Keywords

1 Introduction

Stem cells, which are characterized as immature, self-renewal, and undifferentiated cells that can give rise to many different cell lineages, are expected to open new needed therapeutic avenues [1]. Induced pluripotent stem (iPS) cells are cells reprogrammed from somatic cells via genetic modification to obtain embryonic stem (ES) cell characteristics [2, 3]. Although multipotent adult stem cells such as mesenchymal stem cells (MSCs) have been well investigated for clinical application [4], basic research on pluripotent stem cells, such as ES cells and iPS cells, may lead to further understanding of in vitro tissue/organ development, which in turn could be applied to next-generation therapeutic approaches for whole-tissue/organ regeneration.

Gene delivery to pluripotent stem cells provides a powerful experimental system to investigate the early stages of tissue/organ development. In addition, genetic modification of patient-specific iPS cells, particularly disease-model iPS cells, could facilitate the study of pathological mechanisms and provide new therapeutic approaches in personalized medicine [5]. To obtain efficient and stable transgene expression, various gene delivery methods ranging from viral vectors to plasmid-based transient gene expression have been applied to pluripotent stem cells [6]. In these methods, many types of constitutive promoters have been utilized in the expression vectors, such as the cytomegalovirus (CMV), human elongation factor 1α (EF1α), CMV enhancer/β-actin promoter with β-actin intron (CA), Rous sarcoma virus (RSV) [7], human β-actin (ACTB), phosphoglycerate kinase (PGK) [8], and simian virus 40 early (SV40) [9] promoters. The transcriptional activity of transgenes considerably varies among these promoters depending on the cell type [10]. Notably, the CMV promoter, which is one of the most popular choices for gene delivery vectors because of its strong activity in most cell lines, shows considerably weak transgene activation in stem cells [7, 8, 11–14]; therefore, it is important to select an optimal promoter to obtain the expected transgene expression in iPS cell experiments.

Beyond the constitutive promoter systems described above, regulated control of gene expression has great significance for stem cell research because of the ability to avoid undesirable effects of constitutive transgene expression after cellular differentiation [15]. Several studies have employed tetracycline (tet)-regulated systems to control transgene expression in both ES cells and iPS cells, thereby enabling the transgene function to be explored in a spatiotemporal manner [16–18]. Each gene delivery and expression system has particular advantages and disadvantages depending on the desired outcome of the experimental design. Here, we briefly review representative gene delivery and expression systems from the perspective of their application to iPS cell research.

2 Viral-Based Gene Delivery Systems

Gene delivery systems are classified into two major classes: virus-based vectors and nonviral vectors (Table 11.1). Viruses are suitable for efficient gene delivery experiments because of their ability to penetrate into the cell nucleus and replicate [19]. Viral vectors have been widely used to deliver foreign genes into the cell nucleus because of their high transduction efficiency and capacity for long-term transgene expression [20]. Ideally, the vector should be nontoxic, minimally immunogenic, and capable of highly efficient penetration and delivery to numerous cell types [21]. The principal viral vectors currently in use include adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus [22].

Table 11.1 Comparison of commonly used viral vectors and piggyBac transposon system
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2.1 Adenovirus Vectors

Adenovirus, a 70–100-nm, non-enveloped, double-stranded DNA virus [23], belongs to the family Adenoviridae and is well known to cause respiratory tract infections. Adenoviruses enter mammalian cells via attachment to the Coxsackievirus and adenovirus receptor (CAR) [24]. Adenoviruses rarely integrate with the host genome because their genome is maintained episomally in the cell nucleus. Because of their ability to transduce many cell types, including both dividing and nondividing cells, without genomic integration, adenoviral vectors have been considered as promising delivery systems for gene transduction experiments. However, the transient nature of their transgene expression limits their utility in in vitro research designs, and the toxicity and associated immune responses may hamper their clinical application.

Adenovirus vectors can easily introduce exogenous genes into mouse ES cells [7] and iPS cells [13], and they are also used as effective gene delivery tools for human ES cells and iPS cells [25]. The transient expression mediated by adenovirus vectors is actually an advantage for stem cell research, in that undesirable effects of constitutive transgene expression after cell differentiation can be avoided. Indeed, adenovirally mediated transient expression of Runx2 or PPARγ was shown to efficiently guide mouse iPS cells to differentiate into osteoblasts or adipocytes, respectively [13].

It should be noted that selection of an appropriate constitutive promoter, such as EF1α and CA promoters, is important for effective adenoviral transgene expression in pluripotent stem cells [26]. When used in adenovirus vectors, the CMV and RSV promoters show weak activity in mouse pluripotent stem cells [7, 13] because they are silenced by DNA methylation [27, 28]; therefore these promoters may not be the best choice for adenoviral transduction experiments in iPS cell research.

