**1. Introduction**

The entrance of induced Pluripotent Stem Cells (iPSCs) in the stem cell scene represents a novel approach for studying human diseases and a promising tool for regenerative medicine [1].

The compelling need to overcome ethical and technical issues related to the production and utilization of Embryonic Stem Cells (ESCs) has prompted to search for a method to induce the pluripotency in terminally differentiated cells pushing them to an embryonic-like state.

Several studies, therefore, have been focused on characterizing and isolating unique transcriptional factors expressed by ESCs, presuming that their expression was sufficient to confer to adult cells the peculiar features of pluripotent cells [2]. The hypothesis that genome is not irreversibly modified during the differentiation and that some factors residing in ESCs can confer pluripotency to terminally differentiated nuclei has given a boost to bypass both the practical and ethical concerns

related to the use of ESCs and has paved the way for the development of cuttingedge approaches for tissue regeneration, like cellular reprogramming by artificially inducing the pluripotency [3].

Cell reprogramming consists in converting adult somatic cells in undifferentiated cells defined by an acquired pluripotency, typically showed by ESCs. Many techniques have been developed to achieve the goal since in 2006 Yamanaka and colleagues succeeded in the undertaking challenge of identifying four specific transcription factors (Oct4, SOX2, c-Myc, and KLF4) capable of reprogramming murine or human fibroblasts to embryonic-like cells, and termed them "induced pluripotent stem cells" [4]. The four factors recognized by Yamanaka are involved in multiple mechanisms and are pivotal for the pluripotency of embryonic stem cells, for embryonic development and to determine cell fate [5].

The great potential residing in iPSCs was soon noticeable, primarily for the possibility to obtain stem cell lineages customized for each patient, able to give rise to the needed cell type, then, for the chance to overcome organ shortage difficulties and to avoid invasive medical procedures to treat degenerative diseases [6].

Additionally, iPSCs share several features with ESCs showing similarities for morphology and culturing conditions: they both grow arranging in dome-shaped colonies (**Figure 1**) and need to be cultured in presence of a layer of feeder cells and/or specific cytokines [7]. Furthermore, iPSCs express equal stemness markers showed by ESCs, a common proliferation potential, the capability to self-renew and differentiate into the three fundamental germ layers [8].

It is considerably relevant that iPSCs can also provide effective disease models to investigate cellular and molecular mechanisms involved in the development of pathologies and a platform for toxicological and pharmacological screening [9].

Until the sprawl of cell reprogramming, ESCs were considered the most promising and innovative tool for the research and clinical application in the field of regenerative medicine. Due to their ability to grow indefinitely and to differentiate into cells of the three germ layers while maintaining the pluripotency, ESCs rapidly gained the attention of the scientific community [10]. Despite the tremendous potential they hold for tissue and organ regeneration, at now, the clinical application of ESCs is limited and still faces many obstacles. The use of ESCs, in fact, raises several controversies and the studies focused on the understanding of their biology are strictly regulated or even forbidden in many countries. The reason of such restraint primarily resides in their origin and isolation techniques, as ESCs derive from the inner cell mass of mammalian blastula, the early stage of embryonic development, and common methods for their isolation require the destruction of the embryo, triggering ethical concerns [11].

#### **Figure 1.**

*Skin fibroblast reprogrammed by mRNAs codifying for Oct4, SOX2, c-Myc, KLF4, Nanog, LIN28. Representative images of iPSCs arranging after 24 hours to form a colony (A) that appeared clearly visible ten days later (B). Scale bar is 250* μ*m.*

*Non-integrating Methods to Produce Induced Pluripotent Stem Cells for Regenerative Medicine… DOI: http://dx.doi.org/10.5772/intechopen.95070*

Beside the ethical issue, several other hurdles limit the concrete employment of ESCs, such as the risk of rejection related to their immunogenicity, the challenging conditions to culture and expand ESC lines and then to maintain the undifferentiated state ensuring their stability [12–14]. Additionally, there is a risk for tumor formation if ESCs are not fully addressed into a specific differentiated cell type prior to implantation [15]. Finally, about the therapeutic application of ESCs in human studies, another major concern is the use of non-human and xenogenic materials such as fetal bovine serum for cell culture [16].

However, the pluripotency makes ESCs unique, and this astonishing differentiation capability renders them very attractive to research studies, to the extent that a big effort has been put recently into the search for methods to artificially reproduce their pluripotency (**Figure 2**).

