct families of DNMTs, i.e. DNMT1, DNMT2 and DNMT3, have currently been identified. All are expressed in human liver tissue [58].

DNMT1 is the most abundant DNMT in mammals and mainly methylates hemimethylated GpGs. This ‘maintenance’ DNMT is particulary involved in maintaining DNA methylation patterns during DNA replication [54], [58], [59]. It also shows activity towards unmethylated DNA and plays a role in de novo DNA methylation [60]. DNMT2 is the least distinguished DNMT and lacks the regulatory N-terminal domain present in other DNMT enzyme families [59]. Its associated intrinsic DNMT activity and potential RNA methyltransferase activity suggest a possible role in epigenetic regulation [61], [62]. The DNMT3 family contains three different DNMTs, i.e. DNMT3a, DNMT3b and DNMT3L. DNMT3a and DNMT3b, characterized as de novo DNMTs, mainly methylate unmethylated CpGs and establish new DNA methylation patterns during early embryonal development [54]. They plausibly cooperate with DNMT1 to maintain the DNA methylation pattern [63]. DNMT3L is a methyltransferase-like protein without intrinsic DNMT activity [59], [64]. It interacts with DNMT3a/DNMT3b and directly modulates their catalytic activity [65].

DNMTs play a crucial role in the onset of chromatin remodelling and gene expression regulation. They are responsible for maintaining telomere integrity [66] and methylation pattern acquisition during gametogenesis, embryogenesis and somatic tissue development [67]. Several studies performed in tumor cell lines, including HepG2 cells [68], [69], also indicate the occurrence of cell cycle arrest, apoptosis and cellular differentiation upon DNMT inhibition. Accordingly, it has been suggested that modulation of the expression of specific DNMTs might involve a strategy to target the differentiation status in developing and proliferating and consequently dedifferentiating cells.

Today, a number of synthetic and natural DNMT inhibitors (DNMTi) exist. (i) The group of nucleoside analogue DNMTi contains several structural analogues of deoxycytidine, including 5-azacytidine (azacytidine, 5-AzaC), 5-Aza-2′-deoxycytidine (decitabine, 5-Aza-dC), arabinosyl-5-azacytidine (fazarabine), 5-6-dihydro-5-azacytidine (DHAC) and 2-pyrimidone-1-β-d-riboside (zebularine) [70] (Fig. 2). These analogues, with the exception of zebularine, are modified at the carbon-5 position of the pyrimidine base cytosine [71]. After phosphorylation and incorporation in DNA/RNA, they form covalent bounds with DNA methyltransferases, resulting in passive demethylation upon replication [72]. The use of nucleoside analogue DNMTi in tumor cells results, like HDACi, in cell cycle arrest, induction of apoptosis and differentiation [10], [69]. In addition, latter DNMTi were applied by our group to maintain differentiation in normal primary hepatocytes [73]. This issue will be discussed in the next section.

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• Fig. 2. 

  Nucleoside analogue DNMTi.

(ii) The non-nucleoside analogue DNMTi represent a heterogeneous group of DNMTi enclosing molecules such as derivatives of 4-aminobenzoic acid (procaine and procainamide), the main compound in green tea, (-)-epigallocatechin-3-gallate (EGCG) [74], [75] and psammaplins from the sponge Pseudoceratina purpurea [76]. Procain and procainamide directly bind to CpG-rich DNA, interrupting the interaction between DNMTs and their target DNA sequences. EGCG and psammaplins are both involved in several cellular processes and also affect enzymes other than DNMT [75]. Until now, the DNMTs inhibition mechanisms of EGCG and psammaplins remain unclear.

2.3. Interplay between DNA methylation and histone acetylation 

A tight correlation exists between [gene expression and DNA methylation] and [chromatin structure and DNA methylation]. Suppression of gene expression is frequently associated with methylated DNA and a dense chromatin structure, whereas active transcription is associated with unmethylated DNA and hyperacetylated open chromatin [17] (Fig. 3). Initially, DNA methylation was thought to unidirectionally affect chromatin structure. However, recent data in cancer cells now suggest a mutual interplay between both epigenetic modifications [17]. For example, in several cancer cell lines, combinations of DNMTi and HDACi have synergistic effects on the cellular homeostasis [69], [77], [78]. Accordingly, so far, several ‘cocktails’ are in clinical trials as chemotherapeutics [79]. Also, in vitro, our group discovered a synergistic effect of DNMTi and HDACi with respect to the differentiated phenotype of normal primary cells [73]. This will be further explained in the next section.

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• Fig. 3. 

  Epigenetic control of gene transcription. Inhibition of gene transcription typically corresponds to hypermethylated CpG islands in gene promoter regions and deacetylated histone tails at local chromatin domains. The indirect mechanism of gene silencing may involve binding of methyl-binding proteins (MeCp) to methylated cytosine and subsequent recruitment of HDAC-corepressor (CoR) complexes, resulting in a non-permissive heterochromatin status that blocks binding of transcription factors (TF) and polymerase II RNA complexes (PolII) to target promoter sequences. The direct mechanism may involve the direct interference of TF with HDAC or methylated CpG sites within the promoter. HDAC inhibitors (HDACi) and DNMT inhibitors (DNMTi) modulate the chromatin structure. They create an open, transcriptionally active euchromatin configuration at gene coding and regulatory regions, accessible for TF, thereby facilitating gene transcription. Abbreviations: 5-AzaC, decitabine; M, 5-methyl cytosine at CpGs; SB, sodium butyrate; TSA, trichostatin A; VPA, valproic acid.

In summary, our findings show that transcription of genes, governing maintenance of a cell’s differentiated status in adult life and development, can be accomplished via targeting the expression of DNMTs and/or HDACs with increased chromatin accessibility of transcription factors to their target DNA as a result.

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3. Epigenetic modifiers as potent differentiation-promoting compounds in vitro 

3.1. Acquisition and stabilisation of a differentiated hepatic phenotype in vitro: an interplay between chromatin remodelling and liver-enriched transcription factors 

Of major interest, at least from a pharmaco-toxicological point of view, is the ability of hepatocytes to protect the organism from toxic chemical insults. Hepatocytes dispose of an ingenious multi-step enzymatic clearance system, i.e. xenobiotic biotransformation, and therefore constitute the main cell type of interest for in vitro hepatotoxicity and drug metabolism studies to date [80].

Ex vivo, though, cell–cell and cell–extracellular matrix disruptions, resulting from collagenase perfusion and subsequent oxidative stress response, trigger the activation of several ‘proliferative’ signaling cascades [81]. Unlike hepatocytes in vivo, primary hepatocytes in culture are unable to completely redifferentiate upon proliferation, resulting in a loss of the differentiated phenotype and concomitant deterioration of cytochrome P450 (CYP)-mediated xenobiotic biotransformation capacity [2], [81]. Another essential factor is the substantial decline in LETFs, controlling the transcription of numerous liver-specific genes [3], [4], [5], [53], [81], [82], [83], [84]. Indeed, hepatocyte proliferation and differentiation are predominantly regulated at the transcriptional level [85]. Basically, eukaryotic gene transcription relies on the combinatorial binding of multiple specific trans-acting DNA-binding proteins, i.e. transcription factors, to particular DNA-sequence motifs in regulatory elements of a specific gene. Efficient gene expression is further often determined by interplays between different transcription factors, either adjacently or distantly located on the promoter, and by protein–protein interactions between transcription factors and coactivators/corepressors [7], [13], [82]. Additionally, efficient binding of transcription factors and associated proteins to their cognate DNA-sequences requires a permissive chromatin configuration in order to drive gene expression The dynamic modulation of the chromatin architecture by e.g. DNA methylation and/or covalent histone modifications represents thus a basic machinery for transcriptional activation, repression and derepression [7], [8], [9], [13], [14], [15] (Fig. 3).

In hepatocytes, the liver-enriched transcription factors play an elemental role in hepatocyte-specific gene expression [3], [4], [5], [82], [85], and are as such key regulators of liver development, architecture and physiology. These trans-acting DNA-binding proteins are predominantly, but not exclusively, expressed in liver. It is hypothesized that the coordinated and timely expression of LETFs, in concert with ubiquitously expressed transcription factors such as NF1, Oct-1, Hex and other LETFs, is prerequisite for hepatocyte differentiation and constitutive liver-specific gene expression, including CYP-mediated xenobiotic biotransformation [4], [82], [84], [85]. Evidence is accumulating that recruitment of coactivators/corepressors, able to modulate the local chromatin configuration through post-translational histone modifications, mainly determine their transactivation potential. In this context, the transcriptional activation of LETFs critically depends on the recruitment of co-activator proteins with intrinsic HAT activity, such as CREB-binding protein (CBP), p300/CBP-associated factor (P/CAF) and SRC1, whereas co-repressor complexes containing HDAC negatively regulate liver-specific gene expression [86], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107]. In detail, HNF-4α, directly interacts with SRC1, CBP and p300, resulting in its increased transcriptional activity [86], [87], [88], [89], [90], [91], [92]. The level of upregulation is isoform-dependent [92], [93]. In human hepatoma cells, transactivation of CYP1A1, CYP1A2 and CYP2C9 by HNF-4α relies on the presence of the PPAR-gamma coactivator 1alpha (PGC-1α) [94]. PGC-1α, a key regulator of hepatic gluconeogenesis, lacks HAT-activity, but enables transcription through the assembly of a complex, containing SRC1 and CBP/p300 [95]. Likewise, in differentiating Caco2-cells, Rb strengthens HNF-4-dependent activation of the α-antitrypsin gene through reinforcement of the SRC-coactivator function [96]. Conversely, interaction with SMRT or p53, recruiting HDACs to transcription factors, represses HNF-4α activity [92], [97], [98]. HNF-1α, on the other hand, physically interacts with the HATs CBP/p300, P/CAF, SRC-1, and RAC3 [99], [100]. CBP and PCAF, on one hand, and CBP and p300, on the other hand, synergistically upregulate HNF-1-mediated transactivation [100], [101], whilst association of HNF-1α with HDAC1 – through NCoR– impairs its transcriptional activity. Treatment with the HDACi TSA disrupts latter corepressor complex, enhancing in turn HNF-1α-mediated transcription [102]. A good example of LETFs acting in a cooperative, synergistic regulatory network is the interaction between HNF-6 and HNF-3. In this respect, HNF-6 potentiates HNF-3β transcriptional activity by recruiting p300/CBP HAT proteins, [103] whilst HNF-6-dependent transcription is stimulated by complex formation between HNF-6 and C/EBPα, also recruiting coactivator CBP [104]. The transactivation potential of C/EBPα, in turn, is promoted by direct interaction with either CBP/p300 or Rb [105], [106], [107]. Apparently, this binding to C/EBPα robustly stimulates nucleosomal HAT activity of CBP [108]. C/EBPβ-dependent transactivation is further mediated by direct acetylation through association with the HATs p300 and PCAF [109], [110], [111], [112]. Conversely, interaction between SMRT or subcomponents of the Sin3 complex, e.g. Sin3a, and HDAC1 represses its transcriptional activity [112]. Of particular interest is HNF3 as, in contrast to other LETFs, it directly affects chromatin conformations of numerous hepatic genes such as albumin (ALB) and α-foetoprotein (AFP), likely without interference with intermediary coactivators bearing HAT-activity or ATP-dependent enzymes. More specifically, the C-terminal domain of the protein binds to histones H3/H4 within highly compacted chromatin, creating a local, open nucleosomal domain, which facilitates further interactions between transcription factors, such as GATA4 and other LETFs, and DNA [113], [114], [115]. This HNF3-mediated transcriptional competence is designated as a prerequisite for the onset of liver ontogeny, and more specifically for the developmental activation of genes required for hepatocyte differentiation and function.

3.2. Effect of HDAC and DNMT inhibition on liver-specific gene expression 

An overview of the most important hepatic genes affected by HDACi and DNMTi in various in vitro models is presented in Table 1. Out of these data, it appears that both the cellular origin and the type, concentration and exposure time of epigenetic modifier used, critically determine the final outcome. In transformed cells, including colon cells [116], [117], [118], glioma cells [119], [120], pancreatic cells [121], breast cells [122] and hepatoma cells [11], HDACi-mediated growth arrest is frequently, at least in vitro, associated with induction of differentiation. As such, HDAC inhibition upregulates C/EBPα, HNF-1α, HNF-3α, HNF-3β and HNF-4α levels in various hepatoma cells, resulting in increased CYP expression [11]. Yet, in spite of this beneficial outcome, the contribution of HDACi in long-term in vitro models is hampered by co-occurrence of cell death. Indeed, HDACi, including TSA, butyrate, valproic acid, SAHA, OSU-HDAC42 and ITF2357, induce both in vitro and in vivo apoptosis in hepatoma cells [11], [46], [47], [48]. For example, SAHA reduces, dose- and time-dependently, the viability of HepG2 and Huh6 hepatoma cells as a result of concomitant activation of both extrinsic and intrinsic apoptosis signaling cascades [123]. In contrast, primary hepatocytes are relatively well-resistant against HDACi-induced apoptosis [2], [49], [50], [124], [125]. In this context, exposure of normal primary rat hepatocytes to TSA and 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide (4-Me2N-BAVAH) upregulates C/EBPα and HNF-4α expression [49], which in turn increases CYP protein and activity levels (Fig. 4, Fig. 5), gap junctional communication and ALB secretion, without any evidence of cell death [2], [49], [51], [52], [126], [127]. Moreover, in epidermal growth factor (EGF)-stimulated primary rat hepatocytes, we could demonstrate that TSA and 4-Me₂N-BAVAH even delay the onset of spontaneous apoptosis as evidenced by reduced pro-caspase-3 processing, decreased pro-apoptotic Bid and Bax levels and increased anti-apoptotic Bcl_xL expression [2], [50].

Table 1. Effects of epigenetic modifiers on the expression of important liver genes in various in vitro models.

