Selected Small Molecules as Inducers of Pluripotency

The general idea of regenerative medicine is to fix or replace tissues or organs with live and patient-specific implants. Pluripotent stem cells are capable of indefinite self-renewal and differentiation into all cell types of the body. An easily accessible source of induced pluripotent stem cells (iPSCs) may allow obtaining and culturing tissues in vitro. Many approaches in the methods leading to obtain iPSCs have been tested in order to limit immu-nogenicity and tumorigenesis, and to increase efficiency. One of the approaches causing pluripotency is usage of small molecule compounds. It would be of great importance to assess their specific properties and reveal their new capacity to induce pluripotent stem cells and to improve reprogramming efficiency. Identification of the epigenetic changes during cellular reprogramming will extend our understanding of stem cell biology and many therapeutic applications. In this paper we discuss mainly the nucleotide derivatives, already proven or for now only putative inducers of the cells' pluripotency, that modulate the epigenetic status of the cell.


INTRODUCTION
Cells produced with a renowned method requiring overexpression of four transcription factors: Oct4, Sox2, Klf4, and c-Myc (the so-called Yamanaka factors) have been named induced pluripotent stem cells (iPSCs) (Takahashi & Yamanaka, 2006;Takahashi et al., 2007).They are very similar to embryonic stem cells (ESCs) with respect to morphology, phenotype, transcription and epigenetics (Takahashi & Yamanaka, 2006;Takahashi et al., 2007).Both types of these cells share a similar potency, differentiability and cell division rate.Furthermore, they are able to aggregate into embryoid bodies (Takahashi & Yamanaka, 2006).
There are a number of assays verifying pluripotency of stem cells.The most popular in vivo teratoma assay (Gertow et al., 2007;Wesselschmidt, 2011) is based on injecting potentially pluripotent stem cells into various sites of an immunocompromised mouse body followed by the growth of a tumor.When the injected cells are pluripotent, the tumor demonstrates characteristics of a teratoma, namely the development of differentiated cells originated at all three germ layers (ectoderm, mesoderm, and endoderm) (Brivanlou et al., 2003).The weakness of this assay is lack of standardization, and the time, cost and labor consumption.Moreover, the greatest disadvantage refers to the usage of experimental animals (Hentze et al., 2009;Wesselschmidt, 2011).One of alternative tests is analysis of expression of the pluripotency-associ-ated markers (Fong et al., 2008;Mitsui et al., 2003;Pesce & Scholer, 2001) and exploitation of epigenetic modifications responsible for pluripotency.Another method is based on in vitro embryoid bodies' models of spontaneous and directed differentiation, e. g. cardiac bodies from isolated cardiac cells can be generated that give rise to cardiomyocytes, endothelial cells and smooth muscle cells (Höbaus et al., 2013;Taubenschmid & Weitzer, 2012).To fill the gap between the in vivo and in vitro systems, the in silico models supported by genome wide data sets are used to help identifying characteristic features of pluripotent stem cells in functional genomics (Müller et al., 2008(Müller et al., , 2011;;Williams et al., 2011).An alternative in vivo system uses chicken eggs in which stem cells are transplanted onto chorioallantoic membrane of the chicken embryo and then a tumor similar to teratoma may arise (Durupt et al., 2012;Hagedorn et al., 2005).Another option to study pluripotency is an in situ analysis.In this organotypic model, stem cells are injected into a tissue, such as skin, and then either their development and differentiation or repopulation of cells leaving behind an extracellular matrix by stem cells are observed (Ott & Taylor, 2006;Elliott et al., 2012).
Miscellaneous and numerous methods have evolved to reprogram somatic cells.A lot of improvements in these methods have been made in order to solve problems associated with a derived iPSC line, and thus to limit immunogenicity and tumorigenesis, and increase efficiency (Zhao et al., 2011;Ma et al., 2013).One of the concerns is that the stresses of reprogramming might lead to deleterious DNA mutations in the iPSC lines (Bhutani et al., 2016).Recent studies have demonstrated that reprogramming-based mutations are generally benign and it is improbable to introduce mutational variants that would make cells inadequate for therapy (Bhutani et al., 2016).The acquisition of a stable pluripotent state appears to be difficult to control (Pennarossa et al., 2013).iPSCs and cancer cells share many similarities, like high proliferation rate, immortal cell growth, similarities in gene expression signature, in epigenetic status and chromosomal instability (Bernhardt et al., 2012).
