Sendai virus is a negative sense, mRNA virus belonging to Paramixoviridae family [17]. It is non-pathogenic to humans, and its use as a viral vector has several advantages: (1) being an mRNA virus, it does not enter the nucleus in its lifecycle, thus eliminating the risk of modifying the host genome and/or causing gene silencing by epigenetic changes [18]; (2) it shows a broad tropism, being able to infect several cell types in vitro [19,20,21,22,23]; (3) due to its non-integrating nature, viral genome is diluted to every cell duplication, allowing its removal from the reprogrammed cells; and (4) it allows the production of a large number of proteins, thus allowing multiplicity of infection (MOI) reduction. Sendai viral vectors were successfully used to reprogram fibroblast cells [24], as well as blood [25,26] and renal epithelial cells in the urine [27]. This technique is quite efficient, ranging between 0.01% and 4% in the generation of human iPSCs at 25 days of induction [20,24,25,28,29]. Up to 10 passages or a high-temperature culture (39 °C) might be necessary to remove the viral genome completely [11]; however, an auto-erasable, replication-deficient Sendai virus was recently developed using microRNA-302 which impedes viral replication by blocking the viral RNA-dependent RNA polymerase [30].

Adenovirus is a non-integrating virus [31] that remains in the epichromosomal form in all cell types, except in egg cells [32]. Adenovirus offers a large cargo capacity, a transient expression, and rapid clearance from dividing cells, thus requiring multiple rounds of infection. The reprogramming efficiency is low, 0.001–0.0001% in mouse cells and 0.0002% in human cells, most likely because of the low infection efficiency and the narrow expression window of reprogramming factors [16].

Adeno-associated virus is a non-pathogenic, non-autonomous single-stranded DNA virus, unable to replicate without the presence of a co-infecting helper virus. In its absence, AAV’s genome remains in episomal form within the infected cells, although integration into the host’s genome was reported in fewer than 10% of cases.  For all these reasons, this vector has been used in more than 100 clinical trials [35]. However, the need for multiple rounds of transduction for cell reprogramming, limited transgene capacity (5 kb), and low efficiency (less than 0.01%) [16,35] still limit AAVs’ use as a vector for inducing pluripotency.

mRNA transfection was first used for cell reprogramming by Warren’s group. They overcame several obstacles to transcribe mRNAs to express reprogramming factors efficiently, reaching a 1.4% reprogramming efficiency [36]. Moreover, the addition of Lin28 to the Yamanaka reprogramming factor protocol, valproic acid in the cell culture medium, and a change of O₂ concentration to 5% allowed for an increase in efficiency to 4.4% [36]. 

Regarding the miRNA infection/transfection, several miRNA, such as miR-302b or miR-372, are strongly expressed in ESCs. Their addition to Yamanaka factors increased up to 15-fold reprogramming efficiency for OSKM alone [37]. Interestingly, some miRNAs could reprogram cells at high efficiency even in the absence of co-transfection with OSKM, bringing the efficiency of reprogramming for BJ-1 fibroblasts up to 10% [38].

These two transposons usually consist of a polycistronic transcript containing the OSKM factors joined by 2A peptides, allowing post-translational cleavage of the polyprotein into single reprogramming proteins as well as maintenance of their stoichiometric co-expression [39]. PiggyBac is a transposon, a mobile genetic element, that can be easily inserted and removed from chromosomal TTAA sites in the genome. Using a transposase, it can be integrated and subsequently excised from the genome [11]. When the OSKM factors are cloned into the PiggyBac vector and co-transfected into mouse embryonic fibroblasts (MEFs), reprogramming efficiency ranged from 0.02 to 0.05% of the total transfected cells [40,41]. This technique requires only a single transfection; the transposon can transport substantial cargo and presents low immunogenicity.

An intrinsic feature of the PiggyBac vector is its integration into the host genome. However, it could be cleanly excised from the iPSCs genome. Potential reintegration is conceivable due to the use of the same enzyme for insertion and excision. This reintegration risk forces a tight screening of iPSC clones to confirm the absence of integration and is time-consuming. As previously mentioned, reprogramming efficiency is quite low (0.01–0.1%) but can be improved using sodium butyrate. Regardless, the efficiency remains 50-fold lower than retroviral-mediated reprogramming methods [42].

Sleeping Beauty transposon vector differs from PiggyBac in its ability to integrate randomly into host genomes, thus showing no integration tendency with regard to specific genes and gene-regulatory elements [43].