	Epigenetic modifier	Species	Model	Regulationa	Reference
HDAC inhibition
Phase I biotransformation
CYP1A1	TSA	Rat	Primary hepatocytes	↑	[49]
		Human	Mammary carcinoma MCF-7 cells	↑	[186]
		Human	HeLa cells	↑	[186]
	SAHA	Human	Mammary carcinoma MCF-7 cells	↑	[187]
CYP1A2	TSA	Human	Mammary carcinoma MCF-7 cells	↑	[186]
		Human	HeLa cells	↑	[186]
		Mouse	Primary hepatocytes	↑	[188]
	Butyrate	Mouse	Primary hepatocytes	↑	[188]
CYP1B1	TSA	Human	Mammary carcinoma MCF-7 cells	↑	[186]
		Human	HeLa cells	↑	[186]
		Human	HepG2 hepatoma cells	≈	[191]
CYP2B1/2	TSA	Rat	Primary hepatocytes	↑	[49]
	Valproate	Rat	Primary hepatocytes	↑	[190]
CYP3A4	TSA	Human	HepG2 hepatoma cells	≈	[129]
		Human	HepG2 hepatoma cells	↑	[131]
CYP3A2	TSA	Rat	Primary hepatocytes	↑	[49]
ADH1A	TSA	Human	HepG2 hepatoma cells	≈	[191]
			HeLa cells	≈
ADH1B	TSA	Human	HepG2 hepatoma cells	≈	[191]
			HeLa cells	≈
ADH1C	TSA	Human	HepG2 hepatoma cells	↑	[191]
			HeLa cells	≈
FMO3	TSA	Human	HepG2 hepatoma cells	≈	[192]
Phase II biotransformation
GSTP1	TSA	Human	Hep3B hepatoma cells	≈	[193]
	Butyrate	Human	Colon carcinoma HT29 cells	↑	[194]
	Butyrate	Human	Primary colon cells	≈	[195]
GSTA1/2	Butyrate	Human	Colon carcinoma HT29 cells	↑	[194]
		Human	Primary colon cells	↑	[195]
GSTA4	TSA, MS-275, VPA	Mouse	MC3T3-E1 preosteoblasts	↑	[196]
GSTM2	Butyrate	Human	Colon carcinoma HT29 cells	↑	[194]
GSTT1/2	Butyrate	Human	Colon carcinoma HT29 cells	≈	[194]
		Human	Primary colon cells	↑	[195]
UGT2B7	VPA	Human	Prostate carcinoma LNCaP cells	↑	[197]
UGT2B11	VPA	Human	Prostate carcinoma LNCaP cells	↑	[197]
SULT2B1	TSA	Human	HaCaT keratinocytes	↑	[198]
Ammonia removal	TSA	Human	HepG2 and Huh-7 hepatoma cells	↑	[11]
Albumin synthesis/secretion	TSA	Human	HepG2 and Huh-7 hepatoma cells	↑	[11]
		Rat	Primary hepatocytes	↑	[2], [49]
Gap junctional intercellular communication
Cx32	TSA	Human	Huh-7 hepatoma cells	≈	[199]
		Human	Neural progenitor cells	↑	[200]
		Human	kB nasopharyngeal tumor cells	≈	[201]
		Human	Prostate carcinoma cells	↑	[202]
		Human	Normal prostate epithelial cells	↑	[202]
		Rat	Primary hepatocytes	↑	[52]
	4-Me2N-BAVAH	Rat	Primary hepatocytes	↑	[51]
Cx26	TSA	Human	Huh-7 hepatoma cells	≈	[199]
		Rat	Primary hepatocytes	↓	[52]
	4-Me2N-BAVAH	Rat	Primary hepatocytes	↓	[51]
Cx43	TSA	Human	Huh-7 hepatoma cells	↓	[199]
		Rat	Primary hepatocytes	↑	[52]
	SAHA	Rat	Ras transformed WB-F344 liver epithelial cells	↑	[203]
		Human	Peritoneal mesothelial cells	↑	[203]
		Rat	C6 glioma cells	↑	[204]
	Phenylbutyrate	Human	Glioblastoma cells	↑	[120]
		Human	Glioblastoma cells	↑	[205]
		Human	Neural progenitor cells	↑	[200]
		Human	kB nasopharyngeal tumor cells	↑	[201]
		Rat	Glioma cells	≈	[205]
	Sodium butyrate	Human	kB nasopharyngeal tumor cells	↑	[201]
		Human	Glioblastoma cells	≈	[205]
	4-Me2N-BAVAH	Rat	Primary hepatocytes	↓	[51]
Liver-enriched transcription factors
C/EBPα	TSA	Human	HepG2 hepatoma cells	↑	[10]
		Rat	Primary hepatocytes	↑	[49]
	SAHA	Human	Pancreatic carcinoma PANC-1 cells	↑	[121]
C/EBPβ	Butyrate, TSA	Rat	Intestinal epithelial cell line IEC-6	≈	[206]
C/EBPδ	Butyrate, TSA	Rat	Intestinal epithelial cell line IEC-6	≈	[206]
HNF1α	TSA	Rat	Primary hepatocytes	≈	[49]
HNF1β	Depsipeptide	Human	Papillary thyroid cancer cells	↓	[207]
HNF4α	TSA	Rat	Primary hepatocytes	↑	[49]
Other
Apolipoprotein CIII	TSA	Human	HepG2 and Huh-7 hepatoma cells	↑	[11]
HCFX	TSA	Human	HepG2 and Huh-7 hepatoma cells	↑	[11]
Glutamine synthetase	TSA	Human	HepG2 and Huh-7 hepatoma cells	↑	[11]
DNMT inhibition
Phase I biotransformation
CYP1A1	5-Aza-dC	Human	Mammary carcinoma MCF-7 cells	↑	[186]
		Human	HeLa cells	↑	[186]
CYP1A2	5-Aza-dC	Human	Mammary carcinoma MCF-7 cells	↑	[186]
		Human	HeLa cells	↑	[186]
		Mouse	Primary hepatocytes	≈	[208]
		Mouse	Hepa1c1c7 hepatoma cells	≈	[208]
CYP1B1	5-Aza-dC	Human	Mammary carcinoma MCF-7 cells	↑	[186]
		Human	HeLa cells	↑	[186]
	5-AzaC	Human	HepG2 hepatoma cells	≈	[189]
CYP3A4	5-Aza-dC	Human	HepG2 hepatoma cells	↑	[10]
CYP3A5	5-Aza-dC	Human	HepG2 hepatoma cells	↑	[10]
CYP3A7	5-Aza-dC	Human	HepG2 hepatoma cells	↑	[10]
FMO3	5-Aza-dC	Human	HepG2 hepatoma cells	↑	[192]
Phase II biotransformation
GSTP1	5-Aza-dC	Human	Hep3B hepatoma cells	↑	[193]
	Procainamide	Human	LNCaP prostate cancer cells	↑	[74]
UGT1A6	5-Aza-dC	Human	HepG2 hepatoma cells	↑	[10]
UGT2B15	5-Aza-dC	Human	HepG2 hepatoma cells	↑	[10]
UGT2B28	5-Aza-dC	Human	HepG2 hepatoma cells	↓	[10]
Gap junctional intercellular communication
Cx32	5-Aza-dC	Human	Caki-2 renal cell carcinoma cells	↑	[209]
		Human	Caki-2 renal cell carcinoma cells	↑	[210]
		Human	HK-2 renal tubular cells	≈	[209]
Cx26	5-Aza-dC	Human	Mammary carcinoma cells	↑	[211]
		Human	Mammary carcinoma cells	≈	[212]
		Human	Lung cancer cells	↑	[213]
		Human	Esophageal cells	≈	[214]
Cx43	5-Aza-dC	Human	Esophageal cancer cells	≈	[214]
		Human	Cervical adenocarcinoma cells	↑	[215]
		Human	CNE-1 nasopharyngeal cancer cells	↑	[216]
Liver-enriched transcription factors
C/EBPα	5-Aza-dC	Human	HepG2 hepatoma cells	≈	[10]
C/EBPβ	5-Aza-dC	Human	HepG2 hepatoma cells	↓	[10]
C/EBPγ	5-Aza-dC	Human	HepG2 hepatoma cells	↓	[10]
Other
GLUT2	5-Aza-dC	Mouse	Primary hepatocytes	↑	[217]
		Mouse	Hepa1c1c7 hepatoma cells	↑	[217]
ADH1A	5-Aza-dC	Human	HepG2 hepatoma cells	≈	[191]
			HeLa cells	≈
ADH1B	5-Aza-dC	Human	HepG2 hepatoma cells	↑	[191]
			HeLa cells	≈
ADH1C	5-Aza-dC	Human	HepG2 hepatoma cells	↑	[191]
			HeLa cells	≈
HDAC+DNMT inhibition
Liver-enriched transcription factors
C/EBPα	TSA+5-Aza-dC	Human	HepG2 hepatoma cells	↑	[10]
Phase I biotransformation
ADH1A	TSA+5-Aza-dC	Human	HepG2 hepatoma cells	≈	[191]
			HeLa cells	≈
ADH1B	TSA+5-Aza-dC	Human	HepG2 hepatoma cells	↑	[191]
			HeLa cells	≈
ADH1C	TSA+5-Aza-dC	Human	HepG2 hepatoma cells	↑	[191]
			HeLa cells	≈

^a≈, unchanged; ↑, upregulation; ↓, downregulation.

Abbreviations: ADH, alcohol dehydrogenase; 5-AzaC, 5-Azacytidine; 5-Aza-dC, 5-Aza-2′deoxycytidine, decitabine; C/EBP, CCAAT/enhancer binding protein; Cx, connexin; CYP, cytochrome P450; FMO, flavine monooxygenase; GLUT, glucose transporter; GST, glutathione S-transferase; HNF, hepatocyte nuclear factor; 4-Me₂N-BAVAH, 5-(4-dimethylamniobenzoyl)-aminovaleric acid hydroxamate; SAHA, suberoylanilide hydroxamic acid; SULT, sulfotransferase; TSA, Trichostatin A; UGT, uridine guanyl transferase.

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• Fig. 4. 

  Effects of TSA on phase I CYP-dependent biotransformation activity. Hepatocytes were cultured and remained either unexposed [C] or were exposed to 0.083% (v/v) ethanol as solvent control [SC] or 25μM TSA [T] for 7 days. (A) After 2 [D2], 4 [D4] and 7 days [D7], CYP1A1, CYP2B1 and CYP3A2 protein expression were analysed by means of immunoblotting. In order to control for equal loading of proteins, expression levels of HNF-1α were determined, as latter LETF, in contrast to C/EBPα and HNF-4, is not affected by culture time or exposure to TSA. Representative images for three independent experiments are shown. (B) After 2, 4 and 7 days of culture PROD (CYP2B1)-dependent activities were measured. Data are expressed as percentage of the values found for freshly isolated primary rat hepatocytes, i.e. 7.7±3.1pmol/min/mg microsomal protein. Values represent mean±SD.

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• Fig. 5. 

  Effects of 4-Me₂ N-BAVAH on phase I CYP-dependent biotransformation activity. Cultured hepatocytes were either exposed to 0.05% (v/v) ethanol as a solvent control [C] or to 50μM 4-Me₂N-BAVAH [B] for 7 days. (A) After 4 [D4] and 7 days [D7], CYP1A1, CYP2B1 and CYP3A2 protein expression were analysed by means of immunoblotting. Representative images for three independent experiments are shown. (B) After 4 and 7 days of culture PROD (CYP2B1)-dependent activities were measured. Data are expressed as percentage of the values found for freshly isolated primary rat hepatocytes, i.e. 7.7±3.1pmol/min/mg microsomal protein. Values represent mean±SD (n⩾3). (^∗p<0.05 compared to control values, paired Student’s t-test).

Further indication for the involvement of histone acetylation in the transcriptional activation of hepatic genes is supported by enhanced activity of several LETFs, including HNF-3γ, HNF-4α and C/EBPα, on the promoter of CYP2C8, CYP2C19, CYP3A4, CYP3A5, CYP3A7, CYP7A1 and glucose-6-phosphatase upon exposure to TSA or short chain fatty acids, such as butyrate [57], [128], [129]. In addition, HDAC inhibition is implemented in the aryl hydrocarbon receptor (AhR)-mediated induction of CYP1A1 and CYP1A2 [130], and CYP3A4 induction by rifampicin [131]. Next to genes coding for xenobiotic biotransformation enzymes, HDACi also stimulate the transcription of other liver-specific genes, including phosphoenol-pyruvate carboxykinase [57], HCFX [11], apolipoprotein CIII [11], glucose-6-phosphatase [57] and glutamine synthetase [11]. They also promote ALB synthesis and secretion rate, ammonia removal and gap junctional intercellular communication [2], [11], [50], [51], [52] (Table 1).

DNA methylation marks, on the other hand, are crucial for developmental and tissue-specific transcription of numerous liver-specific genes, including ALB [132], AFP [133], Cx43 [134], Cx32 [134], human CYP2E1 [135], human CYP1A2 [136], rat CYP2D3 and CYP2D5 [137], mouse CYP2D9 [138], mouse CYP2A4 [139], and human SULT1A1 [140]. In addition, in HepG2 cells, CYP3A4, CYP3A5 and CYP3A7 levels were raised upon DNMT inhibition by 5-Aza-dC, whilst C/EBPβ and C/EBPγ were decreased. Combined exposure to TSA and 5-Aza-dC, but not to 5-Aza-dC alone upregulated C/EBPα expression in HepG2 cells [10]. On the other hand, in EGF-stimulated primary rat hepatocytes, both combined and single exposure to 5-Aza-dC and 4-Me2N-BAVAH resulted in dose-dependent inhibition of the DNA replication and improvement of the hepatic functionality/phenotype, as evidenced by enhanced ALB secretion and elevated CYP1A1 protein expression [73]. Yet, upon combined application, lower concentrations of the respective epigenetic modifiers are needed when compared to single treatment in order to observe the same –or even a more pronounced– effect, pointing to a synergetic or even synergistic behaviour of DNMTi and HDACi with respect to important liver-specific processes [73]. Moreover, the interplay between HDACi/DNMTi and the expression of hepatic genes emphasizes a plausible involvement of chromatin remodelling agents in the acquisition/maintenance of a differentiated hepatic phenotype in healthy hepatocytes.

3.3. Epigenetic modifiers: a key factor to (re)program stem cells in vitro? 

In vivo, stem cells inhabit restricted niches within an organ or tissue, directing their self-renewal, differentiation and cell fate [141], [142], [143]. In particular, adult tissue is renewed through asymmetric division of stem/progenitor cells, thereby forming one cell that remains a stem cell and another cell that differentiates into a mature cell type with specialised functions [141], [142], [143]. Batteries of developmental regulatory signaling molecules and transcription factors, including Wnts, fibroblast growth factors, Notch, sonic hedhodge, etc. may play a role [85], [142], [144]. More specifically, the coordinated signaling between stem cells, non-stem niche cells, the scaffold, and integration of stem cell-autonomous characteristics-including a dynamic interplay between transcription, epigenetic control and posttranscriptional regulators represent an interactive system, organized to facilitate cell-fate decisions in a spatio-temporal manner [85], [142], [144]. Identification of these in vivo signaling patterns is crucial for eliciting distinct responses from cultured stem cells and directing lineage-specific cell growth and differentiation in vitro. Lately, evidence is growing that particularly chromatin remodelling or alteration of epigenetic marks, including histone acetylation/methylation and DNA methylation are part of the core machinery required for nuclear reprogramming and cell-fate conversion [9], [12], [13], [14], [15], [145], [146].

Pluripotent ES and more lineage-restricted adult stem/progenitor cells differ in their global gene expression status. Stemness genes, active in pluripotent embryonic stem cells are gradually silenced, whilst lineage-specific genes are switched on upon progression of development [12], [14], [15], [146]. This discrepancy in gene expression profiling might be ascribed to alterations in the nuclear and chromatin architecture, resulting in selective accessibility of transcription factors towards specific DNA-binding sites. Each stage of lineage-directed development is thus featured by a dynamic interplay between unique repertoires of (lineage-specific) transcription factors and epigenetic regulators. This epigenetic code forms the base of the stem cell identity and determines its responsiveness to extrinsic signals at successive developmental stages [9], [82], [85], [114]. Alternatively, extracellular growth factors might directly affect the chromatin status as well and as such facilitate or impede the differentiation competence of stem/progenitor cells [147]. Hence, the hypothesis was concocted that cell fate might be reprogrammed by altering the epigenetic code/marks.