Nonetheless, the efficiency of cell reprogramming remains low, hence, the reprogramming techniques are under intense investigation so as to generate induced pluripotent stem cells ameliorating the efficiency of the process, the quality and safety of the derived cells [17]. The improvement generally targets several aspects of the reprogramming methods: primarily the source of somatic cells, as many studies suggest that some cells are more prone than others to be reprogrammed into certain cell types; [18] reprogramming factor cocktail; [19] the conditions to culture and maintain the iPSCs and, above all, the technique to introduce the reprogramming factors [20].

#### **Figure 2.**

*Human iPSC technology allows, through the introduction of reprogramming factors into adult somatic cells, to obtain pluripotent cells capable of differentiating towards several mature cells which can be used for providing cells for regenerative medicine, for in vitro or in vivo disease modeling, and for screening and developing new drugs. (The figure was prepared with the support of Servier Smart Medical Art, https://smart.servier.com/).*

A major issue related to the production of iPSCs, in fact, is the use of retroviruses to obtain a permanent integration of the reprogramming factors in the host cell genome, leading to teratoma formation due to the residual expression of oncogenes like c-Myc and KLF4 [21].

Different methods are known, at the present, to induce the expression of the reprogramming factors, classified in two major categories: non-viral and viral vectorbased methods [22]. Viral-based methods include integrating viruses like Retrovirus and Lentivirus and non-integrating viruses, such as Adenovirus, Sendai virus [23].

According to several studies, all these methods provide good results in terms of effectiveness of cell reprogramming, hence, the choice of the suitable method strictly depends on the cell type used and on the subsequent applications of the iPSCs obtained [24].

Since from the first studies on reprogramming programs the most common method to generate iPSCs included the employment of retroviruses or lentiviruses to deliver Yamanaka factors [25–27]. Retroviruses integrate into host's genome allowing a satisfying expression of reprogramming factors. The first retrovirus used to deliver specific transcription factors into mouse and human fibroblasts was the Moloney Murine Leukemia Virus (MMLV), capable to infect only actively dividing cells and silent in immature cells such as ESCs [22, 28].

Conversely, the most common lentivirus used as a delivery vector derives from HIV. Usually lentiviruses have higher cloning capacities and infection efficiency than retroviruses. Unlike MMLV based retroviruses, lentiviruses could replicate both in dividing and non-dividing cells. Lentiviruses, with the respect to retroviruses have two safety advantages, the lack of integration near the transcription site of start and the capacity to deliver simultaneously different reprogramming factors in a single construct [29].


#### **Figure 3.**

*The scheme summarizes the major advantages and disadvantages of integrating vs non-integrating methods currently employed for adult somatic cells reprogramming. (The figure was prepared with the support of Servier Smart Medical Art, https://smart.servier.com/).*

*Non-integrating Methods to Produce Induced Pluripotent Stem Cells for Regenerative Medicine… DOI: http://dx.doi.org/10.5772/intechopen.95070*

However both vectors made of retrovirus and lentivirus carry significant risk of insertion mutagenesis during transfection related to their genomic integration; [26] therefore, even if they are properly silenced, viral transgenes can eventually be reactivated during differentiation or during the maturation of iPSCs, with high risk for tumorigenicity [6].

Therefore, while they represent a valuable research tools, they cannot be safely employed in the clinical application (**Figure 3**).

### **2. Introducing non-integrating methods**

The efficiency and safety of generating and using iPSCs show a negative balance, and thus clinical employment of iPSC technology is still waiting for an effective protocol better poising these two fundamental features [30].

Even though the integration of reprogramming factors in iPSCs generated using viral methods offers high efficiency and a good yield, it is risky and represents a strong limitation for further clinical applications. Indeed, the residual expression of Yamanaka factors in the derived cells, such as the oncogene c-Myc, causes several genetic and epigenetic mutations, along with transcriptional abnormalities, despite the silencing of these genes during reprogramming. The integration of the reprogramming factors, in fact, is responsible for disruption of coding regions, promoters, and enhancers/repressors causing the instability of the gene network of the iPSCs obtained. Genetic aberrations are strongly related to cancer onset, hence, the maintenance of genomic stability of iPSCs without dragging integrating viral vectors sequences is highly desirable (**Figure 3**). To overcome all these relevant safety issues newer protocols are currently under development for deriving iPSCs without any integration while addressing the low efficiency showed by earlier reprogramming methods [31].

#### **3. Non-integrating viral-based methods: advantages and disadvantages**

In order to fulfill the above-mentioned requirements several integration-free methods have been developed, many of them employing viruses. It is important to underline that, even if all these methods are classified as "non-integrating", avoiding even a partial and negligible integration of viral genome into the host cells is not possible (**Figures 2** and **3**).