2.2 AAV Vectors

AAV is a nonpathogenic, nonautonomous single-stranded DNA parvovirus that requires a helper virus such as adenovirus or herpes virus for replication. AAV has many serotypes, and among them, AAV2 is well studied and widely used as a gene delivery vector. Without the helper virus, the AAV genome remains episomal in target cells [29, 30]. Genome integration is observed at a low frequency and at a specific site on chromosome 19 [31]. AAV vectors derived from AAV lack viral coding sequences and rarely cause toxic and immune reactions, and they are thus considered as a promising gene delivery system for clinical use. However, the limited packaging capacity of AAV (approximately ~5 kb) is a major limitation of this vector system [32].

One interesting property of AAV is that the inverted terminal repeats (ITRs) of the AAV genome permit AAV vectors to efficiently introduce gene-targeting constructs into homologous chromosomal loci in a cellular genome [33]. This unique property permits gene editing, and efficient gene targeting by AAV vectors has been achieved in human ES cells and iPS cells [34–36]. Damdindorj et al. [37] reported that when used in an AAV vector, the CMV promoter provided stable and robust gene expression in cancer cell lines; however, this promoter does not seem to be preferable for noncancerous cell lines and for the purpose of AAV-based gene targeting. Further studies are needed to identify the most suitable constitutive promoter for the application of AAV vectors to precise genetic manipulation of iPS cells, which could have great scientific and therapeutic potential.

2.3 Retrovirus Vectors

Retroviral vectors are among the most commonly used gene delivery systems for target gene transduction. They possess several advantages compared with other viral vectors, such as high-level transgene expression activity in long-term culture of most dividing somatic cells and their large DNA capacity (9–12 kb) [32]. Retroviral vectors reverse-transcribe their single-stranded RNA genome into DNA that is then integrated into target cell genome. According to their genome organization, retroviruses are broadly divided into two categories: simple onco-retroviruses, such as Moloney murine leukemia virus (MLV), and complex retroviruses including lentiviruses, such as human immunodeficiency virus [23]. Onco-retrovirus-based vectors are not capable of gene transfer to nondividing cells because they rely on cell division for transduction.

Importantly, transgene expression by MLV-based vectors is restricted in undifferentiated pluripotent stem cells by de novo DNA methylation [38, 39] and other mechanisms, such as TRIM28-mediated silencing of the promoter element within the MLV long-terminal repeat (LTR) [40, 41]. A retroviral mutant vector, the murine ES-cell virus (MESV), was developed to facilitate target gene expression in ES cells through its ability to escape immediate silencing and initiate proviral expression [38, 42]; however, the MESV vector is still prone to inactivation during long periods of culture [38]. The self-silencing property of retroviral vectors is advantageous during reprogramming for iPS cell generation because it is necessary for the forced expression of exogenous reprogramming factors to cease once the cell reaches the ES-cell-like state. Indeed, Yamanaka and colleagues first generated iPS cells from mouse and human fibroblasts using MLA-based vectors [2, 3].

However, the self-silencing property of retroviral transduction in pluripotent stem cells would be disadvantageous for molecular studies that require sustained expression of exogenous gene products. In addition, retrovirus vectors have the potential to induce insertional mutagenesis in iPS cells through their random integration into the host genome [43]. These aspects should be carefully considered when designing iPS cell experiments using retroviral vector systems.

2.4 Lentivirus Vectors

In contrast to onco-retroviruses, lentiviruses are transported to the nucleus of the target cells by active transport but do not require cell division for transduction, which allows them to transduce quiescent and nondividing cells [44]. Lentivirus vectors have an ~8-kb packaging capacity that limits the introduction of many genomic DNA sequences, but most cDNA sequences can be accommodated. Given their advantages, lentiviral vectors have become the predominant vectors for gene transduction in many types of cells and in transgenic animals [45].

Similar to other retroviral vectors, lentiviral vectors possess the ability to integrate into the host genome and stably express the delivered target gene. Although insertional mutagenesis by lentiviral vectors is still their major disadvantage, it occurs less frequently for lentiviruses than for onco-retroviruses [46, 47]. To reduce the risk of insertional mutagenesis, non-integrating lentiviral (NIL) vectors, which carry either mutant integrase or mutations in the integrase binding sits, have been developed [48–50]. In this vector system, the lentiviral genome remains episomal in the nucleus, with sustained transgene expression that does not require genome integration [48, 49]; therefore, it is expected to provide a safe and promising gene delivery system for laboratory and clinical use [51].

One important characteristic of lentivirus vectors is their resistance to silencing during propagation and differentiation of ES cells [52]; as a result, they have become widely used for gene transduction in ES cells [53–56]. Hong et al. [12] demonstrated that when used in a lentiviral vector, the EF1α promoter drove robust transgene expression in mouse ES cells from undifferentiated status to fully differentiated status during neuronal differentiation, whereas the CMV promoter activated transgene expression only in late stages of differentiation. Norrman et al. [8] reported that the use of ACTB, EF1α, and PGK promoters in lentiviral vectors permitted stable transgene expression in human ES cells, whereas the CMV promoter was less effective and expression was rapidly downregulated within 7 days.

