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ePaper created 2016-06-09, 15:16:05 | version 1.38.0
Journal of Oral Science & Rehabilitation 32 Volume 2 | Issue 2/2016 H u m a n i n d u c e d p l u r i p o t e n t s t e m c e l l s f r o m h u m a n o r a l m u c o s a based on increased proliferation and stability of the ESC-like morphology. Expression of the ESC-specific surface markers SSEA-3, SSEA-4 andTRA-1-60 in HOF-iPSCswas analyzed using flowcytometry;allthreemarkerswereexpressed (Fig.4). HOF-iPSCs could be maintained beyond 20 passages and still demonstrated ESC-like morphology. In addition, HOF-iPSCs expressed ESC-specific surface markers, such as OCT3/4, SSEA-4,TRA-1-60 andTRA-1-81 (Fig.5).Tumor formation was observed three months after the injection of HOF-iPSCs under the epidermal space in the neck of immunodeficient mice. His- tological examination showed that the tumor contained various tissues, including cartilage (mesoderm), melanocytes (ectoderm), gut-like tube tissue (endoderm) and neural tissue (ecto- derm; Fig. 6). Karyotype analysis of the tested clones showed a normal human karyotype (Fig. 7). Discussion Many strategies have been proposed for the management of large defects in oral tissue or organs such as due to congenital abnormalities, trauma or cancer treatment. Autogenous bone grafts arethe gold standardforsuch reconstruc- tion because of their osteoconductive, osteoin- ductive and nonimmunogenic properties.20, 21 Recently, celltherapyusing stem cells combined with osteoconductive biomaterials or scaffolds has become a promising alternative to autoge- nous bone grafts.22 In order for cell therapy to efficiently treat large defects in oral tissue or organs, it is important to produce a sufficient number of cells that function similarlyto prima- ry islets. IPSCs, referred to as pluripotent stem cells, have been generated via retrovirus-medi- ated introduction of four transcription factors3, 4 and represent a potentiallyunlimited source of cells. IPSCs that can be efficiently generated from tissue easily accessible to dentists have great potential;23 iPSCs have been generated fromvarious oralmesenchymalcells23 andthese cells have been reported to possess higher reprogramming efficiency than skin fibroblasts do.10 Oral mucosal tissue is easily accessible and can be harvested by a simple and safe procedu- re. Oral mucosal wounds are characterized by rapid re-epithelialization and remodeling and are known to heal quickly compared with other skin injuries.This rapid re-epithelialization and remo- deling is due to the increased production of ac- tive MMP-2inoralmucosalfibroblastscompared withskinfibroblasts;MMP-2mayplayanimport- ant role in rapid extracellular matrix reorganiza- tionandscarlesswoundhealing.13,24,25 Therefore, we hypothesized that HOFs generated from pa- tient tissue might provide a superior cell source foriPSCs. Inthe present study,wefoundthatthe endogenous expression level of KLF4 was higher inHOFsthaninESCsorHOF-iPSCs.Endogenous KLF4 has been shown to be expressed in gingival and periodontal fibroblasts derived from oral tissue.12 Miyoshi et al. also found that HOFs ex- press not only KLF4 and C-MYC but also NANOG and OCT4 at low levels, suggesting that HOFs possess a number of epigenetic advantages for reprogramming.13 Integratingvirus-associatedgenotoxicityand tumorformationiniPSCsisofconcernforclinical application.15 Integration-free human iPSCs have been generated using several methods.24, 26–30 Okita et al. used two of their findings to improve reprogramming efficiency using episomal plas- mids;15 iPSC generation is markedlyenhanced by p53 suppression31 and L-MYC is more potent and specific than C-MYC during human iPSC genera- tion.32 In our previous study,17 iPSCs were gene- rated from HGFs using the above-mentioned method.ThegeneratediPSCsexpressedESC-spe- cific markers, as assessed by gene analysis and immunocytochemistry. Embryoid bodies and teratomas were formed from the iPSCs, demon- strating their ability to differentiate into three germlayers.However,50ESC-likecolonieswere obtained only 30 days post-HGF transfection. This lengthy reprogramming process (up to one month) is comparable to that of the standard virus-mediated methodology.33 ThematernalGli-liketranscriptionfactorGlis1 is highly expressed in unfertilized eggs and one- cell-stageembryos.18 Maekawaetal.showedthat Glis1, but not C-MYC, increased iPSC tumorige- nicity and markedly enhanced the generation of iPSCs from both mouse and human fibroblasts whenexpressedtogetherwithOCT3/4,SOX2and KLF4.18 In the present study, we observed 50 co- lonies of human ES-like cells as early as 20 days afterinitialepisomalplasmidvectortransduction. These results demonstrate that Glis1 enhances the efficiency of iPSC generation using episomal plasmid vectors expressing OCT3/4, shRNA against p53, SOX2, KLF4, L-MYC and LIN28. However, iPSC generation from multiple donors willberequiredtoestablishtheapplicationofiPSC technology to biomedical research.