All methods leading to pluripotency induction can be divided into virus-mediated and virus-free (Table 1).
Lentiviruses and retroviruses are vectors that can integrate randomly into the genome of cells and might disrupt active genes or regulatory regions.Such genomic insertions can activate endogenous oncogenes via knock-out of some genes, e. g. oncogene repressor, and lead to cancerogenesis (Baum et al., 2004;Okita et al., 2008).These vectors have been used to create iPSCs from adult human cells -in a retroviral system, the cells were transduced with Oct4, Sox2, Klf4 and c-Myc factors (Takahashi et al., 2007) and in a lentiviral -with Oct3/4, Sox2, Nanog, Lin28 (Yu et al., 2007).To deal with incorporation of viral vector sequences into the iPSC genome, alternative reprogramming systems using non-integrating adenoviruses have been developed, however a significant weakness of these systems is very low efficiency (Stadtfeld et al., 2008;Zhou & Freed, 2009).A better efficiency of pluripotent stem cells induction might be obtained via a system using episomal plasmids delivered by non-integrating Sendai viruses (SeV), where the RNA virus can be easily removed with antibodies, though the cost of this method is much higher than of the other viral methods (Fusaki et al., 2009;Sachamitr et al., 2014).
There are two systems facilitating the removal of genes integrated with the mouse genome or human iP-SCs -the Cre-loxP and PiggyBac transposon systems (Zhou & Zeng, 2013).The first consists of a single viral vector equipped with a cassette of four transcription factors which are flanked by the loxP sites.The Cre-recombinase is delivered to the cell's nucleus by using the Pseudomonas aeruginosa bacteria and then overexpressed.Cre-mediated recombination leads to excision of the DNA sequences between the two loxP repeats (Kaji et al., 2009;Soldner et al., 2009).Another system is based on a transient transposase activity.The reprogramming factors are cloned into a PiggyBac transposon.In the presence of a transiently expressed transposase, this vector can be integrated into the host genome and excised from iPSCs after reprogramming (Kaji et al., 2009;Woltjen et al., 2009, Yusa et al., 2009).
In the non-viral methods of reprogramming, DNA plasmids do not integrate into a genome but are maintained in a cell for a few cell cycles and transiently express reprogramming factors (Okita et al., 2008;Stadtfeld et al., 2008;Yu et al., 2009).An episomal vector system, in turn, is based on the Epstein-Barr Nuclear Antigen-1 that undergoes a permanent extrachromosomal replication in synchrony with the host genome, i.e.only once per cell cycle (Yu et al., 2009;Okita et al., 2011).Another system based on an episomal DNA vector, the minicircles, contains only cDNA of the expressed Yamanaka factors and a eukaryotic promoter (Jia et al., 2010).A self-assembly of complexes that consist of cationic lipids and plasmids or siRNA, with magnetic nanoparticles of iron, has been termed liposomal magnetofection (Mykhaylyk et al., 2010;Park et al., 2012).Such complexes require a magnetic field to transfect vectors into the cells.A different method of reprogramming uses synthetic mRNA encoding the Yamanaka factors, delivered into somatic cells via a cationic lipid vehicle.The mRNA is synthesized using in vitro transcription reactions, treated with modified ribonucleotides and a phosphatase, and the medium is supplemented with an interferon inhibitor which allows for lower cytotoxicity, acquiring high protein expression and improving cell viability (Yu et al., 2007;Hanna et al., 2009).
miRNA play a significant role in reprogramming through epigenetic regulation of chromatin remodeling complexes.Some miRNA clusters participate in control of genes related to maintenance of pluripotency (Subramanyam et al., 2011).It has been demonstrated that miR93, as well as miRNA from the miR302 family, in combination with the Yamanaka factors, can enhance the efficiency of reprogramming (Li et al., 2011;Subramanyam et al., 2011).Furthermore, mir-200, mir-302 and mir-369 could induce pluripotency in human cells (Miyoshi et al., 2011).A cocktail of miR 302-367 very quickly and efficiently reprograms the mouse and human somatic cells to the pluripotent state without additional reprogramming factors (Anokye-Danso et al., 2011;Liao et al., 2011).A genetic modification might be omitted by using methods that do not employ nucleic acids.Delivery of a recombined protein encoded by reprogramming factors into the cells, instead of these factors themselves, is one among those methods (Kim et al., 2009).