#### **3.1 Adenovirus**

Adenovirus is DNA virus that can reprogram cellular metabolism in a variety of ways, like increasing glucose uptake in cells and stimulating the synthesis of lactate, and produce many other metabolic changes related to cancer. Several studies have shown the effectiveness of the adenovirus as a vector to deliver specific differentiation factors to generate iPSCs without integration into host's genome.

The use of Adenovirus as a delivering method for reprogramming factors shows a certain effectiveness, allowing, at the same time, the production of quite safe and integration-free iPSCs. As a matter of fact, Stadtfeld and colleagues have demonstrated that human iPSCs generated from adenovirus are pluripotent and can be differentiated into all three germ layers in vitro and in vivo [32]. Zhou and Freed produced iPSCs from human embryonic fibroblasts using adenoviral vectors expressing c-Myc, KLF4, Oct4, and SOX2, and the iPSCs obtained expressed ESCs specific markers, showed a great differentiation potential and were free of any viral or transgene integration [33]. However, despite the employment of the adenovirus method eliminate the risk for malignant transformation associated with retrovirus or lentivirus, it shows disadvantages, such as the lower efficiency and the shorter expression kinetics requiring repeated transductions to maintain an adequate level of transgene expression [33].

Additionally, not all the cell types are capable to generate iPSCs with this method alone as shown by Okita and colleagues in their studies they were unable to obtain murine hepatocyte iPSCs clones introducing the four reprogramming factors in the adenoviral vector alone, but the entire process required additional transfections of Oct3/4 and KLF4 or Oct3/4 and SOX2 by retrovirus [34].

#### **3.2 Sendai virus**

Sendai virus is an RNA virus that can infect a wide range of cell types either proliferating or quiescent and does not enter the nucleus of host cells. RNA virusderived vectors are considered an attractive tool to vehicle Yamanaka factors, as they show a low risk of genomic insertion and are commonly used to reprogram neonatal and adult fibroblasts, and blood cells. The virus, while replicating, remains in the cytoplasm after infection and can be washed out of the host cells by several passages. Sendai virus shows a high transduction efficiency as confirmed by the expression of transgenes delivered yet detectable within a few hours after transduction, with a maximum expression after 24 hours after transduction. Sendai virus vectors have been largely studied and have emerged for their capability of successfully produce iPSCs with a non- detectable presence of viral RNA reprogramming adult human fibroblasts and circulating T cells [35].

As Sendai virus is an RNA virus it holds the great advantage that does not enter the nucleus of the host cells and allows a highly efficient reprogramming [36]. Further, Sendai virus-based vectors are replication deficient, and their copies became diluted during cell divisions, and eventually virus-free iPSCs are available after about 10 passages. Thus, this reprogramming technique works to obtain iPSCs without introducing changes to genome. However, their relatively short expression strongly limits their use in biological and research application that require longterm manipulation for somatic cell reprogramming.

#### **4. Non-viral-based reprogramming methods**

Due to the highlighted safety issues, it is necessary to develop efficient non-viral reprogramming methods. To generate iPSCs completely free from any viral contamination, researchers have modified and used DNA-based vectors, such as plasmids, episomal vectors, minicircle vectors, piggyBac transposons and non-DNA-based methods to deliver Yamanaka factors as mRNAs, microRNAs and proteins, as well as the direct reprogramming by exosomes (**Figure 2**) [37].

The common denominator of these methods is a much lower reprogramming efficiency as compared to lentiviral vectors-based reprogramming (**Figure 3**).

#### **4.1 DNA-based methods**

They require the use of elements composed of DNA to induce the expression of the reprogramming factors into the target cells. The most used elements include circular DNA vectors of different sizes (episomal vectors and minicircles) and mobile DNA sequences able to move and integrate to different locations within the genome by a cut and paste mechanism (PiggyBac).

*Non-integrating Methods to Produce Induced Pluripotent Stem Cells for Regenerative Medicine… DOI: http://dx.doi.org/10.5772/intechopen.95070*

#### *4.1.1 Episomal vectors*

One of the first integration-free techniques used for cell reprogramming includes the employment of non-replicating or replicating episomal vectors. This method is a quite simple technique not requiring special skills by the operators performing the experiments. Beside these advantage, reprogramming by the means of episomal vectors produces iPSCs still containing fragments of plasmidic DNA, due to the low transfection power that requires multiple transfections to obtain an appropriate expression level of the desired genes in the derived cells. Due to such a low transfection efficacy, the possibility of DNA fragments integration is highly increased, and it is crucial to improve the technique focusing on the reduction of transfection frequency and genetic fragments integration. Reprogramming by episomes is an excellent choice if employing blood cells but needs modification of standard culture conditions to reprogram fibroblasts into iPSCs [34].
