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RNA, these strands are easily removed from infected cells using siRNA (Nishimura
et al. 2011 ). Viral-free vectors were also recently developed to completely remove
viral factors during iPSC production. In the fi rst effort, episomal vectors were devel-
oped to generate iPSCs (Gonzalez et al. 2009 ; Jia et al. 2010 ). However, this method
shows lower effi ciency of successful reprogramming than that achieved using retro-
virus. Another viral-free vector approach uses the piggyBac transposon. Although
some authors have successfully produced iPSCs using the piggyBac transposon,
this technique also generally shows ex tremely low effi ciency (Kaji et al. 2009 ; Yusa
et al. 2009 ). To avoid introducing genetic material, much attention has been recently
focused on introducing reprogramming factors such as RNAs or proteins. For exam-
ple, mRNAs of pluripotent factors (Warren et al. 2010 ) and microRNAs (Anokye-
Danso et al. 2011 ) have been shown to successfully reprogram somatic cells to a
pluripotent state. These RNA-based reprogramming methods avoid both breaks in
existing genes and the reactivation of transgenes. Therefore, these methods hold
much promise as novel iPSC generation methods for clinical use. More recently,
recombinant proteins and small molecule drugs were also reported as successful
means of gene introduction for generating iPSCs. Zhou et al. used recombinant
Oct4, Sox2, Klf4, and c - Myc proteins that were designed with a poly-arginine (11R)
protein transduction domain to aid in penetration into the cytoplasm (Zhou et al.
2009 ). Hou et al. ( 2013 ) reported that pluripotent stem cells can be generated from
mouse somatic cells at a frequency as great as 0.2 % using a combination of seven
small molecule compounds (Hou et al. 2013 ). With viral-free vectors, iPSCs can be
used in clinical applications with a high degree of safety related to genome
stability.
The second issue for clinical grade of iPSCs involves minimizing the invasive-
ness in obtaining donor cells for iPSC production. In early studies, iPSCs were suc-
cessfully produced from human dermal fi broblasts. However, only small fragments
of skin can be collected, and the collection of the skin dermal layer is relatively
invasive. A technique with lower invasion was developed to produce iPSCs from
keratinocytes (Aasen and Izpisua Belmonte 2010 ). Other cells can be obtained by
less invasive techniques and were also considered as suitable sources for iPSC pro-
duction, including dental stem cells (Yan et al. 2010 ) and mesenchymal stromal
cells derived from human third molars (Oda et al. 2010 ), oral gingival cells (Egusa
et al. 2010 ), and oral mucosa fi broblasts (Miyoshi et al. 2010 ). More recently,
peripheral blood cells were successfully used to produce iPSCs (Brown et al. 2010 ;
Loh et al. 2009 ). Collection of peripheral blood is less invasive, and therefore gen-
erating iPSCs from peripheral blood could be one of the most appropriate methods
for establishing iPSCs. With some breakthroughs in techniques, iPSCs could also be
successfully produced from fresh or frozen peripheral blood samples.
Third, animal composition-free culture systems must be implemented to remove
risks related to xenogenic proteins. The highest risk in culture systems is related to
fetal bovine serum supplementation in cell culture medium. The second highest risk
involves the use of murine cells for the feeder layer. Some efforts to use human cells
for feeder layers were initiated (Takahashi et al. 2009 ); however, these techniques
were time consuming and complex. In other studies, Matrigel was used to replace
P.V. Pham et al.