3 Transposon-Based Gene Delivery Systems

As a nonviral gene delivery method, electroporation has been widely used for exogenous gene expression in ES cells [57]; however, a major drawback of this method is low transfection efficiency. Transposon DNA vectors have been recently used for nonviral gene delivery [58]. The piggyBac transposon, a transposon DNA vector identified from the cabbage looper moth Trichoplusia ni [59], has been reported as a highly efficient tool to insert exogenous genes into mammalian cells [60–62]. The piggyBac transposon system is constituted by two basic elements: a 2,472-bp transposon with 13-bp inverted terminal repeats (ITRs) and a 594 amino acid transposase [59, 63].

The piggyBac transposon mediates gene transfer through a cut-and-paste mechanism (Fig. 11.1) [58], where it is inserted into the genomic DNA at TTAA tetranucleotide sites and then integrates with the chromosomal DNA through the activity of the transposase [63]. This system can efficiently deliver DNA fragments sized 9 kb, even up to 14 kb, without significant decreases in transposition efficiency [60]. Another advantage is that the piggyBac transposon can be excised from the original insertion site without leaving any remnant sequence [64]; thus, piggyBac-mediated genetic insertions are reversible. Given its capacity for efficient and reversible gene transfer, the piggyBac transposon system is a promising vector for gene delivery.

Fig. 11.1
figure 1

Mechanism of piggyBac transposition. (a) In the transfected cell, transposase is expressed from the piggyBac transposase expression vector, and it then recognizes and binds to the specific inverted terminal repeats (ITRs) of the transgene vector plasmid and cuts the DNA sequence of the transgene from the original sites. Then, the transgene DNA sequence integrates into the genomic DNA of the target cell. (b) For excision, re-expression of transposase by transfection of the piggyBac transposase expression vector leads to cutting of the transgene at the ITRs in the genomic DNA, which results in removal of the inserted transgene from the genomic DNA

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The piggyBac transposon system can be used to generate transgene-free iPS cells [65–68], which may thus have increased therapeutic utility. Additionally, the piggyBac transposon system has been used for efficient gene delivery to human ES cells, where the insertion can be removed from the ES genome without leaving any insertional mutation as described above [69, 70]. This system has also been used for gene delivery to human iPS cells [71–73]. In dental research, a piggyBac transposon-based gene expression system has been applied to human deciduous tooth dental pulp cell-derived iPS cells to express EGFP and tdTomato transgenes [74].

4 tet-Controlled Transcriptional Regulation System

Control of transgene expression is important for preventing potential adverse effects of the continued overexpression of the transgene. tet-regulated gene expression systems are among the most widely used gene regulation systems [75] and consist of two variants: the tet-off and the tet-on systems (Fig. 11.2) [76, 77]. In the tet-off system, when doxycycline, an analog of tet, is absent, the tet transactivators (tTAs) bind to their target element, a tet-operator sequence (tet response element, TRE) that is upstream of a promoter, to drive transgene expression. Conversely, in the presence of doxycycline, the tTAs cannot bind to the TRE; therefore, transgene expression is hindered. The tet-on system was derived from the tet-off system by inducing random mutations in the tTAs [77]. These mutant reverse transactivators (rtTAs) bind to the TRE in the presence of doxycycline to drive transgene expression, and transgene expression does not occur without doxycycline.

Fig. 11.2
figure 2

Tetracycline (tet)-controlled transcriptional regulation systems. (a) tet-off system: in the presence of doxycycline (Dox), tet transactivators (tTAs) cannot bind to the tet-operator (tetO) sequence to induce target gene (transgene) expression. In the absence of Dox, tTAs bind to the tetO sequence to drive target gene expression. (b) tet-on system: reverse tet transactivators (rtTAs) bind to the tetO in the presence of Dox to induce transgene expression. In contrast, in the absence of Dox, rtTAs cannot bind to the tetO; thus, transgene expression does not occur

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Controlled regulation of transgene expression has great significance for investigating molecular mechanisms of pluripotency and cellular differentiation in stem cells [16], and tet-controlled transcriptional activation systems have thus been applied to pluripotent stem cells [17, 18, 78]. Dox-inducible lentiviral and piggyBac vectors have also been used to direct reprogramming of somatic cells to iPS cells [66, 79]. This system also permits the regulation of transgene expression in iPS cells to drive their differentiation toward specific cell lineages such as myocytes [71–73].

5 Conclusions

Because each gene delivery system possesses its own characteristics, researchers should consider the suitability of the system, rather than technical convenience, for a particular iPS cell experiment. The choice of promoter is also important, especially for experiments in iPS cells. Although little systematic information is available regarding the activity of constitutive promoters in undifferentiated iPS cells, the EF1α and CA promoters, but not the CMV promoter, are expected to be suitable for high levels of stable transgene expression. The piggyBac transposon-based gene delivery system provides several benefits over classic viral and nonviral gene delivery systems. In addition, it can be combined with tet-controlled transcriptional regulation to achieve spatiotemporal control of transgene expression during iPS cell differentiation, which may provide a great impact on iPS cell research.