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Journal of Oral Science & Rehabilitation Volume 2 | Issue 2/2016 27 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 Introduction The successful reprogramming of human and mouse somatic cells into induced pluripotent stem cells (iPSCs) via ectopic overexpression of pluripotency-associated transcription factors is considered a major scientific breakthrough.1–5 Similar to the characteristics of embryonic stem (ES) cells,6–8 human iPSCs can proliferate indef- initely, while retaining pluripotency, and can dif- ferentiate into all cell types found in the body. IPSCs have been generated from dermal fibro- blasts,3 peripheral blood,9 dental pulp cells,10 gingivalfibroblasts,11 periodontalligaments,12 oral mucosa13 and mesenchymal stromal cells.14 Gingival tissue is routinely resected during general dentaltreatments, such astooth extrac- tion, periodontal surgery and dental implanta- tion, and generallytreated as biomedicalwaste.15 Egusa et al. successfully derived iPSCs from human gingival fibroblasts (HGFs) using retro- viral transduction of transcription factors; they also reportedthatthe reprogramming efficiency of mouse gingival fibroblasts was higher than that of dermal fibroblasts.11 However, retroviral integration increases the risk of tumor forma- tion, while integration-free methods decrease this potential risk.15 The development of novel approachesto generating integration-free iPSCs has eliminated the concern of integrating virus-associated genotoxicityin clinical applica- tions.16 Integration-free human iPSCs have been generated using several methods.15 Okita et al. reported a simple method that uses p53 sup- pression and nontransforming L-MYC to gene- rate human iPSCs with episomal plasmid vec- tors.15 Ourrecent studydemonstratedthat iPSCs could be generated from a combination of pri- mary HGFs and an episomal plasmid vector.17 However, the lengthy reprogramming process (up to one month) remains one of the most sig- nificant challenges facing standard virus- mediated methodology. Maekawa et al. reported that the Gli-like transcriptionfactorGlis1(Glisfamilyzincfinger1) markedlyenhancesthe generation ofiPSCsfrom both mouse and human somatic fibroblasts when it is expressed together with three tran- scription factors collectively known as OSK (OCT3/4, SOX2 and KLF4) using retroviraltrans- duction.18 However, little is known regarding whether Glis1 can effectively promote direct reprogramming during iPSC generation using an episomalplasmidvector. Inthe current study, iPSCs were generated by combining primary human oral mucosal fibroblasts (HOFs) with episomal plasmid vectors expressing OCT3/4, short-hairpin RNA (shRNA) against p53, SOX2, KLF4, L-MYC, LIN28 and Glis1. Materials and methods E t h i c a l s t a t e m e n t Approvalforthe sampling ofhuman oralmucosa tissue, establishing iPSCs and genome/gene analysiswasobtainedfromtheEthicsCommittee of Osaka Dental University, Hirakata, Japan (au- thorization No.: 110783; approval date: 30 Sep- tember 2013) and the DNA Recombination Ex- periment Safety Committee of Osaka Dental University (authorization No.: 54; approval date: 18 July 2014). Written informed consent was obtainedfromthe participant.The animalexper- iments followed a protocol approved by the An- imal Committee of Osaka Dental University (au- thorization No.: 14-06002; approvaldate: 8July 2014). C e l l c u l t u r i n g HOFs were established from oral mucosaltissue 3mm in diameter obtained using a skin trephine (derma punch, Maruho, Osaka, Japan) from a 23-year-old Asian male. Human oral mucosal tissuewas placed in 35 mmtissue culture dishes andculturedinDulbecco’sModificationofEagle’s Medium (DMEM) containing 10% fetal bovine serum at 37°C and 5% CO2.11 The medium was replaced every three days. Once the HOFs had proliferated, the tissue was removed. When the cells reached subconfluence, they were dissoci- ated using 0.25% trypsin (Invitrogen, Carlsbad, Calif., U.S.) and transferred to 60mm tissue cul- ture dishes (passage 1). HOFs were regularly passaged at a 1:3 ratio every three to four days. G e n e r a t i o n o f i P S C s f r o m H O F s w i t h e p i s o m a l v e c t o r s One microgram of an expression episomal plas- mid mixture containing pCXLE-hOCT3/4- shp53-F that expresses OCT3/4 and shRNA against p53, pCXLE-hSK that expresses SOX2 and KLF4, pCXLE-hULthat expresses L-MYC and LIN28, and pCXLE-hGlis1 that expresses Glis1 (Addgene, Cambridge, Mass., U.S.) was electro- porated into 6×105 primary HOFs (passage 5) with the Amaxa 4D-Nucleofector (Lonza, Basel, Volume 2 | Issue 2/201627