Another nonviral method that allows avoiding genomic insertions and immunogenicity relies on utilization of small molecule compounds, including RNA-derivatives (Fig. 1).They may improve the quality of reprogramming, such as time and efficiency (Efe and Ding, 2011).It is worth to note that efficiency of reprogramming via such compounds highly depends on the specific cell type (Paull et al., 2015).Because of low mass, which is limited up to 500 Da, they might diffuse freely across the cell membranes (Lipinski, 2004;Dougherty et al., 2012).Given the easiness to synthesize, administer and standardize, as well as cost-effectiveness and simple storage requirements, small molecules are a promising approach to pluripotent cell induction (Hou et al., 2013).However, this method displays some weaknesses, like potential tumorigenicity, mutagenicity, as well as possible targeting of endogenous cell components that are not specific to pluripotency.

INDUCERS OF PLURIPOTENCY
The fundamental mechanism of epigenetics is accommodation of gene expression in response to interactions between the genes and the environment (Morange, 2002).This can be highly manipulated in somatic cells and the cell identity may be reversed to the initial state of development or altered.Most of the small molecules are epigenetic modulators and influence methylation of DNA and histone modifications in the cells (Jaenisch, 2012).Methylation patterns of pluripotency gene promoters should be similar to those found in the embryonic stem cells (Maherali & Hochedlinger, 2009).
There are groups of compounds that are either proven or for now only putative inducers of pluripotency.5-azacytidine and zebularine are cytidine analogues (Fig. 2) and act as DNA methyltransferase inhibitors.5-azacytidine contains a nitrogen atom at position 5, whereas zebularine lacks the amino group at position 4 of the corresponding cytidine.It has been demonstrated that both compounds form covalent bonds with DNMT after incorporation into DNA (Taylor & Jones, 1982;Zhou et al., 2002).5-azacytidine, named also 5-AZ or AZA, may Small molecules cause cellular reprogramming through epigenetic changes, such as DNA methylation, histone modifications, noncoding RNAs and chromatin remodeling.Me, methylation; Ac, acetylation; ncRNAs, noncoding RNAs incorporate into both, DNA and RNA.5-AZ is toxic and unstable under physiological conditions.When incorporated into nucleic acids via the sulfhydryl side chain of the catalytic cysteine residue, these compounds form a stable reaction intermediate.These nucleosides then become suicide substrates for the DNMT enzymes (Lyko & Brown, 2005).It is assumed that the vast majority of azacytidine is incorporated directly into the RNA and the rest (10-20%) is activated and converted by a ribonucleotide reductase into the active nucleotide for DNA methylation inhibition, 5-aza-2'-deoxycytidine-5'-triphosphate (Li et al., 1970;Stresemann & Lyko, 2008).5-azacytidine can substitute for a cytosine, and azacytosineguanine dinucleotides are formed which are recognized by the DNA methyltransferases as natural substrates (Stresemann & Lyko, 2008).As a result, a covalent bond between the carbon-6 of the cytosine and the enzyme is established (Santi et al., 1984;Chen et al., 1991).Substitution of carbon by the nitrogen atom at position 5 in azacytosine precludes the reaction of β-elimination through the carbon-5 atom, and thus DNMT remains covalently bound to DNA and its catalytic function is blocked.Furthermore, such covalent protein-DNA adduct triggers DNA damage signalling and trapped DNMTs are degraded, resulting in depletion of the cellular DNMTs and lost of methylation marks during DNA replication (Stresemann & Lyko, 2008).
