*4.1.2 Minicircle vectors*

Minicircle vectors were first developed as smaller alternates to episomal vectors with a higher efficiency of transfection. They are circular, non-viral DNA elements that have been freed from a prokaryotic vector containing sequences of interest i.e. Oct3/4, SOX2, Nanog, LIN28, Green Fluorescent Protein (GFP). Expression of minicircle-coded genes occurs in both dividing and non-dividing cells with high efficiency, and typically yield higher expression levels of desired proteins, as they are less likely to be inactivated and silenced by cellular mechanisms targeting foreign nucleic acids [38, 39].

#### *4.1.3 PiggyBac transposons*

In PiggyBac transposon reprogramming, transgene sequence can be removed from integration site without changing host's DNA. It requires only the inverted terminal repeats, flanking a transgene and transient expression of the transposase to catalyze insertion or excision events [40]. All the mentioned methods show disadvantages, for example, the Sendai virus is effective on all cell types, but requires a lot of passages to obtain iPSCs. The PiggyBac method could represent an attractive alternate but studies in human cells are still limited and weak [40].

#### **4.2 Non-DNA-based methods**

Most common reprogramming strategies are based on the use of DNA. All these techniques are effective in achieving a successful reprogramming of the somatic cell, but they are considered less safe as some fragments of the DNA employed can integrate into the host cells genome due to the repeated treatments required to obtain the appropriate expression level of the desired genes. To avoid this safety issues, several groups focused on the development of protocols including non-DNA based methods for reprogramming, such as the use of mRNAs, microRNAs, or recombinant proteins (**Figures 2** and **3**).

Beside reprogramming, and subsequent differentiation into desired cell type, several authors have recently reported the possibility of trans-differentiation, or conversion of one cell type to another one, while bypassing the iPSC-state. An example is the direct conversion of myocardial scar fibroblasts (MSFs) to cardiomyocytes by infection of human MSFs with a lentivirus vector encoding the potent cardiogenic transcription factor myocardin [41].

The direct reprogramming, in fact, is a procedure by which a mature, fully differentiated cell is converted into another cell type, completely or partially bypassing an intermediate pluripotent state. The direct reprogramming is an interesting new approach of regenerative medicine allowing to overcome the numerous problems related to the use of stem cells. Additionally, it has a low risk for genetic alterations and tumor development, as the reprogramming by this technique avoids risky genetic manipulation and the use of viruses or other strategies causing the residual integration of exogenous genetic material.

#### *4.2.1 mRNAs*

High-efficiency, synthetic mRNA-based reprogramming was recently described [42]. Synthetic mRNAs codifying for Yamanaka factors are modified to overcome innate antiviral responses. Since mRNA is translated to protein in the cytoplasm, it does not enter the nucleus, minimizing chance of unwanted modifications of hosts DNA. This method appears to work fast and efficiently, but the major disadvantage is that mRNA is degraded in few days. As such, repeated transfection is required for successful reprogramming [42, 43].

The direct delivery of synthetic mRNAs for the conversion of adult mature cells into iPSCs is an example of direct reprogramming. An effective protocol including the employment of this method was proposed by Warren et al. The idea of delivering mRNA directly raised from the possibility of random DNA fragment integration when DNA is used to derive iPSCs. This procedure is based on the in vitro transcription by the means of templates previously amplified by molecular biology techniques to encode the four Yamanaka reprogramming factors. A strong limitation related to the employment of this procedure is due to the multiple administrations required to gain an adequate protein expression levels, therefore the entire reprogramming process consists in a daily mRNA transfection and the derivation of iPSCs can take up to 18 days. Nonetheless, the transfection of human dermal fibroblast with Yamanaka's reprogramming factors combined with Nanog and LIN28, from Thomson's approach, have been reported as inducing the arrangement of cells in colonies as early as 24 h after the first transfection (**Figure 1**) [18, 19]. To increase the efficiency of the technique, the delivery of mRNAs is combined with hypoxic culture conditions that seem to double the efficiency of reprogramming. However, direct cell reprogramming mediated by mRNA is risky, as the numerous and repeated administrations of them to ensure a high expression level of proteins of interest can eventually trigger the activation of c-Myc, with a high risk for tumor development. A pivotal improvement for this procedure could target the frequency of mRNAs administration and the activation of the oncogene c-Myc.

#### *4.2.2 MicroRNAs*

MicroRNAs are small molecules of non-coding RNA primarily involved in gene expression regulation at both transcriptional and post-transcriptional level; in particular, they are responsible for gene silencing. Several studies have reported that including microRNAs in the traditional procedures employed for reprogramming can positively impact the efficiency of the process. Equally to other procedures not requiring DNA, reprogramming by microRNAs produces iPSCs free from exogenous DNA integration, but the needing of multiple administrations makes the procedure complicated and time consuming [44].

#### *4.2.3 Recombinant proteins*

To overcome the issue related to the introduction of exogenous DNA into derived iPSCs, another approach consists in the employment of recombinant proteins as

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

reprogramming factors. Protein-based reprogramming carries the advantage that it does not cause any genetic changes. As already mentioned, current methods of protein-based reprogramming are less efficient that lentiviral delivery of Yamanaka factors [42, 45]. Typically, synthesized in bacteria, Yamanaka factors are modified so that they express basic amino acids or other transport peptides enabling to cross the cell membrane [4].

