81increased the expression of endogenous DNMT3β, Nanog, Oct4, Rex1, and Sall4.
The cells were cultured in human ESC medium, and small aggregates positive for
Oct4 protein and alkaline phosphatase activity were detected within 30 days (Plews
et al. 2010 ). Another published approach is based on highly reproducible RNA that
uses a single, synthetic self-replicating VEE-RF RNA replicon. The replicon con-
sists of four essential factors (Klf-4, Oct4, and Sox2, with GLIS1 or c-Myc) with
high-level expression before regulated RNA degradation. iPSCs were successfully
generated from adult or newborn human fi broblasts by transfection of a single
VEE-RF RNA. These transfected cells expressed all the hallmarks of stem cells,
including global gene expression profi les, cell-surface markers, and in vivo pluripo-
tency, with differentiation into all three germ layers (Yoshioka et al. 2013 ). These
studies demonstrate that mRNA transfection is a promising approach to activate
pluripotency genes in differentiated cells. However, one limitation of this approach
is the short half-life of RNA.
Together, these studies show that several successful methods have been estab-
lished for the generation of iPSCs. The fi nal application or the required yield of
iPSCs may help dictate the appropriate strategy.
4.2 Production of iPSCs with Clinical Grade
4.2.1 iPSCs Can Be Produced in Clinical Conditions
Differing from preclinical studies, clinical-grade iPSCs must be produced in a Good
Manufacturing Practice (GMP)-compliant manner that minimizes the risk of viruses
and infection as well as modifi cations during the iPSC production process. Three
issues must be addressed to satisfy the clinical grade of iPSCs.
First, gene delivery vehicles must be improved to minimize genome instability of
iPSCs. In the initial effort to produce iPSC, retroviruses vectors were used to carry
transgenes to target cells (Kitamura et al. 2003 ; Takahashi et al. 2007a , b ). Lentiviral
vectors were subsequently used to increase the effi ciency of infection compared
with retrovirus vectors (Blelloch et al. 2007 ; Yu et al. 2007 ). Both retroviral and
lentiviral vectors can cause genomic integration, and these integration events can
activate oncogenesis in iPSC-derived cells (Okita et al. 2007 ). As a greater concern,
the transgenes have the potential to interfere with functional genes. Therefore, some
recent efforts have aimed to generate iPSCs without genomic insertions. Adenovirus
vectors have been the subject of current focus as these vectors integrate into the
genome of target cells at extremely low frequencies (Harui et al. 1999 ). A recent
study used a Cre-deletable lentivirus system to produce iPSCs (Hanna et al. 2007 ).
However, although these systems can avoid transgene reactivation, there is a risk of
introduction of gene breaks near the insertion site (Nagy 2000 ). Finally, scientists
have successfully developed a transgene system without gene disruption near the
insertion site and reactivation of transgenes using the Sendai virus (Fusaki et al.
2009 ). Moreover, because the Sendai virus genome is negative-sense single-stranded
4 New Trends in Clinical Applications of Induced Pluripotent Stem Cells