In this regard, a recent breakthrough was achieved by Takahashi and Yamanaka, who successfully reprogrammed mouse embryonic/adult fibroblasts to ES-like stem cells, referred to as induced pluripotent cells (iPS), via viral mediated transduction of Oct4, Sox2, c-Myc and Klf4 [148]. These four transcription factors act as core regulators of the transcriptional circuitry to maintain pluripotency in ES. The theory exists that their ectopic expression in lineage-committed somatic cells induces alterations in the histone code and DNA methylation status of stemness genes such as Oct4, Sox2 and Nanog [146], [148]. Although the underlying mechanism remains elusive, latter realization emphasizes the tight epigenetic control of transcriptional machinery regulating pluripotency and lineage-specific differentiation. More specifically, on/off switch of cell fate reprogrammation and transcription of lineage-specific genes may be poised by dynamic open/closed configuration of histone and nuclear architecture at specific binding sites for transcription factors.

Since HDACi and/or DNMTi upregulate the transactivation potential of liver-enriched transcription factors in a plethora of hepatoma cells and primary hepatocytes [cf. Table 1], being key regulators of liver embryogenesis and liver-specific gene expression in particular, addition of HDACi and/or DNMTi to stem/progenitor cells, preferentially co-conditioned with hepatogenic growth factors and cytokines, is thought to comprise a potential strategy for driving differentiation programs and more specifically for directing hepatic differentiation of stem/progenitor cells. An overview of currently applied epigenetics-based strategies for in vitro hepatic differentiation of ES and MSC is given in Table 2.

Table 2. Detailed epigenetics-based strategies for in vitro differentiation of ES and MSC-like cells into hepatic cells.

Origin	Hepatic differentiation conditions					Hepatic features		Refs.
	Cell density	Cell–matrix/cell–cell interaction	Serum	Growth factors-cytokines nonepigenetic additives	Differentiation-inducing agents	RNA+protein level	Functionality level
EMBRYONIC STEM CELLS
				Hepatic progenitor cells
	NS	Gelatin	15% FBS	(3)D11–17/23: 10ng/ml HGF until confluent	(1)D0–4: 0.8% DMSO	AFP, α1AT, CK18/19		[182]
					(2)D4–10: 2.5mM SB	GGT, HNF3 β, DPPIV
mES(D3)				Hepatocytes
	NS	Collagen type I	10% FBS	4)D17/23–23/29: insulin, dex nicotinamide		ALB, G6P↑, TAT↑	Glycogen storage
				20ng/ml EGF			ALB secretion
				10ng/ml HGF
				(5)from D23/29: 10ng/ml OSM, dex
hES	NS	PAU-coating, nonwoven PTFE	NS	D0–3:100ng/ml bFGF	D3–11:1% DMSO	ALB	Lidocaine	[173]
				D3–11:100ng/ml HGF			metabolism
				D11–14:dex			ALB and urea
							production
hEBs		Matrigel	20%FBS		5mM SB	−: AFP	Glycogen storage	[152]
						+: ALB, α1AT, CK8/18	Inducible
							CYP450 activity
mES (D3)	1×104cells/cm2	D0–10:Gelatin collagen type I polystyrene	D0–10: 20% FBS		D0–4: 1% DMSO		Glycolysis
					D4–10: 2.5mM SB		Glycogen storage
							Urea production	[183]
						+: ALB, K18, DPPIV ADH, CYP3A13, CYP27A1	CYP activity
	1×104cells/cm2	D0–10: gelatin from D11: 5% polyacrylamide	D0–10:20% FBS from D11:/		D0–4: 1% DMSO	-: AFP	Glycogen storage	[184]
	D11: subculture at 15×103cells/cm2				D4-10: 2.5mM SB		↑urea production
					From D11:/		↑ALB secretion
mES (D3)	Phase I
	Pre-differentiation=no confluence	Gelatin	15%FBS	(3) from D10 until confluence: 10ng/ml HGF	(1)D0–4: 0.8% DMSO	+: CK19, ALB, α1AT HNF3β		[182]
					(2)D4–10: 2.5mM SB	-: SSEA1, AFP
	Phase II	Collagen type I	10%FBS	(4) Upon subculture: D0–6: insulin, dex nicotinamide,		+: AFP, ALB, CK18, α1AT HNF3β, HNF4, TAT	Glycogen storage
	Differentiation upon confluence and subculture			20ng/ml EGF		-: CK19	ALB secretion
				10ng/ml HGF
				(5) From D6–12/18:
				(5) From D6–12/18: 10ng/ml OSM, dex
hES	Differentiation onset pon 50–70% confluence D3/5:1/2 split	Matrigel	D0–3/5:/D3/5–D10/12:SR from D10/12: 8.3% FBS	D0–3/5: 100ng/ml activin A**	D0-D1/2:1mM SB	D0–3/5: CXCR4, HNF3β, Sox17	Glycogen storage ALB/fibrinogen/fibronectin/A2M secretion inducible CYP activity	[153]
				From D10/12:insulin, hydrocortisone, 10ng/ml HGF, 20ng/ml OSM
					D1/2–3/5: 0.5mM SB	D3/5–D10/12: HNF4α↑, HNF1↑, TTR↑
					D3/5–D10/12: 1% DMSO	AFP
						From D10/12:ALB, ApoF, CAR, TO, TAT↑, CYP3A4/7, CYP2C9/19
mES Monkey ES	mES:109cells/cm3 lumen V	Organoid culture in hollow fibers	20% FBS		D9: 1mM SB	+: CPS↑	ALB secretion ammonia removal	[174]
	monkey ES: 4×103cells/cm3 lumen V
MESENCHYMAL(-LIKE) STEM/PROGENITOR CELLS
BM (tibias+femora, C57/BL6 mice)	1×104MSC/cm2 +5.2×104 liver cells/cm2	Coculture with nonparenchymal liver cells on collagen	>coculture: 20% FBS	12h before coculture: dex	12h before coculture:: 5mM 5-AzaC	ALB, CK18/8/19, TAT		[156]
			Upon coculture: 5% liver failure patients sera	Upon coculture: nicotinamide, insulin, dex, 50ng/ml HGF, 20ng/ml OSM	Upon coculture: 1% DMSO
ADSC (abdominoplasty, 19–55 years human adults; P3–5)	2.5–3×10−4cells/cm2	FN	/	10ng/ml HGF	From D10 on: supplement of 0.1% DMSO	AFP, ALB	LDL uptake urea production	[161]
				10ng/ml OSM, ITS
				10ng/ml EGF, dex
BM (iliac crest, human adults)	100–200cells/cm2					Cx32, HepPar1, CYP3A4, CPS CK18, ALB, PEPCK	Glycogen storage Urea secretion	[185]
ADSC (subcutaneous/peritoneal, female donor)	Differentiation onset upon 100% confluence	NS	2% FCS	D1:40ng/ml HGF, 20ng/ml EGF	D0:preincubation with 20km 5-AzaC	Cx32, CYP3A4, CPS CK18, ALB, PEPCK, DPPIV	Glycogen storage Urea secretion	[168]
BM (iliac crest and femora, human)
BM (healthy human; P4–5)	22×103MSC/cm2 differentiation onset upon 100% confluence	Collagen type I	Predifferentiation:2% FBS	D0-2: 10ng/ml FGF4	From D6 on: supplement of 1μM TSA	ALB↑, CK18↑, HNF1α↑, MRP2↑ C/EBPα↑	ALB secretion↑, inducible CYP activity Urea secretion	[154]
				D3–5: 20ng/ml HGF from D6 on:ITS, dex 20ng/ml HGF
ADSC (peritonial, Fischer 344 rats)	200–300cells/cm2 Differention onset: upon 95% confluence:	FN		(2)D1: hepatocyte growth medium	D0: preincubation with 20μM 5-AzaC	AFP, ALB, CK18/19, CYP1A1, HepPar1, Cx32, DPPIV, PCK1	Glycogen storage	[155]
							Urea production
UCB (hTERT retroviral infected)	2.1×103cells/cm2	NS	D1–22:10% FBS	D1–22: 10ng/ml FGF2	D0: preincubation with 1μM5-AzaC	ALB, C/EBPα/β↑, CYP1A1/2, PEPCK↑, wnt↓	Glycogen storage	[167]
				20ng/ml HGF			Urea secretion
				20ng/ml OSM
BM (tibias+femora, SD rats)	22×103MSC/cm2 differentiation onset upon 100% confluence	Collagen type I	Predifferentiation:2% FBS	D0–2: 10ng/ml FGF4	From D6 on: supplement of 1μM TSA	AFP↑, ALB↑, CK18↑, HNF1α↑, HNF3β	ALB secretion↑	[169]
				D3–5: 20ng/ml HGF from D6 on:ITS, dex 20ng/ml HGF

Abbreviations: α1AT, alpha1-antitrypsin; ADH, alcohol dehydrogenase; ADSC, adipose tissue-derived stem cells; AFP, alpha-fetoprotein; ALB, albumin; A2M, α2macroglobulin; ApoF, apolipoprotein factor; 5-AzaC, 5-azacitidine; bFGF, basic fibroblast growth factor; BM, bone marrow; CAR, constitutive androstane receptor; C/EBP, CCAAT enhancer binding protein; CK, cytokeratin; CPS CPS-1, carbamyl phosphate synthetase; Cx, connexin; CYP, cytochrome P450-dependent monooxygenases; dex, dexamethasone; DMSO, dimethylsulfoxide; DPPIV, dipeptidylpeptidase IV; EGF, epidermal growth factor; ES, embryonic stem cells; FBS, fetal bovine serum; FCS, fetal calf serum; FGF, fibroblast growth factor; FN, fibronectin; GGT, γ-glutamyltransferase; G6P, glucose-6-phosphatase; h, human; HepPar1, hepatocyte paraffin 1; HGF, hepatocyte growth factor; HNF, hepatocyte nuclear factor; ITS, insulin-transferrin-selenious acid; LDL, low density lipoprotein; m, mouse/murine; MRP, multidrug resistance protein; MSC, mesenchymal stem cells; NS, not specified; OSM, oncostatin M; P, passage; PAU, poly-amino-urethane; PEPCK, phosphoenol-pyruvate carboxykinase; PTFE, polytetrafluoroethylene; SB, sodium byturate; SD, Sprague–Dawley; Sox17, Sry-related HMG box transcription factor; SR, serum replacement; SSEA, stage-specific embryonic antigen; TAT, tyrosine amino-transferase; TERT, telomerase reverse transcriptase; TO, tryptophan-2,3-dioxygenase; TSA, trichostatin A; TTR, transthyretin; UCB, umbilical cord blood; 1.,2.,3; indicate order of serial steps.

↓, downregulation; ↑, upregulation; −, negative; +, positive.

3.3.3.1. Pluripotent embryonic stem cells 

ES harbour a unique pluripotent versatility compared with fetal and adult multi/bipotent stem/progenitor cell populations. They posses the unrestricted capacity to form cell types of the three germ layers, including neuroectodermal cells, cardiomyocytes and hepatocytes. Yet, spontaneous ES differentiation is encountered by lack of organization and inherent heterogeneity [149], [150], [151]. Introduction of chromatin remodelling agents, biologically-derived signals such as purified growth factors, or other lineage-selective agents, though, could enrich for specific cell populations [150]. For example, exposure to 5mM sodium butyrate enriches ES cultures for 10–15% to pure hepatic cells [152]. Priming with alternating concentrations of sodium butyrate (0.5–1mM) in the presence of Activin A even results in 10–70% enrichment [153]. Basically, combined application of epigenetic modification and stepwise exposure to cytokine stimuli considerably contribute to homogeneity of the end-population and acquisition of hepatic functionality [153].

3.3.3.2. Multipotent mesenchymal stem cells 

In recent years, evidence has been provided that MSC(-like) cells from various sources (bone marrow/adipose tissue/placenta/umbilical cord) could occasionally overcome lineage borders and differentiate into endodermal (hepatocytes) and ectodermal (neural cells) cell types upon coordinated in vitro stimulation [154], [155], [156], [157], [158], [159], [160]. New insights into the underlying mechanisms indicate that next to lineage-specific cytokines/growth factors (their concentrations, mode of presentation, and order of application) [144], alterations of the epigenetic traits and chromatin code of specific gene regulatory regions are essential for bypassing cell fate determinism and reprogramming cell fate [9], [13], [15], [145], [146]. In this context, we found as first that addition of 1μM TSA to cultured human bone marrow MSC, pre-treated for 6 days with hepatogenic-stimulating agents, triggers their ‘trans’differentiation into cells with similar phenotypic and functional characteristics as primary hepatocytes [154]. In line with our results, Seo et al. showed enhanced hepatic differentiation upon addition of 0.1% dimethylsulfoxide to human adipose tissue-derived stem cells (ADSC), prestimulated for 10 days with a mixture of hepatogenic cytokines [161]. Likewise, neuroectodermal and cardiomyocyte direction could be accomplished via co-exposure to HDACi along with neural stimulating agents [162] and shear stress [163], [164], respectively. Recently also DNMTi, either alone or in combination with HDACi, were introduced to alter cell fate [155], [165], [166], [167]. Basically, DNMTi function as preconditioning agents prior to hepatic differentiation [155], [167], [168], whereas HDACi act as stimulants during or post-differentiation [154], [156], [161], [169]. In general, chromatin remodelling seems thus a potential innovative strategy to overcome cell fate determinism, cross lineage borders and favour lineage-specific differentiation. We expect that this field will emerge in the upcoming years.

Next to successful differentiations, also failures have been reported. For example, 1mM valproic acid (VPA), 100nM TSA and 1μM sodium butyrate failed to promote oligodendrocyte or astrocyte differentiation in rat neural progenitors under respective stimulating conditions [162], whilst they could trigger differentiation into neural cells in a neural stimulating microenvironment [162]. In addition, Jori and group reported that 2mM VPA, but not 50nM TSA, could stimulate neural transition of MSC [170]. On the other hand, we found that functional hepatic differentiation of bone marrow MSC was especially successful upon exposure of 1μM TSA to 6 days preconditioned cells.

Successful cell fate manipulation thus highly relies on (i) the microenvironment (cell–cell contact, cell densities), (ii) the appropriate type of epigenetic modifier and (iii) optimal fine-tuning of its dose and timing – onset and duration – of exposure [154], [161], [169], [171]. The suitability of HDACi and/or DNMTi to promote hepatic (trans)differentiation requires a delicate balance between (i) proliferation and differentiation, (ii) biological activity, pharmacokinetic properties and toxicological characteristics, and finally (iii) apoptosis and cell survival. At least in some cases, failure of lineage-specific differentiation could be ascribed to inaccurate timing of exposure and dosage of chromatin modulating agents. Basically, although not generally [153], pre-stimulation of the cells towards the intended selected direction prior to introduction of HDACi, may comprise a key determinant to cross lineage boundaries and achieve promoted transdifferentiation into a specific lineage by means of HDAC inhibition [154], [161], [164], [169], [172], [173], [174]. An up-to-date overview of both successful and failed epigenetics-induced cellular (re)programmations of progenitor cells is given in Table 3.

