79Several viral vector strategies were used to obtain iPSCs, including retrovirus,
lentivirus, adenovirus, and Sendai virus approaches. The fi rst iPSCs were estab-
lished through the constitutively active retroviral transduction of c-Myc, Sox2,
Klf4, and Oct4 (Takahashi and Yamanaka 2006 ). In this system, the active retroviral
vector is stably integrated into the genome. Lentiviral vectors are more effective
than retroviral vectors because of the ability to infect different somatic cell types;
furthermore, lentiviruses can be used to express polycistronic cassettes encoding all
four reprogramming factors, thus increasing reprogramming effi ciency (Maherali
et al. 2007 ). Optimization of retroviruses or lentiviruses has helped increase the
effi ciency of infection of mammalian cells (Kumar et al. 2015 ). The pantropic
vesicular stomatitis virus G protein was used as a substitute for viral transduction of
a number of derived cells (Kumar et al. 2015 ). However, the viral approach has
three major disadvantages. First, this system has a limited cloning capacity with a
maximum of 7 kb. The second limitation is the induction of immune response by
the host upon infection. The third limitation involves potential genotoxic effects.
Retroviruses and lentiviruses do not randomly integrate in the genome but show a
signifi cant preference for promoter and exonic regions, which may compromise the
regulation of endogenous genes (Kumar et al. 2015 ). In the case of lentiviral vec-
tors, the inert drug doxycycline can regulate expression, decrease continued trans-
gene expression, and help select fully reprogrammed iPSCs, because cells that
depend on the expression of exogenous factors promptly stop proliferating when
doxycycline is removed (Brambrink et al. 2008 ). The iPSCs formed by retroviruses
or lentiviruses had the absence of integration sites as an indispensable result, which
indicates that insertional mutagenesis has a supportive function during iPSC pro-
duction. To avoid insertional mutagenesis, viruses that do not integrate into the
genome or that can be subsequently removed are required, such as adenovirus or
Sendai virus (Ban et al. 2011 ).
The derived nonviral iPSCs have been highly recommended for regenerative
medicine. Advantages of this approach include the ability to insert DNA into host
cells without integration and no lack of limitation of DNA size in the transferring
process. Nonviral methods include transposons, “minicircle” DNAs, episomal vec-
tors, plasmid vectors, mRNAs, small molecules, transposons, and recombinant pro-
teins such as Sleeping Beauty and piggyBac (Stadtfeld and Hochedlinger 2010 ). A
recent study described nonintegrating episomal vector-derived human iPSCs. After
episome removal, iPSCs are derived that are completely defi cient of transgene
sequences and vectors and resemble the proliferative and developmental potential
of human ESCs. These results indicate that reprogramming human somatic cells
does not require the continued presence of exogenous reprogramming factors or
genomic integration and eliminates one obstacle to the clinical application of human
iPSCs (Yu et al. 2009 ). The use of “minicircle” DNA (Chabot et al. 2013 ), a vector
that is capable of high expression in cells and is free of bacterial DNA, was success-
ful in producing transgene-free adult human adipose stem cell-derived iPSCs (Jia
et al. 2010 ). One strategy that will augment reprogramming uses small molecules as
core reprogramming factors. Two approaches were explored in determining
conditions for replacing viral transduction of oncogenic transcription factors and
4 New Trends in Clinical Applications of Induced Pluripotent Stem Cells