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and remove transgenic elements in a precise and site-specific manner without leaving any
permanent genetic modification to cellular genomic DNA. Indeed, this can be achieved with
the PiggyBac transposon system. 49,50 Utilizing PiggyBac transposons with four different
transgene inserts (KLF4, OCT4, c-MYC, and SOX2), Woltjen et al. 49,50 managed to
successfully reprogram human fibroblasts into iPSC and excise these transgenic elements
from the cellular genome, without leaving any permanent trace of genetic alteration. 49,50
Another strategy to avoid the use of recombinant DNA would be to utilize cell-permeable
recombinant proteins for cellular reprogramming. 124 The existence of specific peptide
sequences that confer cell-penetrating ability is well known, and are commonly referred to
as protein transduction domains. 158 160 Zhou et al. 124 fused a polyarginine (11R) protein
transduction domain to four recombinant transcription factors (KLF4, OCT4, c-MYC, and
SOX2), and these were utilized to successfully reprogram murine embryonic fibroblasts to
iPSC. Using a similar approach, Kim et al. 161 were able to derive iPSC from human
fibroblasts with recombinant transcription factors fused to polyarginine protein
transduction domains.
Success in cellular reprogramming with cell-permeable proteins was quickly followed by the
first successful derivation of iPSC through transfection with chemically modified mRNA. 125
There are two major challenges in utilizing RNA transfection in cellular reprogramming. The
first challenge is the relative instability and short half-life of RNA within the cell. The second
challenge is that there exists an innate antiretroviral response within mammalian cells
against foreign RNA that triggers cellular apoptosis. Warren et al. 125 extended the stability
and half-life of synthetic mRNA with a 5 0 -guanine cap, 162,163 while the innate antiretroviral
response within mammalian cells against foreign RNA was overcome through chemical
modifications to the synthetic mRNA. This included substitution of uridine with
pseudouridine, 164 and substitution of cytidine with 5-methylcytidine. 165 Additionally, the
B18R protein was supplemented into the culture media during RNA transfection to suppress
the interferon-1 pathway that leads to cellular apoptosis upon introduction of foreign RNA
into the cell. 166 This is achieved by the B18R protein acting as decoy receptor for type I
interferon, 166 and is absolutely crucial for maintaining cell viability during cellular
reprogramming with RNA transfection. By utilizing chemically modified mRNA
corresponding to the four Yamanaka transcription factors (KLF4, OCT4, c-MYC, and SOX2),
together with the supplementation of B18R within the culture medium, Warren et al. 125
were able to successfully reprogram human fibroblasts to iPSC at higher efficiencies
compared with previous techniques utilizing recombinant DNA.
173
Although the derivation of iPSC from differentiated somatic cells is an exciting development
in the field of stem cells and regenerative medicine, a pertinent question is whether it is
absolutely necessary to set the developmental clock back to the embryonic state for
therapeutic applications in regenerative medicine. It is possible that the developmental clock
may instead be reset halfway to a less immature developmental stage that is more directly
applicable to therapeutic applications, that is, the transit amplifying progenitor stage. 167,168
Some recent studies have even demonstrated direct lineage conversion from one
differentiated phenotype to another through the recombinant expression of transcription
factors. Most notably, murine fibroblasts have been converted directly into
cardiomyocytes, 169 neurons, 170,171 and hepatocytes 172,173 ; while human fibroblasts have
been converted directly into neurons 174 176 and hematopoietic progenitors. 177
Nevertheless, it is unlikely that future regenerative medicine applications would bypass the
stem cell or progenitor cell stages completely. This is because terminally differentiated
somatic phenotypes have limited proliferative capacity, and transplantation/transfusion
therapy would certainly require large quantities of cells for individual patients. Extensive
proliferation in situ or ex vivo is only possible at either the stem cell or progenitor
cell stages.
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