Table 3. Accomplished and failed HDAC and DNMT inhibitor-induced in vitro and in vivo differentiation of stem cells into various cell types.

Epigenetic modifier	Culture conditions	Stem cell type	Intended cell type	Observed features	Reference
(A) Successful (‘trans’)differentiation
HDAC inhibitor
TSA∗	1μM TSA	Human bone marrow MSC	Hepatocyte-like cells	Increased Alb, CK18, HNF1α, MRP2, C/EBPα expression and Alb secretion	[154]
	Exposure from day 6 of differentiation onwards
	Hepatic stimulating medium
TSA∗	1μM TSA	Rat MPC	Hepatocyte-like cells	Increased AFP, HNF, Alb, CK18, C/EBPα, CYPs expression and Alb secretion	[169]
	Exposure from day 6 of differentiation onwards
	Hepatic stimulating medium
DMSO∗	0.1% DMSO	Human ADSC	Hepatocyte-like cells	Alb and AFP expression, urea production	[161]
	Exposure from D10 of differentiation
	Hepatic stimulating medium
DMSO∗	(1) 1% DMSO	Human ES	Hepatocyte-like cells	Alb expression, lidocaine metabolism, Alb and urea production	[173]
	Exposure from D3–11 of differentiation
	Hepatic stimulating medium
DMSO+sodium butyrate∗	(1) 1% DMSO	Murine ES (D3)	Hepatocyte-like cells	Alb, CK18, DPPIV, ADH, CYP expression,	[183], [184]
	Exposure from D0–4 of differentiation
	(2) 2.5mM sodium butyrate
	Exposure from D4–10 of differentiation
DMSO+sodium butyrate∗	(1) 0.8% DMSO	Murine ES (D3)	Hepatocyte-like cells	AFP, ALB, α1AT, CK18, CK19, GGT, HNF3b, G6P, TAT and DPPIV expression, glycogen storage, ALB secretion	[182]
	Exposure from D0–4 of differentiation
	(2) 2.5mM sodium butyrate
	Exposure from D4–10 of differentiation
	From D11: hepatic stimulating medium
Sodium butyrate∗	1mM Sodium butyrate	urine and monkey ES	Hepatocyte-like cells	Alb expression, urea and Alb secretion	[174]
	Exposure from D9 of culture
	Unspecified differentiation medium
Sodium butyrate+DMSO∗	(1) 1mM sodium butyrate	Human ES	Hepatocyte-like cells	Expression of CYPs, HNF3b, HNF4a, HNF1a,b, TTR, AFP, Alb, ApoF, CAR, TO, TAT, glycogen storage, Alb/fibrinogen/fibronectin/A2M secretion, CYP activity	[153]
	Exposure upon 50–70% confluence for 24–48h
	(2) 0.5mM sodium butyrate
	Exposure from D1-2 of differentiation for 48-72h
	(3) 1% DMSO
	Exposure from D3 to D5 of differentiation upon subculture for 7days
	Hepatic stimulating medium
TSA∗+shear stress	10ng/ml TSA	ES (129/Ola derived)	Cardiomyocytes	Increased acetylated GATA-4, β-MHC, Nkx2.5 and ANF expression	[164]
	Shear stress: concentration not specified
	24h exposure after 7 days of differentiation
	Unspecified differentiation medium
TSA∗+shear stress	32nM TSA	Murine ES (D3)	Cardiovascular cells	Increased VEGFR2, PECAM, SMA, SM-2C, α-SA, etc. expression	[163]
	Exposure from onset of differentiation
	Shear stress: concentration not specified
	1–24h exposure to TSA pre-incubated cells
	Unspecified differentiation medium
TSA∗	80nM TSA	Embryos cloned from cow donor cells	Blastocysts	Change in morphology	[172]
	24h exposure after 7 days of differentiation
	Non-stimulating medium
TSA∗	100nM TSA	Rat neural progenitors	Neural cells	Increased Tuj1 or MAP2ab expression	[162]
VPA∗	0.3–1mM VPA
Sodium butyrate∗	1μM Sodium butyrate
	4 days exposure upon onset of differentiation
	Neural stimulating medium
VPA+DMSO∗	2mM VPA, 2% DMSO	Rat bone marrow MSC	Neurons and astrocytes	Increased expression of NSE and NF,	[170]
	Exposure from differentiation onwards			AchE activity
	Neural stimulating medium
VPA∗	0.5–3mM VPA	Human bone marrow MSC	Osteoblasts	Increased osterix, osteopontin BMP-2 and Runx2 expression	[218]
	Exposure during first 3 days of differentiation and first 4 days prior to differentiation
DNMT inhibitor
5-AzaC∗,$	20μM 5-AzaC	Rat ADSC	Hepatocyte-like cells	Expression of DPPIV, Alb, CYP1A1, HepPar1, AFP, CK19, PCK1, CK18	[155]
	Exposure for 24h upon 95%confluence prior to
	onset of differentiation
	Upon 24h: Hepatic stimulating medium
5-AzaC∗,$	20μM 5-AzaC	Human ADSC	Hepatocyte-like cells	Expression of Alb, CYP3A4, HepPar1, CK18, Cx32, PEPCK, CPS	[168], [185]
	Exposure for 24h prior to hepatic stimulation onset of differentiation at 100% confluency	Human bone marrow MSC		and Cx32, urea production, glycogen storage
	Hepatic stimulating medium
5-AzaC∗,$	1μM 5-AzaC	Human umbilical cord blood MSC	Hepatocyte-like cells	Expression of Alb, C/EBPa, CYP1A1/2, glycogen storage	[167]
	Exposure for 24h prior to hepatic stimulation
	Hepatic stimulating medium
HDAC+DNMT inhibitor
TSA+5-Aza-dC$	150nM TSA+500nM 5-Aza-dC	Murine neurosperes	Haematopoietic cells	Expression of CD45, CD3, CD19, MAC1, CD4, CD8	[166]
	2days exposure prior to transplantation
	Neural stem cell stimulating medium
TSA+5-Aza-dC$	5ng/ml TSA+500nM 5-Aza-dC	Human CD34+ HSC	CD34+/CD90+ HPC	Marrow repopulating capability, self-renewing potential	[165]
	5-Aza-dC: exposure from 16 to 48h cultivation in medium promoting exit of G0/G1 phase
	TSA: exposure from 48h cultivation in medium promoting terminal differentiation
DMSO+AzaC∗	1% DMSO+5mM 5-AzaC	Murine bone marrow MSC	Hepatocyte-like cells	Expression of Alb, TAT, CK18/8, CK19	[156]
	AzaC: exposure for 12h before coculture
	DMSO: exposure throughout culture time
	Hepatic stimulating medium
	Coculture with nonparenchymal liver cells
(B) Failure of (‘trans’)differentiation
HDAC inhibitor
TSA+VPA+DMSO∗	50nM TSA, 2mM VPA, 2% DMSO	Rat bone marrow MSC	Neurons and astrocytes	Decreased expression of NSE and NF, Decreased AchE activity	[170]
	Exposure upon D1 of differentiation onwards
	Neural stimulating medium
Butyrate∗	2mM Butyrate	Mononuclear cells from murine/human peripheral blood/bone marrow/spleen	Endothelial cells	Failure of HoxA9, VEGFR2 expression	[219]
TSA∗	1–2.5μM TSA
MS-275∗	3–10μM MS-275
	Exposure upon onset of differentiation
	Endothelial stimulating medium
TSA∗	5 or 10nM TSA	Murine ES	Embryoid bodies	Steady alkaline phosphatase activity	[220]
	Exposure upon onset of differentiation
	Unspecified differentiation medium
VPA∗	0.3–1mM VPA	Rat neural progenitors	Astrocytes, oligodendrocytes	Failure of Rip and GFAP expression	[162]
TSA∗	100nM TSA
Sodium butyrate∗	1μM Sodium butyrate
	Exposure for 4 days upon onset of differentiation
	Oligodendrocyte and astrocyte stimulating medium
VPA∗	0.5–2mM VPA	Mouse 3T3-L1 preadipocytes	Adipocytes	Reduced PPARγ, SREBP1a, C/EBPα expression	[221]
TSA∗	3nM TSA	Primary human preadipocytes
	Exposure after 2 days of confluence
	Differentiation inducing medium
(C) Time-specified related failure/ accomplishment of (‘trans’)differentiation
HDAC inhibitor
VPA$	300mg/kg VPA	Pups neural progenitor cells	Oligodendrocytes		[171]
	Injection: every 12h, for 4 times
	- In postnatal weeks 1–2			- Suppression of myelination
	- From postnatal week 3 onwards			- No suppression of myelination
TSA∗	5–400nM TSA	Mouse myoblasts	Myotubes		[222]
Sodium butyrate∗	0.1–10mM Sodium butyrate
VPA∗	0.1–10mM VPA
	Differentiation inducing medium
	- Exposure from onset of differentiation			- Prevention of muscle differentiation
	- Exposure after onset of differentiation			- Progression of terminal muscle differentiation and prevention of reorganisation of constitutive heterochromatin
TSA∗	0.1, 1 and 10ng/ml TSA	Rat oligo-dendrocyte progenitors	oligodendrocytes		[223]
	Pre-treated neuroectodermal stimulating medium
	- Exposure up to to 12–24h upon mitogen removal			- Steady state expression of A2B5, no oligodendrocyte maturation
	- Exposure after 24h upon mitogen removal			- Failure of GAIC and PLP expression, oligodendrocyte maturation (20)

Abbreviations: α1AT, alpha1-antitrypsin; AChE, acetylcholinesterase; ADH, alcohol dehydrogenase; ADSC, adipose tissue-derived stromal cells; AFP, alpha-fetoprotein; ALB, albumin; A2M, alpha-2 macroglobulin; ANF, atrial natriuretic factor; ApoF, apolipoprotein; α-SA, alpha-sarcomeric actin; AzaC, 5-azacytidine; 5-Aza-dC, decitabine 5-Aza-2′deoxycytidine; β-MHC, beta-myosin heavy chain; BMP-2, bone morphogenic protein; CAR, constitutive androstane receptor; CD, cluster of differentiation; C/EBP, CCAAT enhancer binding protein; CK, cytokeratin; CPS, carbamyl phosphate synthetase; Cx, connexin; CYP, cytochrome P450; DMSO, dimethylsulfoxide; DPPIV, dipeptidyl peptidase IV; DNMT inhibitor, DNA methyltransferase inhibitor; ES, embryonic stem cells; GAlC, galactocerebroside; GFAP, glial fibrillary acidic protein; HDAC inhibitor, histone deacetylase inhibitor; HNF, hepatocyte-nuclear factor; HSC, haematopoietic stem cells; Hox, homeobox; LDL, low density lipoprotein; MAC1, macrophage antigen-1; MAP, microtubule-associated protein; MPC, mesenchymal progenitor cells; MRP, multidrug resistance protein; MSC, mesenchymal stem cells; NF, neurofilament; Nkx2.5, NK2 transcription factor related locus 5; NSE, neuron specific enolase; PCK1, phosphoenolpyruvate carboxykinase; PeB, peripheral blood; PECAM, platelet and endothelial cell adhesion molecule; PEPCK, phosphoenol-pyruvate carboxykinase; PLP, proteolipid protein; PPARγ, peroxisome proliferator-activated receptor gamma; Rip, oligodendrocyte marker; Runx2, transcription factor vertebrate homologue of the Drosophila runt gene 2; SMA, smooth muscle actin; SREBP, sterol regulatory element binding protein; SS, shear stress; TAT, tyrosine aminotransferase; TO, tryptophan dioxygenase; TSA, trichostatin A; TTR, transthyretin; VEGFR2, vascular endothelial growth factor receptor type 2; VPA, valproic acid.

The differentiation of embryonic/fetal hepatoblasts into adult hepatocytes in vivo basically implies consecutive expression of early (HNF-3β, AFP, transthyretin), midlate (HNF-1α, HNF-4α, ALB, cytokeratin 18) and late (tryptophan-2,3-dioxygenase, tyrosine amino-transferase, C/EBPα, CYPs) markers [3], [5], [82], [85], [113], [114], [142], [144], [175], [176]. Most metabolic and detoxifying enzymes only become functional during the terminal step of liver organogenesis, i.e. peri/postnatal. Therefore, functional assays for enzymes, related to specific functions of the adult liver [80], [177], must be carried out in order to state the mature status of resultant stem cell-derived hepatocyte-like cells. At present, functional analysis is particularly focused on ALB-secretion, urea metabolism and glycogen uptake. Little attention has been paid to other metabolic functions, including CYP450-dependent enzymatic activity and responsiveness to prototype inducers such as phenobarbital (human: CYP2B6, CYP3A4, rat CYP2B1/2), rifampicin (CYP3A4) and 3-methylcholantrene (human and rat CYP1A1/2). Bearing in mind that inducible CYP-dependent activity is considered to be a key determinant of the functional hepatic phenotype [80], [178], characterization should preferably comprise the above mentioned metabolic functionality assays. Alternatively, metabolic profiling using well-known molecules (e.g. paracetamol) might shed light on their potency as in vitro models for preclinical toxicological screening of drug candidates. Recently, Hay and colleagues, in fact succeeded to produce a nearly (≈71%) pure population of functional, metabolism competent hepatocytes out of human ES via fine-tuned preconditioning with HDACi and growth factor stimuli [153]. The resultant cells display active and inducible CYP isozymes, capable of converting various substrates, e.g. midazolam, bufuralol, phenacetin, tolbutamide and rifampicine, to their respective metabolites, thereby supporting their potential use as preclinical in vitro systems for toxicity screening of drugs [153].

The ultimate demonstration of hepatic functionality is no doubt in vivo transplantation of ex vivo generated hepatic cells in (immunodeficient) animal models suffering from liver injury. Sgodda and coworkers recently confirmed the functional integration of hepatic cells derived from MSC, that were primed with 5-AzaC prior to combined exposure to hepatocyte growth factor and EGF [155]. Despite seemingly irrefutable evidence that stem/progenitor cells could contribute to liver reconstitution, caution should be taken with production of false positives due to application of inaccurate labelling techniques [179]. Also, one should keep in mind that, apart from generating fully functional stem-cell derived hepatocytes, other mechanisms including the bystander effect, fusion, partial transdifferentiation and horizontal gene transfer [180], [181] might be responsible.

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4. Conclusion 

During preclinical drug development, early screening of promising drug candidates with respect to their metabolism, pharmacokinetics and potential (toxic) interactions is encouraged to diminish the number of failures at later stages. In vitro techniques, including hepatocyte-based in vitro models, are currently being applied. Hepatocytes in culture, however, enter the cell cycle and irreversibly dedifferentiate. Their limited viability and major loss of xenobiotic biotransformation capacity strongly limit their applicability. Understanding how to control differentiation and proliferation of these primary cells is a key-challenge. Next to targeting the differentiated status of adult primary hepatocytes, stem/progenitor cell technology has been proposed as an alternative to produce functional hepatocytes. Until recently the mechanisms governing lineage-directed and terminal differentiation in these stem/progenitor cells and adult primay cells, respectively, largely have remained unknown. In this review, we provide a better understanding of the intracellular regulation of directed and established liver-specific gene transcription.