5-azacytidine improves reprogramming efficiency by 3 folds (with an effective concentration of about 2 µM in mouse embryonic fibroblasts; MEFs) (Huangfu et al., 2008a;Mikkelsen et al., 2008).There are cases when some cells become trapped in partially reprogrammed states and show DNA hypermethylation at pluripotencyrelated loci.In such cases, 5-AZ enables to complete the iPSCs reprogramming (Huangfu et al., 2008a;Mikkelsen et al., 2008).Five µM concentration of 5-AZ boosts and may increase efficiency of reprogramming during late stages of this process in a doxycycline-inducible Oct4 expression screening system, in the presence of a cocktail that consist of valproic acid, CHIR99021, RepSox and tranylcypromine (Polo et al., 2012;Hou et al., 2013).
Besides its effects on reprogramming, 5-azacytidine has been also proved to participate in transdifferentiation events from one cell type to another.It participates in conversion of murine fibroblasts into adipocytes and bone cells, of mesenchymal stromal cells and fibroblasts into haematopoietic cells, of adult skin fibroblasts and granulose cells into highly permissive state and towards different cell lineages and phenotypes, of fibroblasts into insulin-secreting cells, of human granulosa cells into muscle cells with human recombinant vascular endothelial growth factor, and in transformation of adipose-derived stem cells into myoblasts (Taylor & Jones, 1979;Tamada et al., 2006;Pennarossa et al., 2013;Brevini et al., 2014;Wang et al., 2014).
Zebularine is a stable hydrophilic cytidine analogue with the depleted 4-amino group, and acts as a DNMT inhibitor and was formerly developed as a cytidine deaminase inhibitor (Zhou et al., 2002;Nakamura et al., 2013).It forms tight covalent complexes between the DNMT enzymes and DNA substituted with zebularine, which could lead to a compositional change in the DNMT protein, and thus it is conceivable that DNMTs can be then degraded via the ubiquitination system (Hurd et al., 1999;You & Park, 2012).Zebularine has been shown to exhibit low toxicity in mice (Cheng et al., 2003;Yoo et al., 2004, Cheng et al., 2004).This compound preferentially targets cancer cells (Andersen et al., 2010).It has been demonstrated that zebularine decreased the levels of DNMT1, DNMT3a, DNMT3b in cholangiocarcinoma, hepatocellular carcinoma cells bladder, cervical, and breast cancer cells (Cheng et al., 2004;Fandy, 2009;You & Park, 2012;Nakamura et al., 2013;Nakamura et al., 2015).
Zebularine is a proven inducer of pluripotency.It has been demonstrated to participate in reprogramming of the yak fibroblasts for cloning (Xiong et al., 2013).
Histone methyltransferases (HMTs) transfer methyl groups from the S-Adenosyl methionine (SAM) specifically onto either lysine or arginine residues of the H3 and H4 histones.There are two suggested mechanisms of the SAH hydrolase inhibition -either via oxidation of NAD + to NADH (type I -reversible), or via cova-  lent binding to the active site by an inhibitor with a nucleophilic residue (type II -irreversible) (Wolfe & Borchardt, 1991).
Naturally occurring neplanocin A, an analogue of adenosine with the oxygen atom substituted by carbon-5, and its derivative DZNep are effective inhibitors of the S-adenosylhomocysteine (SAH) hydrolase (Tam et al., 2015).However, both of these compounds are toxic, which is a result of phosphorylation of the C-5' primary hydroxyl group (Wolfe & Borchardt, 1991).Neplanocin A has been demonstrated to be metabolized via conversion into a 5′-triphosphate (Montgomery et al.;1982, Saunders et al., 1985)).
A neplanocin A analogue that lacks nitrogen at position 3, 3-deazaneplocin A (DZNep), acts as a SAH hydrolase inhibitor.It can productively deplete cellular levels of the EZH2 complex, effectively and selectively inhibit trimethylation of lysine 27 of histone H3 (H3K27me3) and lysine 20 of histone H4 (H4K20me3), and induce apoptosis in cancer cells (Chiang, 1998;Gordon et al., 2003;Tan et al., 2007).This compound has been shown to exhibit a minimal toxicity in vivo (Bray et al. 2000).
