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ePaper created 2016-06-09, 15:16:05 | version 1.38.0
Journal of Oral Science & Rehabilitation 26 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 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 Accelerated generation of human induced pluripotent stem cells from human oral mucosa using episomal plasmid vectors and maternal transcription factor Glis1 Takahiro Kashiwagi,a Yoshiya Hashimoto,b Masahiro Tanakac & Shunsuke Babaa a Department of Oral Implantology, Osaka Dental University, Hirakata, Japan b Department of Biomaterials, Osaka Dental University, Hirakata, Japan c Department of Fixed Prosthodontics and Occlusion, Osaka Dental University, Hirakata, Japan C o r r e s p o n d i n g a u t h o r : Dr. Yoshiya Hashimoto yoshiya@cc.osaka-dent.ac.jp H o w t o c i t e t h i s a r t i c l e : Kashiwagi T, Hashimoto Y, Tanaka M, Baba S. Accelerated generation of human induced pluripotent stem cells from human oral mucosa using episomal plasmid vectors and maternal transcription factor Glis1. J Oral Science Rehabilitation. 2016 Jun;2(2):26–35. Abstract O b j e c t i v e Induced pluripotent stem cells (iPSCs) possess high pluripotencyand dif- ferentiation potential and mayconstitute a possible source ofautologous stem cells for clinical applications. However, the lengthy reprogramming process (upto one month) remains one ofthe most significant challenges facing standard virus-mediated methodology. The Gli-like transcription factor Glis1 is highly expressed in unfertilized eggs and one-cell-stage embryos. In this study, iPSCs were generated using a combination of pri- maryhumanoralmucosalfibroblasts(HOFs)andepisomalplasmidvectors expressing transcription factors, including Glis1. M a t e r i a l s a n d m e t h o d s HOFs were established from oral mucosal tissue 3mm in diameter from a 23-year-old Asian male using a skin trephine. Human iPSCs were gen- erated from the established HOFs using the following episomal plasmid vectors: pCXLE-hOCT3/4-shp53-F that expresses OCT3/4 and short- hairpin RNA (shRNA) against p53, pCXLE-hSK that expresses SOX2 and KLF4, pCXLE-hUL that expresses L-MYC and LIN28, and pCXLE-hGlis1 that expresses Glis1. R e s u l t s Fifty colonies of human embryonic stem (ES)-like cells were observed as early as 20 days after initial episomal plasmid vector transduction. The resulting cell lines shared several characteristics with human ES cells, includingmorphology,pluripotency-associatedgeneandproteinmarkers, karyotypeanalysisandtheabilitytodifferentiateinvivointoallthreegerm layers. C o n c l u s i o n Our method, combining the use of HOFs and episomal plasmid vectors expressing OCT3/4, shRNA against p53, SOX2, KLF4, L-MYC, LIN28 and Glis1, offers a powerful tool for safely and rapidly generating bona fide humaniPSCsandfacilitatestheapplicationofiPSCtechnologytobiomed- ical research. K e y w o r d s iPSC, integration-free plasmid vector, Glis1, human oral mucosal tissue.