In vivo, a constellation of intra- and extra-cellular signaling pathways is known to govern the balance of growth and differentiation in all cells. Stem cells, their differentiated progeny-including adult hepatocytes, and elements of their microenvironment make up a structural, (epi)genetic controlled machinery that coordinates normal homeostatic functioning of these cells [3], [5], [85], [113], [114], [142], [143], [144]. By extensively reviewing the literature, we realised that next to the reconstruction of extracellular communication pathways in vivo (lineage-specific factors, including growth factors, cytokines, hormones, glucocorticoids, cell–cell and cell–matrix interactions), interference at the intracellular level via chromatin remodelling agents might involve a strategy to control lineage-specific gene expression, and consequently (i) the multilineage differentiation potency of stem cells and (ii) the acquisition of a differentiated genotype in adult cells [9], [12], [13], [14], [15], [82], [85], [114], [49], [52], [128], [129], [135], [137], [145], [146]. Gene expression is in fact largely regulated by epigenetic modifications of DNA and chromatin on genomic regulatory and coding regions. In general, acetylation of core histones is associated with transcriptional activation, whereas DNA methylation is associated with gene silencing [17], [20]. However, as reviewed in Table 1, Table 2, Table 3, differential effects are observed depending on the factors studied e.g. the origin of the cells, the property evaluated, the type of epigenetic modifier used and the exposure time. Nevertheless, from the presented data it appears that in several stem cells-derived and primary hepatic-based models both HDACi and DNMTi are potent modulators of liver-specific functions and cellular contacts, and as such could significantly contribute to the acquisition and maintenance of the hepatocyte-specific geno/phenotype in culture.

Up to now, HDACi and DNMTi are mostly applied separately, though, their combined exposure is advantageous [73] and is expected to gain in importance in the following years. Still, more in depth studies are needed to unravel the tangled web of epigenetic marks and lineage-specific transcription, governing the multipotent status of stem cells and the terminal differentiated geno/phenotype of adult cell types.

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Acknowledgements 

This work was supported by the Fund for Scientific Research – Flanders (FWO), the Research Council (OZR) of the Vrije Universiteit Brussel (Belgium), European Community’s Sixth Framework Programme (FP6/2005-2011) under Grant agreement No 037499, the European Community’s Seventh Framework Programme (FP7/2007-2013) under Grant agreement No 20161, ISRIB (Brustem) and BELSPO (IAP HEPRO).