Through structure and activity relationship (SAR) analysis, as well as correlation of physicochemical properties, it has been identified D9, a neplanocin A analogue that lacks hydroxymethyl group at position 4'.As an analogue of DZNep, it shows a comparable cellular activity with DZNep, about 20 fold less toxicity in mice and could potentially affect reprogramming (Jiang et al., 2015;Tam et al., 2015).D9 has been reported to induce suppression of histone methylation marks, such as H3K27me3 and H4K20me3, and to a lesser extent on H3K4me3 and H3K79me2, and had only little effects on H3K9me2 and H3K9me3 (Jiang et al., 2015).
3-Deazaadenosine (DZA) is an adenosine analogue lacking the nitrogen atom at position 3 and also acts as a SAH hydrolase inhibitor and leads to a rapid loss of H3K4 trimethylation in the ESC, followed by ESCs differentiation and death (Shyh-Chang et al., 2013).DZA, in the presence of valproic acid, CHIR99021, RepSox, tranylcypromine and forskolin participates in the reprogramming induction (Hou et al., 2013).
Inhibition of the catalytic activity of the H3K79 histone methyltranferase (Dotl, disruptor of telomeric silencing-like) is key to reprogramming.Mono-, di-, and trimethylation of H3K79 are all entirely catalyzed by Dot1l (Nguyen & Zhang, 2011).EPZ004777 is a 7-dezaze with added urea and phenyl fragments.This small molecule inhibits Dot1l which is followed by a decrease in the H3K79me2 levels, at concentrations ranging from 1 μM to 10 μM (Onder et al., 2012), and affects the iPSC reprogramming (Lin et al., 2009).By using EPZ004777 in mouse and human fibroblasts, the yields of four transcription factors-mediated induction of pluripotency increased by 3-4 folds (Onder et al., 2012).The iPSCs generated through the Dot1l inhibition show all the hallmarks of pluripotency.They have exhibited characteristic ESC morphology, have differentiated into all three germ layers in vitro, as well as in teratomas.The Dot1l inhibition substitutes for Klf4 and c-Myc (Onder et al., 2012).

PERSPECTIVES
The potential of nucleic acid derivatives to develop medical treatment of degenerative diseases and advance the field of regenerative medicine should profoundly increase in the near future.Such compounds may target specific signaling pathways and mechanisms and trigger pluripotent stem cells induction, thus they are effective tools for cell manipulation and development of therapeutic approaches for regenerative medicine (Ma et al., 2013, Chin et al., 2009;Nie et al., 2012;Hou et al., 2013;Jung et al., 2014).Usage of small molecules may lead to development of cell-based therapies and modelling of diseases via the production of patient-specific stem cells (Tang et al., 2016).Because reprogramming efficiency in vitro depends on the specific donor cell type and culture conditions, an appropriate usage of their combinations under proper conditions is needed.
Revealing and studying the influence of new nucleic acid derivative compounds on reprogramming is riveting and might lead to understanding the mechanisms underlying their activity.
Recently, there was a big progress in the small molecules application, however, many limitations that do not allow the use of such compounds in clinical settings in a large scale still remain.Modifications of particular structural sites or substitutes in derivatives of nucleic acids or other natural compounds influence the modulating activities of these small molecules, especially their inhibiting activity.Further pharmacological studies will provide data allowing identifying the optimal pluripotency induction conditions.Molecular mechanisms underlying the activity of small molecule compounds need to be fully elucidated.Insight into the epigenetic changes during pluripotent stem cell induction and further chemical and pharmacological studies would improve understanding of the stem cell biology and the major mechanisms and pathways involved in the cell reprogramming, as well as support the development of potential therapeutic approaches (Bojarski, 2006;Frye, 2010).

Figure 2 .
Figure 2. Chemical structures of cytidine and its analogues.

Figure 3 .
Figure 3.Chemical structures of adenosine and its analogues and derivative.

Table 1 . Summary of the iPSCs strategies Table
includes established methods for iPSCs derivation.They involve viral and nonviral approaches with their advantages and disadvantages.