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growth. The next generation of bioengineered stem cells will likely include
specialized properties for improved stress tolerance, streamlined differen-
tiation capacity, and increased engraftment/survival to improve regenera-
tive potential.
6.2.1.1  First-Generation Technology
Retroviral and lentiviral approaches offered the initial methodology
that launched the field and established the technological basis of nuclear
reprogramming with rapid confirmation across integrating vector sys-
tems [18,22,23,31,44-50]. The risk of oncogenic genes and insertional muta-
genesis inherent to stable genomic integration have been recognized as
potential limitations from the outset of this technology. However, distinct
advantages of the retroviral-based vector systems have generated critical
insights to the mechanisms of reprogramming. Retroviral and lentiviral
systems have built-in sequences that silence the process of transcription on
pluripotent induction, thus temporarily restricting the persistent exposure
to ectopic gene expression at the time of reinduction of pluripotency. This
allows an essential observation to be made in that successful self-mainte-
nance of the pluripotent state was possible without long-term transgene
expression. This made it possible to envision systems for transient pro-
duction of stemness-related genes without integration into the genome to
improve the safety and efficacy of nuclear reprogramming. The first proof
of principle was achieved by nonintegrating viral vector systems, such as
adenovirus [51], and confirmed by repeated exposure to extrachromosomal
plasmid-based transgenes [52]. Importantly, these reports demonstrated
that expression of stemness-related factors was required for only a limited
time frame until the progeny developed autonomous self-renewal, estab-
lishing nuclear reprogramming as a bioengineered process that resets a
sustainable pluripotent cell fate independent of permanent genomic modi-
fications. The inherent inefficiency of nonintegrated technologies has, how-
ever, hindered broader applicability and stimulated the search for more
efficient methodology.
6.2.1.2  Second-Generation Technology
Nonviral approaches capable of high-efficiency production have advanced
iPS cell technology toward clinical applications [53,54]. These approaches are
dependent on short sequences of mobile genetic elements that can be used to
integrate transgenes into host cell genomes and provide a genetic tag to “cut
and paste” flanked genomic DNA sequences [55]. The piggyBac (PB) system
couples enzymatic cleavage with sequence-specific recognition using a trans-
poson/transposase interaction to ensure high-efficiency removal of flanked
DNA without residual footprint. This technology achieves a traceless trans-
genic approach in which nonnative genomic sequences, transiently required
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