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References 

1. Papeleu P, Vanhaecke T, Rogiers V. Histone deacetylase inhibition: a differentiation therapy for cultured primary hepatocytes. Curr Enz Inhib. 2006;2:91–104
   • View In Article
2. Papeleu P, Loyer P, Vanhaecke T, Elaut G, Geerts A, Guguen-Guillouzo C, et al. Trichostatin A induces cell cycle arrests but does not induce apoptosis in primary cultures of mitogen-stimulated rat hepatocytes. J Hepatol. 2003;39:374–382
   • View In Article
   • Abstract
   • Full Text
   • Full-Text PDF (968 KB)
   • CrossRef
3. Kyrmizi I, Hatzis P, Katrakili N, Tronche F, Gonzalez FJ, Talianidis I. Plasticity and expanding complexity of the hepatic transcription factor network during liver development. Genes Dev. 2006;20:2293–2305
   • View In Article
   • MEDLINE
   • CrossRef
4. Akiyama TE, Gonzalez FJ. Regulation of P450 genes by liver-enriched transcription factors and nuclear receptors. Biochim Biophys Acta. 2003;1619:223–234
   • View In Article
   • MEDLINE
5. Costa RH, Kalinichenko VV, Holterman AX, Wang X. Transcription factors in liver development, differentiation, and regeneration. Hepatology. 2003;38:1331–1347
   • View In Article
   • MEDLINE
   • CrossRef
6. Turner BM. Reading signals on the nucleosome with a new nomenclature for modified histones. Nat Struct Mol Biol. 2005;12:110–112
   • View In Article
   • MEDLINE
   • CrossRef
7. Wolffe AP, Guschin D. Review: chromatin structural features and targets that regulate transcription. J Struct Biol. 2000;129:102–122
   • View In Article
   • MEDLINE
   • CrossRef
8. Wang GG, Allis CD, Chi P. Chromatin remodeling and cancer, part I: covalent histone modifications. Trends Mol Med. 2007;13:363–372
   • View In Article
   • CrossRef
9. Collas P. Epigenetic states in stem cells. Biochim Biophys Acta. 2008;[ahead of print]
   • View In Article
10. Dannenberg LO, Edenbergh HJ. Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation. BMC Genomics. 2006;7:181
    • View In Article
    • MEDLINE
    • CrossRef
11. Yamashita Y, Shimada M, Harimoto N, Rikimaru T, Shirabe K, Tanaka S, et al. Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells. Int J Cancer. 2003;103:572–576
    • View In Article
    • MEDLINE
    • CrossRef
12. Atkinson S, Armstrong L. Epigenetics in embryonic stem cells: regulation of pluripotency and differentiation. Cell Tissue Res. 2008;331:23–29
    • View In Article
    • CrossRef
13. Reik W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature. 2007;447:425–432
    • View In Article
    • CrossRef
14. Yeo S, Jeong S, Kim J, Han JS, Han YM, Kang YK. Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells. Biochem Biophys Res Commun. 2007;359:536–542
    • View In Article
    • MEDLINE
    • CrossRef
15. Perry P, Sauer S, Billon N, Richardson WD, Spivakov M, Warnes G, et al. A dynamic switch in the replication timing of key regulator genes in embryonic stem cells upon neural induction. Cell Cycle. 2004;3:1645–1650
    • View In Article
16. Moggs JG, Goodman JL, Trosko JE, Roberts RA. Epigenetics and cancer: implications for drug discovery and safety assessment. Toxicol Appl Pharmacol. 2004;196:422–430
    • View In Article
    • MEDLINE
    • CrossRef
17. Szyf M. DNA methylation and demethylation as targets for anticancer therapy. Biochemistry (Mosc). 2005;70:533–549
    • View In Article
    • MEDLINE
    • CrossRef
18. Allis CD, Berger SL, Cote J, Dent S, Jenuwien T, Kouzarides T, et al. New nomenclature for chromatin-modifying enzymes. Cell. 2007;131:633–636
    • View In Article
    • CrossRef
19. Weidle UH, Grossmann A. Inhibition of histone deacetylases: a new strategy to target epigenetic modifications for anticancer treatment. Anticancer Res. 2000;20:1471–1485
    • View In Article
    • MEDLINE
20. Grunstein M. Histone acetylation in chromatin structure and transcription. Nature. 1997;389:349–352
    • View In Article
    • MEDLINE
    • CrossRef
21. Yang XJ, Seto E. HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention. Oncogene. 2007;26:5310–5318
    • View In Article
    • CrossRef
22. Xu WS, Parmigiani RB, Marks PA. Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene. 2007;26:5541–5552
    • View In Article
    • CrossRef
23. Minucci S, Pellici PG. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer. 2006;6:38–51
    • View In Article
    • MEDLINE
24. Smith CL. A shifting paradigm: histone deacetylases and transcriptional activation. Bioessays. 2008;30:15–24
    • View In Article
    • CrossRef
25. Gray SG, Ekström TJ. The human histone deacetylase family. Exp Cell Res. 2001;262:75–83
    • View In Article
    • MEDLINE
    • CrossRef
26. Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone deacetylase inhibitors. Nature Rev Drug Discov. 2006;5:769–784
    • View In Article
27. Mariadason JM. HDACs and HDAC inhibitors in colon cancer. Epigenetics. 2008;3:28–37
    • View In Article
    • CrossRef
28. Grozinger CM, Hassig CA, Schreiber SL. Three proteins define a class of human histone deacetylases related to yeast Hda1p. Proc Natl Acad Sci USA. 1999;96:4868–4873
    • View In Article
29. Finnin MS, Donigian JR, Cohen A, Richon VM, Rifkind RA, Marks PA, et al. Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors. Nature. 1999;401:188–193
    • View In Article
    • MEDLINE
    • CrossRef
30. Yang XJ, Grégoire S. Class II histone deacetylases: from sequence to function, regulation, and clinical implication. Mol Cell Biol. 2005;25:2873–2884
    • View In Article
    • MEDLINE
    • CrossRef
31. Zhang CL, McKinsey TA, Olsen EN. Association of class II histone deacetylases with heterochromatin protein 1: potential role for histone methylation in control of muscle differentiation. Mol Cell Biol. 2002;22:7302–7312
    • View In Article
    • MEDLINE
    • CrossRef
32. Martin M, Kettmann R, Dequiedt F. Class IIa histone deacetylases: regulating the regulators. Oncogene. 2007;26:5450–5467
    • View In Article
    • CrossRef
33. Vega RB, Matsuda K, Oh J, Barbosa AC, Yang X, Meadows E, et al. Histone deacetylase 4 controls chondrocyte hypertrophy during skeletogenesis. Cell. 2004;119:555–566
    • View In Article
    • MEDLINE
    • CrossRef
34. Bolger TA, Yao TP. Intracellular trafficking of histone deacetylase 4 regulates neuronal cell death. J Neurosci. 2005;25:9544–9553
    • View In Article
    • CrossRef
35. Boyault C, Sadoel K, Pabion M, Khochbin S. HDAC6, at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination. Oncogene. 2007;26:5468–5476
    • View In Article
    • CrossRef
36. Schroeder TM, Kahler RA, Li X, Westendorf JJ. Histone deacetylase 3 interacts with runx2 to repress the osteocalcin promoter and regulate osteoblast differentiation. J Biol Chem. 2004;279:41998–42007
    • View In Article
    • MEDLINE
    • CrossRef
37. Puri PL, Iezzi S, Stiegler P, Chen TT, Schiltz RL, Muscat GE, et al. Class I histone deacetylases sequentially interact with MyoD and pRb during skeletal myogenesis. Mol Cell. 2001;8:885–897
    • View In Article
38. Wilson AJ, Byun DS, Popova N, Murray LB, L’Italien K, Sowa Y, et al. Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer. J Biol Chem. 2006;281:13548–13558
    • View In Article
    • MEDLINE
    • CrossRef
39. Farooq M, Sulochana KN, Pan X, To J, Sheng D, Gong Z, et al. Histone deacetylase 3 (hdac3) is specifically required for liver development in zebrafish. Dev Biol. 2008;317:336–353
    • View In Article
    • CrossRef
40. Knutson SK, Chyla BJ, Amann JM, Bhaskara S, Huppert SS, Hiebert SW. Liver-specific deletion of histone deacetylase 3 disrupts metabolic transcriptional networks. EMBO J. 2008;27:1017–1028
    • View In Article
    • CrossRef
41. Wang AG, Seo SB, Moon HB, Shin HJ, Kim DH, Kim JM, et al. Hepatic steatosis in transgenic mice overexpressing human histone deacetylase 1. Biochem Biophys Res Commun. 2005;330:461–466
    • View In Article
    • MEDLINE
    • CrossRef
42. Wang AG, Kim SU, Lee SH, Kim SK, Seo SB, Yu DY, et al. Histone deacetylase 1 contributes to cell cycle and apoptosis. Biol Pharm Bull. 2005;28:1966–1970
    • View In Article
    • MEDLINE
    • CrossRef
43. Jung M. Inhibitors of histone deacetylase as new anticancer agents. Curr Med Chem. 2001;8:1505–1511
    • View In Article
    • MEDLINE
44. Johnstone RA. The evolution of inaccurate mimics. Nature. 2002;418:524–526
    • View In Article
    • MEDLINE
    • CrossRef
45. Duvic M, Vu J. Vorinostat: a new oral histone deactylase inhibitor approved for cutaneous T-cell ymphoma. Expert Opin Investig Drugs. 2007;16:1111–1120
    • View In Article
    • CrossRef
46. Lu YS, Kashida Y, Kulp SK, Wang YC, Wang D, Hung JH, et al. Efficacy of a novel histone deacetylase inhibitor in murine models of hepatocellular carcinoma. Hepatology. 2007;46:1119–1130
    • View In Article
    • CrossRef
47. Herold C, Ganslmayer M, Ocker M, Hermann M, Geerts A, Hahn EG, et al. The histone-deacetylase inhibitor Trichostatin A blocks proliferation and triggers apoptotic programs in hepatoma cells. J Hepatol. 2002;36:233–240
    • View In Article
    • Full-Text PDF (113 KB)
    • CrossRef
48. Wakabayashi K, Saito H, Kaneko F, Nakamoto N, Tada S, Hibi T. Gene expression associated with the decrease in malignant phenotype of human liver cancer cells following stimulation with a histone deacetylase inhibitor. Int J Oncol. 2005;26:233–239
    • View In Article
    • MEDLINE
49. Henkens T, Papeleu P, Elaut G, Vinken M, Rogiers V, Vanhaecke T. Trichostatin A, a critical factor to maintain differentiation in primary cultures of rat hepatocytes. Toxicol Appl Pharmacol. 2007;218:64–71
    • View In Article
    • MEDLINE
50. Papeleu P, Wullaert A, Elaut G, Henkens T, Vinken M, Laus G, et al. Inhibition of NF-kappaB activation by the histone deacetylase inhibitor 4-Me2N-BAVAH induces an early G1 cell cycle arrest in primary hepatocytes. Cell Prolif. 2007;40:640–655
    • View In Article
    • CrossRef
51. Vinken M, Henkens T, Snykers S, Lukaszuk A, Tourwé D, Rogiers V, et al. The novel histone deacetylase inhibitor 4-Me2N-BAVAH differentially affects cell junctions between primary hepatocytes. Toxicology. 2007;236:92–102
    • View In Article
    • MEDLINE
    • CrossRef
52. Vinken M, Henkens T, Vanhaecke T, Papeleu P, Geerts A, Van Rossen E, et al. Trichostatin A enhances gap junctional intercellular communication in primary cultures of adult rat hepatocytes. Toxicol Sci. 2006;91:484–492
    • View In Article
    • MEDLINE
    • CrossRef
53. Baylin SB. DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol. 2005;2:4–11
    • View In Article
    • MEDLINE
    • CrossRef
54. Rice KL, Hormaeche I, Licht JD. Epigenetic regulation of normal and malignant hematopoiesis. Oncogene. 2007;26:6697–6714
    • View In Article
    • CrossRef
55. Costello JF, Plass C. Methylation matters. J Med Genet. 2001;38:285–303
    • View In Article
56. Métivier R, Gallais R, Tiffoche C, Le Peron C, Jurkowska RZ, Carmouche RP, et al. Cyclical DNA methylation of a transcriptionally active promoter. Nature. 2008;452:45–50
    • View In Article
    • CrossRef
57. Massillon D, Arinze IJ, Xu C, Bone F. Regulation of glucose-6-phosphatase gene expression in cultured hepatocytes and H4IIE cells by short-chain fatty acids: role of hepatic nuclear factor-4alpha. J Biol Chem. 2003;278:40694–40701
    • View In Article
    • MEDLINE
    • CrossRef
58. Park HJ, Yu E, Shim YH. DNA methyltransferase expression and DNA hypermethylation in human hepatocellular carcinoma. Cancer Lett. 2006;233:271–278
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (150 KB)
    • CrossRef
59. Pradhan S, Esteve PO. Mammalian DNA (cytosine-5) methyltransferases and their expression. Clin Immunol. 2003;109:6–16
    • View In Article
    • MEDLINE
    • CrossRef
60. Bestor TH. The DNA methyltransferases of mammals. Hum Mol Genet. 2000;9:2395–2402
    • View In Article
    • MEDLINE
    • CrossRef
61. Kuhlmann M, Borisova BE, Kaller M, Larsson P, Stach D, Na J, et al. Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi. Nucleic Acids Res. 2005;33:6405–6417
    • View In Article
    • CrossRef
62. Jurkowski TP, Meusburger M, Phalke S, Helm M, Nellen W, Reuter G, et al. Human DNMT2 methylates tRNAAsp molecules using a DNA methyltransferase-like catalytic mechanism. RNA. 2008;14:1663–1670
    • View In Article
    • CrossRef
63. Liang G, Chan MF, Tomigahara Y, Tsai YC, Gonzales FA, Li E, et al. Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. Mol Cell Biol. 2002;22:480–491
    • View In Article
    • MEDLINE
    • CrossRef
64. Klose RJ, Bird AP. Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci. 2006;31:89–97
    • View In Article
    • MEDLINE
    • CrossRef
65. Suetake I, Shinozaki F, Miyagawa J, Takeshima H, Tajima S. DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction. J Biol Chem. 2004;279:27816–27823
    • View In Article
    • MEDLINE
    • CrossRef
66. Gonzalo S, Jaco I, Fraga MF, Chen T, Li E, Esteller M, et al. DNA methyltransferases control telomere length and telomere recombination in mammalian cells. Nat Cell Biol. 2006;8:416–424
    • View In Article
    • MEDLINE
    • CrossRef
67. Turek-plewa J, Jagodzinski PP. The role of mammalian DNA methyltransferases in the regulation of gene expression. Cell Mol Biol Lett. 2005;10:631–647
    • View In Article
    • MEDLINE
68. Qiu W, Zhou B, Zou H, Liu X, Chu PG, Lopez R, et al. Hypermethylation of growth arrest DNA damage-inducible gene 45 beta promotor in human heptocellualr carcinoma. Am J Pathol. 2004;165:1689–1699
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (1091 KB)
    • CrossRef
69. Zhu WG, Otterson GA. The interaction of histone deacetylase inhibitors and DNA methyltransferase inhibitors in the treatment of human cancer cells. Curr Med Chem Anticancer Agents. 2003;3:187–199
    • View In Article
    • MEDLINE
    • CrossRef
70. Laird PW. Cancer epigenetics. Hum Mol Genet. 2005;14:R65–R76
    • View In Article
    • CrossRef
71. Yoo CB, Cheng JC, Jones PA. Zebularine: a new drug for epigenetic therapy. Biochem Soc Trans. 2004;32:910–912
    • View In Article
    • MEDLINE
    • CrossRef
72. Stresemann C, Lyko F. Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine. Int J Cancer. 2008;123:8–13
    • View In Article
    • CrossRef
73. Rogiers V, Vanhaecke T, De Rop E, Fraczek J. Title of invention: stabilisation of the phenotypic properties of isolated primary cells. International patent number: PCT/EP2008/056706.
    • View In Article
74. Lin X, Asgari K, Putzi MJ, Gage WR, Yu X, Cornblatt BS, et al. Reversal of GSTP1 CpG island hypermethylation and reactivation of pi-class glutathione S-transferase (GSTP1) expression in human prostate cancer cells by treatment with procainamide. Cancer Res. 2001;61:8611–8616
    • View In Article
    • MEDLINE
75. Villar-Garea A, Fraga MF, Espada J, Esteller M. Procaine is a DNA-demethylating agent with growth-inhibitory effects in human cancer cells. Cancer Res. 2003;63:4984–4989
    • View In Article
    • MEDLINE
76. Piña IC, Gautschi JT, Wang GY, Sanders ML, Schmitz FJ, France D, et al. Psammaplins from the sponge Pseudoceratina purpurea: inhibition of both histone deacetylase and DNA methyltransferase. J Org Chem. 2003;68:3866–3873
    • View In Article
    • MEDLINE
    • CrossRef
77. Belinsky SA, Klinge DM, Stidley CA, Issa JP, Herman JG, March TH, et al. Inhibition of DNA methylation and histone deacetylation prevents murine lung cancer. Cancer Res. 2003;63:7089–7093
    • View In Article
    • MEDLINE
78. Klisovic MI, Maghraby EA, Parthun MR, Guimond M, Sklenar AR, Whitman SP, et al. Depsipeptide (FR 901228) promotes histone acetylation, gene transcription, apoptosis and its activity is enhanced by DNA methyltransferase inhibitors in AML1/ETO-positive leukemic cells. Leukemia. 2003;17:350–358
    • View In Article
    • MEDLINE
    • CrossRef
79. Brueckner B, Lyko F. DNA methyltransferase inhibitors: old and new drugs for an epigenetic cancer therapy. Trends Pharmacol Sci. 2004;25:551–554
    • View In Article
    • MEDLINE
    • CrossRef
80. Vanhaecke T, Rogiers V. Hepatocyte cultures in drug metabolism and toxicological research and testing. In: Phillips IR, Shephard EA, editors. Cytochrome P450 Protocols. Methods in molecular biology. 2nd ed. Totowa, NJ: Humana Press Inc.; 2006. p. 209–27.
    • View In Article
81. Elaut G, Henkens T, Papeleu P, Snykers S, Vinken M, Vanhaecke T, et al. Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures. Curr Drug Metab. 2006;7:629–660
    • View In Article
    • MEDLINE
    • CrossRef
82. Schrem H, Klempnauer J, Borlak J. Liver-enriched transcription factors in liver function and development. Part I: the hepatocyte nuclear factor network and liver-specific gene expression. Pharmacol Rev. 2002;54:129–158
    • View In Article
    • MEDLINE
    • CrossRef
83. Padgham CR, Boyle CC, Wang XJ, Raleigh SM, Wright MC, Paine AJ. Alteration of transcription factor mRNAs during the isolation and culture of rat hepatocytes suggests the activation of a proliferative mode underlies their de-differentiation. Biochem Biophys Res Commun. 1993;197:599–605
    • View In Article
    • MEDLINE
    • CrossRef