Some studies have led to the development of different methods to isolate, purify, and then deliver reprogramming factors in form of recombinant proteins [33, 45].

The reprogramming mediated by recombinant proteins is challenging and need several improvements. The synthesis of a consistent amount of proteins is quite hard and requires specific skills that make the technique ineffective for a number of laboratories.

#### *4.2.4 Exosomes*

The modern trend for cell reprogramming consists in the direct conversion of a cell into another by the means of exosomes containing a cocktail of reprogramming factors for a specific purpose, named reprosomes. With the respect to the iPSCs, reprogramming cells by exosomes seems to be more likely for clinical applications, as it requires easier procedures and the risk for tumor formation and mutations is low [46].

Exosomes are nanovesicles with a size ranging between 30 and 200 nm. They are secreted by all cell types and circulate in many body fluids, from where they can be easily isolated. After the discovery that exosomes are able to transfer molecules of biological relevance, like mRNA, miRNA and proteins to one cell to another eliciting phenotypical changes, several studies are ongoing to define their potential as an integration-free method for cellular reprogramming. Despite several advantages offered by the use of exosomes, like the easy extraction method, the reduction of immunological host response and the possibility to reprogram cells without genetical manipulation, their effective employment is still under investigation and the procedures for their isolation and characterization are still limited by a low efficiency and a poor specificity [47, 48].

#### **5. Conclusions**

The improvement of non-integrating methods is now the target for cell reprogramming to derive iPSCs. In fact, these methods do not require the incorporation of viral genome into the host cells, avoiding the risk of tumor development. The safety of these methods, that makes the derived cells more appealing for clinical applications, is a common strong point, although beside the above-mentioned specific issues related to each method, the common major weakness is represented by a general low efficiency respect to the traditional integrating approaches. The original protocol proposed by Yamanaka for generating iPSCs from adult somatic cells was based on the insertion of only four factors: octamer-binding transcription factor-3/4 (OCT3/4), SRY-related high-mobility group (HMG)-box protein-2 (SOX2), Myc, and Kruppel-like factor-4 (KLF4), or Nanog and LIN28 instead of Myc and KLF4 [4]. A major obstacle in cellular reprogramming, beside the risk of tumor formation due to the integrating methods, is the very low efficiency of the reprogramming procedure, strictly related to several factors, such as the type of cell to be reprogrammed, the method of delivering the reprogramming factors and culture conditions. Although the non-integrating methods offer a safer way to produce iPSCs for further clinical application, it is crucial to focus on the enhancement of the efficiency of the existing and ongoing protocols. In this respect, several strategies have been developed, such as the employment of promoters or enhancers boosting the reprogramming of somatic cells have being developed. Regulatory genes involved in proliferation and cell cycle modulators represent a valid example among the approaches proposed, although if on one side they allow a better yield, on the other they have the disadvantage of being potentially tumorigenic [49, 50].

Additional candidates investigated for their ability to increase up to 100 folds the efficiency of reprogramming, due to their capability of remodeling chromatin, are small molecules and inhibitor factors, such as valproic acid (VPA) and histone deacetylase (HDAC) inhibitor. Further, the use of VPA together with hypoxic conditions greatly boosts the efficiency of reprogramming [51–53]. The remodeling of chromatin induces a dynamic modification of chromatin architecture that allows the access to the condensed DNA by proteins involved in transcriptional regulation mechanism and responsible for the modulation of the gene expression in the cells [54].

Other factors heavily impacting on the efficiency of reprogramming are culture conditions, the possible employment of supporting feeder cells, and the composition of culture medium [55, 56]. It is well documented that reprogramming under hypoxic conditions of 5% O2 instead of the atmospheric 21% O2 increases the reprogramming efficiency of mouse embryonic fibroblasts (MEFs) and human dermal fibroblasts. The presence of a layer of feeder cells is extremely important to support cells during the reprogramming procedures, as feeder cells are responsible for the secretion of growth factors essential for cell survival. Usually, mouse feeder cells are used to support the growth and culture of iPSCs, but they must be removed before the use in clinical applications. Basically, feeder cells consist in a layer of growth-arrested cells unable to divide, which provides extracellular secretions to help other cells to proliferate. However, the use of animal derived feeder cells rises safety issues for the clinical applications due to the contamination of pathogens cross-transfer. To overcome this limitation, the use of Matrigel, a mixture of extracellular matrix proteins such as laminin, collagen and fibronectin, and supplemented with a medium conditioned by feeder cells, as substitute supporting layer is widely popular to produce and support iPSCs [19, 57–58].

A successful reprogramming also depends on the choice of the proper cell type to reprogram. The original protocol proposed by Yamanaka included the use of fibroblasts, first from mouse, then from humans, and these cells still remain the favorite cell type, primarily for the easiness of harvesting by skin biopsy. However, even among the different types of fibroblasts several studies highlighted that they are not reprogrammable with the same efficiency [18]; hence, other cell sources need to be found. In fact, the specific promptness of cell to be reprogrammed is strictly related to the endogenous expression of some reprogramming factors and from the starting differentiation state. Currently, there are different strategies, which allow choosing the appropriate cell source, the delivery method, and the system to boost the efficiency of cell reprogramming to derive iPSCs in the safer manner. Nevertheless, all these techniques need to be strongly boosted in order to be considered useful for a clinical application of the derived iPSCs.:

### **Conflict of interest**

The authors declare no conflict of interest.

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