Episomal plasmids ensure a technically simple procedure, a stable transgene expression due to their self-replication, and a low immunogenicity, allowing their removal by culturing cells in the absence of drug selection [44]. Due to their vulnerability to exonucleases, episomal vectors have an extremely low reprogramming efficiency, primarily due to the short expression time in the cells. It is possible to overcome this issue by repeating transfections daily; however, the reprogramming efficiency remains unsatisfactory (0.0003–0.0006%) [45]. The inclusion of NANOG, LIN28, and LT (SV40 large T gene) as reprogramming factors enhanced the efficiency 100 times, making it comparable to viral-based methods [46]. Instead of using a single plasmid for every Yamanaka factor, which is laborious and less efficient, as only a few cells receive all the plasmids, some groups use polycystronic plasmids to obtain a “3+1 delivery” of the reprogramming factors (with Oct4, Klf4, and Sox2, carried by one plasmid, and c-Myc by the other) [15], while other groups rely on one single plasmid to deliver all four genes [47], under the control of a constitutively active CAG promoter. However, these last methods do not ensure an adequate stoichiometric co-expression of the multiple reprogramming factors. The use of picornaviral 2A self-cleaving peptides to link reprogramming factors, when used as a polycistronic construct [48], can partially ameliorate the balance of the expression of the four genes. However, there is still the chance that polycistronic plasmid could produce an unbalanced expression of each reprogramming factor [49]. This issue, together with the large size of the plasmid, hampers the efficiency of plasmid-based reprogramming [16].

Minicircle vectors are supercoiled DNA episomal vectors similar to a standard plasmid but containing only the eukaryotic promoter and the cDNA(s) of the genes to be expressed. Their small size, resistance to cleavage, extremely low immunogenicity, and high transfection efficiency make them a good tool for cell reprogramming [16], despite a very low reprogramming efficiency (0.005%) and long reprogramming time (14–18 days). Thus, multiple rounds of transfection are required, causing a reduction in cell viability [50]. In order to improve the efficiency of reprogramming, various researchers used electropulsation, included additional reprogramming factors and/or microRNAs, used small molecules, and included hypoxic conditions [51,52,53,54]. Tight screening of the clones is necessary to exclude the integration of transgene sequences.

Liposomal magnetofection is a non-viral technique that allows the delivery of nucleic acids in cultured cells by mixing nucleic acid and magnetic nanoparticles in cationic lipids. The lipids are concentrated on the surface of the cells using a magnetic field [55]. This technique has little chance of genomic integration, requires only a single transfection, and has low immunogenicity. There are rare cases of genomic integration. Consequently, screening iPSC clones is necessary to confirm the absence of integration [56]. Moreover, its reprogramming efficiency is between 0.032% and 0.040% after 8 days [16].

Although the bioactive forms of reprogramming proteins can be synthesized by prokaryotic or eukaryotic systems, the main hurdle for reprogramming is their limited capability to cross the cell membrane. To overcome this obstacle, the protein approach takes advantage of the HIV-TAT protein (a protein transduction domain) in delivering recombinant proteins. This technique allows the introduction of proteins into cells from the external environment without permeabilization agents [57]. The efficiency of this procedure is quite low, at 0.006% of mouse fibroblasts [58] and 0.001% of human fibroblasts [9,59]. To improve the reprogramming efficiency, some authors supplemented the culture media with valproic acid (VPA), with 0.006% of cells induced to pluripotency after 30–35 days [58].

Protein transduction domains later became a method to deliver not only proteins but also other macromolecules. Those included peptide nucleic acids (PNA), antisense, short-interfering ribonucleic acids (siRNA), liposomes, iron nanoparticles, and plasmids [57].

Despite the methods mentioned above for reprogramming, the highest degree of safety at the cost of low reprogramming efficiency is represented by iPSC generation through the use of small molecules to obtain chemically induced pluripotent stem cells (CiPSCs).

Hou et al. developed a combination of six small molecules (obtained after an intense work of screening of more than 10,000 compounds). They included several cAMP agonists (Forskolin, Rolipram, and Prostaglandin E2) and epigenetic modulators (sodium butyrate, 3-deazaneplanocin A (DZNep), 5-Azacytidine, and RG108) to generate chemically induced iPSCs (CiPSCs). Interestingly, they found that small molecule (sm) iPSCs could be generated using only one gene of the OSKM, namely Oct4, with the addition of CHIR99021, tranylcypromine (VC6T), VPA, and 616452 [8,60]. Compared to ESCs, CiPSCs have similar doubling time, gene expression profiles and differentiation ability, and they generate teratomas and chimeric mice [8,61]. Moreover, it is intriguing that different chemical cocktails are needed to induce other source cells [60]. To date, current chemical reprogramming efficiency is only 0.2%, with an induction time of more than 36 days that was recently reduced to 16 days [62,63].