84. Rodriguez-Antona C, Donato MT, Boobis A, Edwards RJ, Watts PS, Castell JV, et al. Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica. 2002;32:505–520
    • View In Article
    • MEDLINE
    • CrossRef
85. Zaret KS, Watts J, Xu J, Wandzioch E, Smale ST, Sekiya T. Pioneer factors, genetic competence, and inductive signaling: programming liver and pancreas progenitors from the endoderm. Cold Spring Harb Symp Quant Biol. 2008;[ahead of print]
    • View In Article
86. Kistanova E, Dell H, Tsantili P, Falvey E, Cladaras C, Hadzopoulou-Cladaras M. The activation function-1 of hepatocyte nuclear factor-4 is an acidic activator that mediates interactions through bulky hydrophobic residues. Biochem J. 2001;356:635–642
    • View In Article
    • MEDLINE
    • CrossRef
87. Green VJ, Kokkotou E, Ladias JA. Critical structural elements and multitarget protein interactions of the transcriptional activator AF-1 of hepatocyte nuclear factor 4. J Biol Chem. 1998;273:29950–29957
    • View In Article
    • MEDLINE
    • CrossRef
88. Wang JC, Stafford JM, Granner DK. SRC-1 and GRIP1 coactivate transcription with hepatocyte nuclear factor 4. J Biol Chem. 1998;273:30847–30850
    • View In Article
    • MEDLINE
    • CrossRef
89. Sladek FM, Ruse MD, Nepomuceno L, Huang SM, Stallcup MR. Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1. Mol Cell Biol. 1999;19:6509–6522
    • View In Article
    • MEDLINE
90. Yoshida E, Aratani S, Itou H, Miyagishi M, Takiguchi M, Osumu T, et al. Functional association between CBP and HNF4 in trans-activation. Biochem Biophys Res Commun. 1997;241:664–669
    • View In Article
    • MEDLINE
    • CrossRef
91. Dell H, Hadzopoulou-Cladaras M. CREB-binding protein is a transcriptional coactivator for hepatocyte nuclear factor-4 and enhances apolipoprotein gene expression. J Biol Chem. 1999;274:9013–9021
    • View In Article
    • MEDLINE
    • CrossRef
92. Torres-Padilla ME, Sladek FM, Weiss MC. Developmentally regulated N-terminal variants of the nuclear receptor hepatocyte nuclear factor 4alpha mediate multiple interactions through coactivator and corepressor-histone deacetylase complexes. J Biol Chem. 2002;277:44677–44687
    • View In Article
    • MEDLINE
    • CrossRef
93. Torres-Padilla ME, Weiss MC. Effects of interactions of hepatocyte nuclear factor 4alpha isoforms with coactivators and corepressors are promoter-specific. FEBS Lett. 2003;539:19–23
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (157 KB)
    • CrossRef
94. Martinez-Jimenez CP, Castell JV, Gomez-Lechon MJ, Jover R. Transcriptional activation of CYP2C9, CYP1A1, and CYP1A2 by hepatocyte nuclear factor 4alpha requires coactivators peroxisomal proliferator activated receptor-gamma coactivator 1alpha and steroid receptor coactivator 1. Mol Pharmacol. 2006;70:1681–1692
    • View In Article
    • MEDLINE
    • CrossRef
95. Puigserver P, Adelmant G, Wu Z, Fan M, Xu J, O’Malley B, et al. Activation of PPARgamma coactivator-1 through transcription factor docking. Science. 1999;286:1368–1371
    • View In Article
    • MEDLINE
    • CrossRef
96. Batsche E, Desroches J, Bilodeau S, Gauthier Y, Drouin J. Rb enhances p160/SRC coactivator-dependent activity of nuclear receptors and hormone responsiveness. J Biol Chem. 2005;280:19746–19756
    • View In Article
    • MEDLINE
    • CrossRef
97. Ruse MD, Privalsky ML, Sladek FM. Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4alpha1: modulation by the F domain. Mol Cell Biol. 2002;22:1626–1638
    • View In Article
    • MEDLINE
    • CrossRef
98. Maeda Y, Seidel SD, Wei G, Liu X, Sladek FM. Repression of hepatocyte nuclear factor 4alpha tumor suppressor p53: involvement of the ligand-binding domain and histone deacetylase activity. Mol Endocrinol. 2002;16:402–410
    • View In Article
    • MEDLINE
    • CrossRef
99. Ban N, Yamada Y, Someya Y, Miyawaki K, Ihara Y, Hosokawa M, et al. Hepatocyte nuclear factor-1alpha recruits the transcriptional co-activator p300 on the GLUT2 gene promoter. Diabetes. 2002;51:1409–1418
    • View In Article
    • MEDLINE
    • CrossRef
100. Soutoglou E, Papafotiou G, Katrakili N, Talianidis I. Transcriptional activation by hepatocyte nuclear factor-1 requires synergism between multiple coactivator proteins. J Biol Chem. 2000;275:12515–12520
     • View In Article
     • MEDLINE
     • CrossRef
101. Dohda T, Kaneoka H, Inayoshi Y, Kamihira M, Miyake K, Iijima S. Transcriptional coactivators CBP and p300 cooperatively enhance HNF-1alpha-mediated expression of the albumin gene in hepatocytes. J Biochem. 2004;136:313–319
     • View In Article
     • MEDLINE
     • CrossRef
102. Soutoglou E, Viollet B, Vaxillaire M, Yaniv M, Pontoglio M, Talianidis I. Transcription factor-dependent regulation of CBP and P/CAF histone acetyltransferase activity. EMBO J. 2001;20:1984–1992
     • View In Article
     • MEDLINE
     • CrossRef
103. Rausa FM, Tan Y, Costa RH. Association between hepatocyte nuclear factor 6 (HNF-6) and FoxA2 DNA binding domains stimulates FoxA2 transcriptional activity but inhibits HNF-6 DNA binding. Mol Cell Biol. 2003;23:437–449
     • View In Article
     • MEDLINE
     • CrossRef
104. Yoshida Y, Hughes DE, Rausa FM, Kim IM, Tan Y, Darlington GJ, et al. C/EBPalpha and HNF6 protein complex formation stimulates HNF6-dependent transcription by CBP coactivator recruitment in HepG2 cells. Hepatology. 2006;43:276–286
     • View In Article
     • MEDLINE
     • CrossRef
105. Erickson RL, Hemati N, Ross SE, MacDougald OA. p300 coactivates the adipogenic transcription factor CCAAT/enhancer-binding protein alpha. J Biol Chem. 2001;276:16348–16355
     • View In Article
     • MEDLINE
106. Chen PL, Riley DJ, Chen Y, Lee WH. Retinoblastoma protein positively regulates terminal adipocyte differentiation through direct interaction with C/EBPs. Genes Dev. 1996;10:2794–2804
     • View In Article
     • MEDLINE
     • CrossRef
107. Charles A, Tang X, Crouch E, Brody JS, Xiao ZX. Retinoblastoma protein complexes with C/EBP proteins and activates C/EBP-mediated transcription. J Cell Biochem. 2001;83:414–425
     • View In Article
     • MEDLINE
     • CrossRef
108. Chen CJ, Deng Z, Kim AY, Blobel GA, Lieberman PM. Stimulation of CREB binding protein nucleosomal histone acetyltransferase activity by a class of transcriptional activators. Mol Cell Biol. 2001;21:476–487
     • View In Article
     • MEDLINE
     • CrossRef
109. Mink S, Haenig B, Klempnauer KH. Interaction and functional collaboration of p300 and C/EBPbeta. Mol Cell Biol. 1997;17:6609–6617
     • View In Article
     • MEDLINE
110. Cui TX, Piwien-Pilipuk G, Huo JS, Kaplani J, Kwok R, Schwartz J. Endogenous CCAAT/enhancer binding protein beta and p300 are both regulated by growth hormone to mediate transcriptional activation. Mol Endocrinol. 2005;19:2175–2186
     • View In Article
     • MEDLINE
     • CrossRef
111. Cesena TI, Cardinaux JR, Kwok R, Schwartz J. CCAAT/enhancer-binding protein (C/EBP) beta is acetylated at multiple lysines: acetylation of C/EBPbeta at lysine 39 modulates its ability to activate transcription. J Biol Chem. 2007;282:956–967
     • View In Article
     • MEDLINE
112. Wiper-Bergeron N, Salem HA, Tomlinson JJ, Wu D, Hache RJ. Glucocorticoid-stimulated preadipocyte differentiation is mediated through acetylation of C/EBPbeta by GCN5. Proc Natl Acad Sci USA. 2007;104:2703–2708
     • View In Article
113. Shim EY, Woodcock C, Zaret KS. Nucleosome positioning by the winged helix transcription factor HNF3. Genes Dev. 1998;12:5–10
     • View In Article
     • MEDLINE
     • CrossRef
114. Cirillo LA, Lin FR, Cuesta I, Friedman D, Jarnik M, Zaret KS. Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol Cell. 2002;9:279–289
     • View In Article
115. Chaya D, Hayamizu T, Bustin M, Zaret KS. Transcription factor FoxA (HNF3) on a nucleosome at an enhancer complex in liver chromatin. J Biol Chem. 2001;276:44385–44389
     • View In Article
     • MEDLINE
     • CrossRef
116. Lea MA, Ibeh C, Shah N, Moyer MP. Induction of differentiation of colon cancer cells by combined inhibition of kinases and histone deacetylase. Anticancer Res. 2007;27:741–748
     • View In Article
     • MEDLINE
117. Mayo C, Lloreta J, Real FX, Mayol X. In vitro differentiation of HT-29 M6 mucus-secreting colon cancer cells involves a trychostatin A and p27(KIP1)-inducible transcriptional program of gene expression. J Cell Physiol. 2007;212:42–50
     • View In Article
     • MEDLINE
     • CrossRef
118. Hinnebusch BF, Meng S, Wu JT, Archer SY, Hodin RA. The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation. J Nutr. 2002;132:1012–1017
     • View In Article
     • MEDLINE
119. Gensert JM, Baranova OV, Weinstein DE, Ratan RR. CD81, a cell cycle regulator, is a novel target for histone deacetylase inhibition in glioma cells. Neurobiol Dis. 2007;26:671–680
     • View In Article
     • MEDLINE
     • CrossRef
120. Asklund T, Appelskog IB, Ammerpohl O, Ekström TJ, Almqvist PM. Histone deacetylase inhibitor 4-phenylbutyrate modulates glial fibrillary acidic protein and connexin 43 expression, and enhances gap-junction communication, in human glioblastoma cells. Eur J Cancer. 2004;40:1073–1081
     • View In Article
     • Abstract
     • Full Text
     • Full-Text PDF (541 KB)
     • CrossRef
121. Kumagai T, Wakimoto N, Yin D, Gery S, Kawamata N, Takai N, et al. Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (Vorinostat, SAHA) profoundly inhibits the growth of human pancreatic cancer cells. Int J Cancer. 2007;121:656–665
     • View In Article
     • MEDLINE
     • CrossRef
122. Munster PN, Troso-Sandoval T, Rosen N, Rifkind R, Marks PA, Richon VM. The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces differentiation of human breast cancer cells. Cancer Res. 2001;61:8492–8497
     • View In Article
     • MEDLINE
123. Emanuele S, Lauricella M, Carlisi D, Vassallo B, D’Anneo A, Di Fazio P, et al. SAHA induces apoptosis in hepatoma cells and synergistically interacts with the proteasome inhibitor Bortezomib. Apoptosis. 2007;12:1327–1338
     • View In Article
     • MEDLINE
     • CrossRef
124. Armeanu S, Pathil A, Venturelli S, Mascagni P, Weiss TS, Gottlicher M, et al. Apoptosis on hepatoma cells but not on primary hepatocytes by histone deacetylase inhibitors valproate and ITF2357. J Hepatol. 2005;42:210–217
     • View In Article
     • Full-Text PDF (238 KB)
     • CrossRef
125. Papeleu P, Vanhaecke T, Elaut G, Vinken M, Henkens T, Snykers S, et al. Differential effects of histone deacetylase inhibitors in tumor and normal cells-what is the toxicological relevance?. Crit Rev Toxicol. 2005;35:363–378
     • View In Article
     • MEDLINE
     • CrossRef
126. Henkens T, Vinken M, Lukaszuk A, Tourwé D, Vanhaecke T, Rogiers V. Differential effects of hydroxamate histone deacetylase inhibitors on cellular functionality and gap junctions in primary cultures of mitogen-stimulated hepatocytes. Toxicol Lett. 2008;178:37–43
     • View In Article
     • CrossRef
127. Vanhaecke T, Henkens T, Kass GE, Rogiers V. Effect of the histone deacetylase inhibitor trichostatin A on spontaneous apoptosis in various types of adult rat hepatocyte cultures. Biochem Pharmacol. 2004;68:753–760
     • View In Article
     • MEDLINE
     • CrossRef
128. Bort R, Gomez-Lechon MJ, Castell JV, Jover R. Role of hepatocyte nuclear factor 3 gamma in the expression of human CYP2C genes. Arch Biochem Biophys. 2004;426:63–72
     • View In Article
     • MEDLINE
     • CrossRef
129. Rodrı´guez-Antona C, Bort R, Jover R, Tindberg N, Ingelman-Sundberg M, Gómez-Lechón MJ, et al. Transcriptional regulation of human CYP3A4 basal expression by CCAAT enhancer-binding protein alpha and hepatocyte nuclear factor-3 gamma. Mol Pharmacol. 2003;63:1180–1189
     • View In Article
     • MEDLINE
     • CrossRef
130. Haarmann-Stemmann T, Bothe H, Kohli A, Sydlik U, Abel J, Fritsche E. Analysis of the transcriptional regulation and molecular function of the Aryl Hydrocarbon Receptor Repressor in human cell lines. Drug Metab Dispos. 2007;35:2262–2269
     • View In Article
     • CrossRef
131. Kim JY, Ahn MR, Kim DK, Sheen YY. Histone deacetylase inhibitor stimulate CYP3A4 proximal promoter activity in HepG2 cells. Arch Pharm Res. 2004;27:407–414
     • View In Article
     • MEDLINE
132. Tronche F, Rollier A, Bach I, Weiss MC, Yaniv M. The rat albumin promoter: cooperation with upstream elements is required when binding of APF/HNF1 to the proximal element is partially impaired by mutation or bacterial methylation. Mol Cell Biol. 1989;9:4759–4766
     • View In Article
     • MEDLINE
133. Petropoulos CJ, Yaswen P, Panzica M, Fausto N. Methylation of the alphafetoprotein gene in cell populations isolated from rat livers during carcinogenesis. Nucleic Acids Res. 1985;13:8105–8118
     • View In Article
     • MEDLINE
134. Piechocki MP, Burk RD, Ruch RJ. Regulation of connexin32 and connexin43 gene expression by DNA methylation in rat liver cells. Carcinogenesis. 1999;20:401–406
     • View In Article
     • MEDLINE
     • CrossRef
135. Vieira I, Sonnier M, Cresteil T. Developmental expression of CYP2E1 in the human liver. Hypermethylation control of gene expression during the neonatal period. Eur J Biochem. 1996;238:476–483
     • View In Article
     • MEDLINE
136. Hammons GJ, Yan-Sanders Y, Jin B, Blann E, Kadlubar FF, Lyn-Cook BD. Specific site methylation in the 5′-flanking region of CYP1A2 interindividual differences in human livers. Life Sci. 2001;69:839–845
     • View In Article
     • MEDLINE
     • CrossRef
137. Matsunaga E, Gonzalez FJ. Specific cytosine demethylations within the first exons of the rat CYP2D3 and CYP2D5 genes are associated with activation of hepatic gene expression during development. DNA Cell Biol. 1990;9:443–452
     • View In Article
     • MEDLINE
     • CrossRef
138. Yokomori N, Kobayashi R, Moore R, Sueyoshi T, Nehishi M. A DNA methylation site in the male-specific P450 (Cyp 2d-9) promoter and binding of the heteromeric transcription factor GABP. Mol Cell Biol. 1995;15:5355–5362
     • View In Article
     • MEDLINE
139. Yokomori N, Nishio K, Aida K, Negishi M. Transcriptional regulation by HNF-4 of the steroid 15alpha-hydroxylase P450 (Cyp2a-4) gene in mouse liver. J Steroid Biochem Mol Biol. 1997;62:307–314
     • View In Article
     • MEDLINE
     • CrossRef
140. Kwon MS, Kim SJ, Lee SY, Jeong JH, Lee ES, Kang HS. Epigenetic silencing of the sulfotransferase 1A1 gene by hypermethylation in breast tissue. Oncol Rep. 2006;15:27–32
     • View In Article
     • MEDLINE
141. Benayahu D, Akavia UD, Shur I. Differentiation of bone marrow stroma-derived mesenchymal cells. Curr Med Chem. 2007;14:173–179
     • View In Article
     • MEDLINE
     • CrossRef
142. Theise ND. Gastrointestinal stem cells. III. Emergent themes of liver stem cell biology: niche, quiescence, self-renewal, and plasticity. Am J Physiol Gastrointest Liver Physiol. 2006;290:G189–G193
     • View In Article
     • MEDLINE
     • CrossRef
143. Naveiras O, Daley GQ. Stem cells and their niche: a matter of fate. Cell Mol Life Sci. 2006;63:760–766
     • View In Article
     • MEDLINE
     • CrossRef
144. Moore KA, Lemischka IR. Stem cells and their niches. Science. 2006;311:1880–1885
     • View In Article
     • CrossRef
145. Kondo T. Epigenetic alchemy for cell fate conversion. Curr Opin Genet Dev. 2006;16:502–507
     • View In Article
     • MEDLINE
     • CrossRef
146. Zardo G, Cimino G, Nervi C. Epigenetic plasticity of chromatin in embryonic and hematopoietic stem/progenitor cells: therapeutic potential of cell reprogramming. Leukemia. 2008;22:1503–1518
     • View In Article
     • CrossRef
147. Song MR, Ghosh A. FGF2-induced chromatin remodeling regulates CNTF-mediated gene expression and astrocyte differentiation. Nat Neurosci. 2004;7:229–235
     • View In Article
     • MEDLINE
     • CrossRef
148. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676
     • View In Article
     • MEDLINE
     • CrossRef
149. Sathananthan AH, Trounson A. Human embryonic stem cells and their spontaneous differentiation. Ital J Anat Embryol. 2005;110:151–157
     • View In Article
     • MEDLINE
150. Lavon N, Benvenisty N. Study of hepatocyte differentiation using embryonic stem cells. J Cell Biochem. 2005;96:1193–1202
     • View In Article
     • MEDLINE
     • CrossRef
151. Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cell lines. Stem Cells. 2001;19:193–204
     • View In Article
     • MEDLINE
     • CrossRef
152. Rambhatla L, Chiu CP, Kundu P, Peng Y, Carpenter MK. Generation of hepatocyte-like cells from human embryonic stem cells. Cell Transplant. 2003;12:1–11
     • View In Article
     • MEDLINE
     • CrossRef
153. Hay DC, Zhao D, Fletcher J, Hewitt ZA, McLean D, Urruticoechea-Uriguen A, et al. Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo. Stem Cells. 2008;26:894–902
     • View In Article
     • CrossRef
154. Snykers S, Vanhaecke T, De Becker A, Papeleu P, Vinken M, Van Riet I, et al. Chromatin remodeling: a key-factor in the endogenic differentiation of human mesenchymal stem cells. BMC Dev Biol. 2007;7:1–15
     • View In Article
     • MEDLINE
     • CrossRef
155. Sgodda M, Aurich H, Kleist S, Aurich I, König S, Dollinger MM, et al. Hepatocyte differentiation of mesenchymal stem cells from rat peritoneal adipose tissue in vitro and in vivo. Exp Cell Res. 2007;313:2875–2886
     • View In Article
     • CrossRef
156. Yamazaki S, Miki K, Hasegawa K, Sata M, Takayama T, Makuuchi M. Sera from liver failure patients and a demethylating agent stimulate transdifferentiation of murine bone marrow cells into hepatocytes in coculture with nonparenchymal liver cells. J Hepatol. 2003;39:17–23
     • View In Article
     • Abstract
     • Full Text
     • Full-Text PDF (1573 KB)
     • CrossRef
157. Kang XQ, Zang WJ, Bao LJ, Li DL, Song TS, Xu XL, et al. Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes. World J Gastroenterol. 2005;11:7461–7465
     • View In Article
     • MEDLINE
158. Ong SY, Dai H, Leong KW. Hepatic differentiation potential of commercially available human mesenchymal stem cells. Tissue Eng. 2006;12:3477–3485
     • View In Article
     • MEDLINE
     • CrossRef
159. Kwak DH, Yu K, Kim SM, Lee DH, Kim SM, Jung JU, et al. Dynamic changes of gangliosides expression during the differentiation of embryonic and mesenchymal stem cells into neural cells. Exp Mol Med. 2006;38:668–676
     • View In Article
     • MEDLINE
160. Park KS, Lee YS, Kang KS. In vitro neuronal and osteogenic differentiation of mesenchymal stem cells from human umbilical cord blood. J Vet Sci. 2006;7:343–348
     • View In Article
     • MEDLINE
     • CrossRef
161. Seo MJ, Suh SY, Bae YC, Jung JS. Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo. Biochem Biophys Res Commun. 2005;328:258–264
     • View In Article
     • MEDLINE
     • CrossRef
162. Hsieh J, Nakashima K, Kuwabara T, Mejia E, Gage FH. Histone deacetylase inhibition-mediated neuronal differentiation of multipotent adult neural progenitor cells. Proc Natl Acad Sci USA. 2004;101:16659–16664
     • View In Article
163. Illi B, Scopece A, Nanni S, Farsetti A, Morgante L, Biglioli P, et al. Epigenetic histone modification and cardiovascular lineage programming in mouse embryonic stem cells exposed to laminar shear stress. Circ Res. 2005;96:501–508
     • View In Article
     • CrossRef
164. Kawamura T, Ono K, Morimoto T, Wada H, Hirai M, Hidaka K, et al. Acetylation of GATA-4 is involved in the differentiation of embryonic stem cells into cardiac myocytes. J Biol Chem. 2005;280:19682–19688
     • View In Article
     • MEDLINE
     • CrossRef
165. Milhem M, Mahmud N, Lavelle D, Araki H, DeSimone J, Saunthararajah Y, et al. Modification of hematopoietic stem cell fate by 5aza 2′deoxycytidine and trichostatin A. Blood. 2004;103:4102–4110
     • View In Article
     • MEDLINE
     • CrossRef
166. Schmittwolf C, Kirchhof N, Jauch A, Dürr M, Harder F, Zenke M, et al. In vivo haematopoietic activity is induced in neurosphere cells by chromatin-modifying agents. EMBO J. 2005;24:554–566
     • View In Article
     • MEDLINE
     • CrossRef
167. Yoshida Y, Shimomura T, Sakabe T, Ishii K, Gonda K, Matsuoka S, et al. A role of Wnt/beta-catenin signals in hepatic fate specification of human umbilical cord blood-derived mesenchymal stem cells. Am J Physiol Gastrointest Liver Physiol. 2007;293:G1089–G1098
     • View In Article
     • CrossRef
168. Stock P, Staege MS, Müller LP, Sgodda M, Völker A, Volkmer I, et al. Hepatocytes derived from adult stem cells. Transplant Proc. 2008;40:620–623
     • View In Article
     • Abstract
     • Full Text
     • Full-Text PDF (544 KB)
     • CrossRef
169. De Kock J, Vanhaecke T, Rogiers V, Snykers S. Chromatin remodelling, a novel strategy to expedite the hepatic differentiation of adult bone marrow stem cells in vitro. AATEX. 2008;14:605–611
     • View In Article
170. Jori FP, Napolitano MA, Melone MA, Cippolaro M, Cascino A, Altucci L, et al. Molecular pathways involved in neural in vitro differentiation of marrow stromal stem cells. J Cell Biochem. 2005;94:645–655
     • View In Article
     • MEDLINE
     • CrossRef
171. Shen S, Li J, Cassaccia-Bonnefil P. Histone modifications affect timing of oligodendrocyte progenitor differentiation in the developing rat brain. J Cell Biol. 2005;169:577–589
     • View In Article
     • MEDLINE
     • CrossRef
172. Enright BP, Sung LY, Chang CC, Yang X, Tian XC. Methylation and acetylation characteristics of cloned bovine embryos from donor cells treated with 5-aza-2′-deoxycytidine. Biol Reprod. 2005;72:944–948
     • View In Article
     • MEDLINE
     • CrossRef
173. Soto-Gutierrez A, Navarro-Alvarez N, Rivas-Carrillo JD, Chen Y, Yamatsuji T, Tanaka N, et al. Differentiation of human embryonic stem cells to hepatocytes using deleted variant of HGF and poly-amino-urethane-coated nonwoven polytetrafluoroethylene fabric. Cell Transplant. 2006;15:335–341
     • View In Article
     • MEDLINE
     • CrossRef
174. Mizumoto H, Aoki K, Nakazawa K, Ijima H, Funatsu K, Kajiwara T. Hepatic differentiation of embryonic stem cells in HF/organoid culture. Transplant Proc. 2008;40:611–613
     • View In Article
     • Abstract
     • Full Text
     • Full-Text PDF (90 KB)
     • CrossRef
175. Cascio S, Zaret KS. Hepatocyte differentiation initiates during endodermal-mesenchymal interactions prior to liver formation. Development. 1991;113:217–225
     • View In Article
     • MEDLINE
176. Shiojiri N. Enzymo- and immunocytochemical analyses of the differentiation of liver cells in the prenatal mouse. J Embryol Exp Morphol. 1981;62:139–152
     • View In Article
     • MEDLINE
177. Kmiec Z. Cooperation of liver cells in health and disease. Adv Anat Embryol Cell Biol. 2001;161:1–151
     • View In Article
178. Rozga J. Liver support technology–an update. Xenotransplantation. 2006;13:380–389
     • View In Article
     • MEDLINE
     • CrossRef
179. Pearson H. Stem-cell tagging shows flaws. Nature. 2006;439:519
     • View In Article
     • CrossRef
180. Brulport M, Schormann W, Bauer A, Hermes M, Elsner C, Hammersen FJ, et al. Fate of extrahepatic human stem and precursor cells after transplantation into mouse livers. Hepatology. 2007;46:861–870
     • View In Article
     • CrossRef
181. Hengstler JG, Brulport M, Schormann W, Bauer A, Hermes M, Nussler AK, et al. Generation of human hepatocytes by stem cell technology: definition of the hepatocyte. Expert Opin Drug Metab Toxicol. 2005;1:61–74
     • View In Article
     • MEDLINE
     • CrossRef
182. Zhou QJ, Xiang LX, Shao JZ, Hu RZ, Lu YL, Yao H, et al. In vitro differentiation of hepatic progenitor cells from mouse embryonic stem cells induced by sodium butyrate. J Cell Biochem. 2007;100:29–42
     • View In Article
     • MEDLINE
     • CrossRef
183. Sharma NS, Shikhanovich R, Schloss R, Yarmush ML. Sodium butyrate-treated embryonic stem cells yield hepatocyte-like cells expressing a glycolytic phenotype. Biotechnol Bioeng. 2006;94:1053–1063
     • View In Article
     • MEDLINE
     • CrossRef
184. Li L, Sharma N, Chippada U, Jiang X, Schloss R, Yarmush ML, et al. Functional modulation of ES-derived hepatocyte lineage cells via substrate compliance alteration. Ann Biomed Eng. 2008;36:865–876
     • View In Article
     • CrossRef
185. Aurich I, Mueller LP, Aurich H, Luetzkendorf J, Tisljar K, Dollinger MM, et al. Functional integration of human mesenchymal stem cell-derived hepatocytes into mouse livers. Gut. 2007;56:405–415
     • View In Article
     • MEDLINE
     • CrossRef
186. Nakajima M, Iwanari M, Yokoi T. Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells. Toxicol Lett. 2003;144:247–256
     • View In Article
     • MEDLINE
     • CrossRef
187. Hooven LA, Mahadevan B, Keshava C, Johns C, Pereira C, Desai D, et al. Effects of suberoylanilide hydroxamic acid and trichostatin A on induction of cytochrome P450 enzymes and benzo[a]pyrene DNA adduct formation in human cells. Bioorg Med Chem Lett. 2005;15:1283–1287
     • View In Article
     • MEDLINE
     • CrossRef
188. Jin B, Ryu DY. Regulation of CYP1A2 by histone deacetylase inhibitors in mouse hepatocytes. J Biochem Mol Toxicol. 2004;18:131–132
     • View In Article
     • MEDLINE
     • CrossRef
189. Shehin SE, Stephenson RO, Greenlee WF. Transcriptional regulation of the human CYP1B1 gene. Evidence for involvement of an aryl hydrocarbon receptor response element in constitutive expression. J Biol Chem. 2000;275:6770–6776
     • View In Article
     • MEDLINE
     • CrossRef
190. Rogiers V, Akrawi M, Vercruysse A, Phillips IR, Shephard EA. Effects of the anticonvulsant, valproate, on the expression of components of the cytochrome-P-450-mediated monooxygenase system and glutathione S-transferases. Eur J Biochem. 1995;231:337–343
     • View In Article
     • MEDLINE
     • CrossRef
191. Dannenberg LO, Chen HJ, Tian H, Edenberg HJ. Differential regulation of the alcohol dehydrogenase 1B (ADH1B) and ADH1C genes by DNA methylation and histone deacetylation. Alcohol Clin Exp Res. 2006;30:928–937
     • View In Article
     • MEDLINE
     • CrossRef
192. Klick DE, Shadley JD, Hines RN. Differential regulation of human hepatic flavin containing monooxygenase 3 (FMO3) by CCAAT/enhancer-binding protein beta (C/EBPbeta) liver inhibitory and liver activating proteins. Biochem Pharmacol. 2008;76:268–278
     • View In Article
     • CrossRef
193. Bakker J, Lin X, Nelson WG. Methyl-CpG binding domain protein 2 represses transcription from hypermethylated pi-class glutathione S-transferase gene promoters in hepatocellular carcinoma cells. J Biol Chem. 2002;277:22573–22580
     • View In Article
     • MEDLINE
     • CrossRef
194. Ebert MN, Klinder A, Peters WH, Schäferhenrich A, Sendt W, Scheele J, et al. Expression of glutathione S-transferases (GSTs) in human colon cells and inducibility of GSTM2 by butyrate. Carcinogenesis. 2003;24:1637–1644
     • View In Article
     • MEDLINE
     • CrossRef
195. Pool-Zobel BL, Selvaraju V, Sauer J, Kautenburger T, Kiefer J, Richter KK, et al. Butyrate may enhance toxicological defence in primary, adenoma and tumor human colon cells by favourably modulating expression of glutathione S-transferases genes, an approach in nutrigenomics. Carcinogenesis. 2005;26:1064–1076
     • View In Article
     • MEDLINE
     • CrossRef
196. Schroeder TM, Nair AK, Staggs R, Lamblin AF, Westendorf JJ. Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors. BMC Genomics. 2007;8:362
     • View In Article
     • CrossRef
197. Valentini A, Biancolella M, Amati F, Gravina P, Miano R, Chillemi G, et al. Valproic acid induces neuroendocrine differentiation and UGT2B7 up-regulation in human prostate carcinoma cell line. Drug Metab Dispos. 2007;35:968–972
     • View In Article
     • MEDLINE
     • CrossRef
198. Lee YC, Higashi Y, Luu C, Shimizu C, Strott CA. Sp1 elements in SULT2B1b promoter and 5′-untranslated region of mRNA: Sp1/Sp2 induction and augmentation by histone deacetylase inhibition. FEBS Lett. 2005;579:3639–3645
     • View In Article
     • Abstract
     • Full Text
     • Full-Text PDF (313 KB)
     • CrossRef
199. Yamashita Y, Shimada M, Harimoto N, Tanaka S, Shirabe K, Ijima H, et al. cDNA microarray analysis in hepatocyte differentiation in Huh7 cells. Cell Transplant. 2004;13:793–799
     • View In Article
     • MEDLINE
     • CrossRef
200. Khan Z, Akhtar M, Asklund T, Juliusson B, Almqvist PM, Ekström TJ. HDAC inhibition amplifies gap junction communication in neural progenitors: potential for cell-mediated enzyme prodrug therapy. Exp Cell Res. 2007;313:2958–2967
     • View In Article
     • CrossRef
201. Hattori Y, Fukushima M, Maitani Y. Non-viral delivery of the connexin 43 gene with histone deacetylase inhibitor to human nasopharyngeal tumor cells enhances gene expression and inhibits in vivo tumor growth. Int J Oncol. 2007;30:1427–1439
     • View In Article
     • MEDLINE
202. Hernandez M, Shao Q, Yang XJ, Luh SP, Kandouz M, Batist G, et al. A histone deacetylation-dependent mechanism for transcriptional repression of the gap junction gene cx43 in prostate cancer cells. Prostate. 2006;66:1151–1161
     • View In Article
     • MEDLINE
     • CrossRef
203. Ogawa T, Hayashi T, Tokunou M, Nakachi K, Trosko JE, Chang CC, et al. Suberoylanilide hydroxamic acid enhances gap junctional intercellular communication via acetylation of histone containing connexin 43 gene locus. Cancer Res. 2005;65:9771–9778
     • View In Article
     • MEDLINE
     • CrossRef
204. Ammerpohl O, Thormeyer D, Khan Z, Appelskog IB, Gojkovic Z, Almqvist PM, et al. HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells. Biochem Biophys Res Commun. 2004;324:8–14
     • View In Article
     • MEDLINE
     • CrossRef
205. Robe PA, Jolois O, N’Guyen M, Princen F, Malgrange B, Merville MP, et al. Modulation of the HSV-TK/ganciclovir bystander effect by n-butyrate in glioblastoma: correlation with gap-junction intercellular communication. Int J Oncol. 2004;25:187–192
     • View In Article
     • MEDLINE
206. Desilets A, Gheorghiu I, Yu SJ, Seidman EG, Asselin C. Inhibition by deacetylase inhibitors of IL-1-dependent induction of haptoglobin involves CCAAT/Enhancer-binding protein isoforms in intestinal epithelial cells. Biochem Biophys Res Commun. 2000;276:673–679
     • View In Article
     • MEDLINE
     • CrossRef
207. Xu J, Hershman JM. Histone deacetylase inhibitor depsipeptide represses nicotinamide N-methyltransferase and hepatocyte nuclear factor-1beta gene expression in human papillary thyroid cancer cells. Thyroid. 2006;16:151–160
     • View In Article
     • MEDLINE
     • CrossRef
208. Jin B, Park DW, Nam KW, Oh GT, Lee YS, Ryu DY. CpG methylation of the mouse CYP1A2 promoter. Toxicol Lett. 2004;152:11–18
     • View In Article
     • MEDLINE
     • CrossRef
209. Hirai A, Yano T, Nishikawa K, Suzuki K, Asano R, Satoh H, et al. Down-regulation of connexin 32 gene expression through DNA methylation in a human renal cell carcinoma cell. Am J Nephrol. 2003;23:172–177
     • View In Article
     • MEDLINE
     • CrossRef
210. Hagiwara H, Sato H, Ohde Y, Takano Y, Seki T, Ariga T, et al. 5-Aza-2′-deoxycytidine suppresses human renal carcinoma cell growth in a xenograft model via up-regulation of the connexin 32 gene. Br J Pharmacol. 2008;153:1373–1381
     • View In Article
     • CrossRef
211. Tan LW, Bianco T, Dobrovic A. Variable promoter region CpG island methylation of the putative tumor suppressor gene Connexin 26 in breast cancer. Carcinogenesis. 2002;23:231–236
     • View In Article
     • MEDLINE
     • CrossRef
212. Singal R, Tu ZJ, Vanwert JM, Ginder GD, Kiang DT. Modulation of the connexin26 tumor suppressor gene expression through methylation in human mammary epithelial cell lines. Anticancer Res. 2000;20:59–64
     • View In Article
     • MEDLINE
213. Chen Y, Hühn D, Knösel T, Pacyna-Gengelbach M, Deutschmann N, Peterson I. Downregulation of connexin 26 in human lung cancer is related to promoter methylation. Int J Cancer. 2005;113:14–21
     • View In Article
     • MEDLINE
     • CrossRef
214. Loncarek J, Yamasaki H, Levillain P, Milinkevitch S, Mesnil M. The expression of the tumor suppressor gene connexin 26 is not mediated by methylation in human esophageial cancer cells. Mol Carcinog. 2003;36:74–81
     • View In Article
     • MEDLINE
     • CrossRef
215. King TJ, Fukushima LH, Donlon TA, Hieber AD, Shimabukuro KA, Bertram JS. Correlation between growth control, neoplastic potential and endogenous connexin43 expression in HeLa cell lines: implications for tumor progression. Carcinogenesis. 2000;21:311–315
     • View In Article
     • MEDLINE
     • CrossRef
216. Yi Zc, Wang H, Zhang GY, Xia B. Downregulation of connexin 43 in nasopharyngeal carcinoma cells is related to promoter methylation. Oral Oncol. 2007;43:898–904
     • View In Article
     • Abstract
     • Full Text
     • Full-Text PDF (315 KB)
     • CrossRef
217. Jin B, Seong JK, Ryu DY. Tissue-specific and de novo promoter methylation of the mouse glucose transporter 2. Biol Pharm Bull. 2005;28:2054–2057
     • View In Article
     • MEDLINE
     • CrossRef
218. Cho HH, Park HT, Kim YJ, Bae YC, Suh KT, Jung JS. Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors. J Cell Biochem. 2005;96:533–542
     • View In Article
     • MEDLINE
     • CrossRef
219. Rössig L, Urbich C, Brühl T, Dernbach E, Heeschen C, Chavakis E, et al. Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells. J Exp Med. 2005;201:1825–1835
     • View In Article
     • MEDLINE
     • CrossRef
220. Lee JH, Hart SR, Skalnik DG. Histone deacetylase activity is required for embryonic stem cell differentiation. Genesis. 2004;38:32–38
     • View In Article
     • MEDLINE
     • CrossRef
221. Lagace DC, Nachtigal MW. Inhibition of histone deacetylase activity by valproic acid blocks adipogenesis. J Biol Chem. 2004;279:18851–18860
     • View In Article
     • MEDLINE
     • CrossRef
222. Terranova R, Sauer S, Merkenschlager M, Fisher AG. The reorganisation of constitutive heterochromatin in differentiating muscle requires HDAC activity. Exp Cell Res. 2005;310:344–356
     • View In Article
     • MEDLINE
     • CrossRef
223. Marin-Husstege M, Muggironi M, Liu A, Casaccia-Bonnefil P. Histone deacetylase activity is necessary for oligodendrocyte lineage progression. J Neurosci. 2002;22:10333–10345
     • View In Article