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ePaper created 2016-06-09, 15:16:05 | version 1.38.0
Journal of Oral Science & Rehabilitation 30 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 K a r y o t y p e a n a l y s i s Chromosome G-band analysiswas performed at Nihon Gene Research Laboratories (Sendai, Japan). At least 15 metaphases were analyzed. Results 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 u s i n g e p i s o m a l p l a s m i d v e c t o r s Three lines of HOFs were established from the oral mucosa of the 23-year-old Asian male (Fig.1). Homogeneousfibroblasts emergedfrom the oral mucosal tissue one week after the start of culturing. HOFs were exponentially expanded up to 30 passages; cells were counted at each passage and plated at 1.5×104 cells/cm2 . Colo- nies with a flat human ESC-like morphology and non-ESC-like colonies were counted at around day 20 after HOF transfection with episomal plasmid vectors expressing human OCT3/4, shRNA against p53, SOX2, KLF4, L-MYC, LIN28 and Glis1. The average number of ESC-like colo- nies from three experiments was 54.7 ± 3.05, withareprogrammingefficiencyofapproximately 1%;theaveragenumberofnon-ESC-likecolonies was 25.3±3.21 (Table 3). A number of colonies obtained from the HOF cells were mechanically picked at passage 1. Severaldays later,fourESC- like colonies were selected and expanded. All colonies were similar to ESCs in morphology and proliferative capacity and were named “HOF-iPSCs”. E x p r e s s i o n o f E S C - s p e c i f i c m a r k e r g e n e s i n H O F - i P S C s HOF-iPSCs were selected for characterization from amongthe picked clones after23 passages based on their higher level of proliferation and stabilityofthe ESC-like morphology.The expres- sion of the ESC-specific marker genes OCT3/4, NANOG, SOX2, TERT, KLF4 and C-MYC in HOF-iPSCswasanalyzedusingqRT-PCR(Fig.3). Expression ofNANOG and SOX2was significant- ly higher and that of C-MYC and TERT was lower inKhES-1cellscomparedwiththatinHOF-iPSCs (Figs. 3b–e). No significant difference was ob- served between KhES-1 and HOF-iPSCs for OCT3/4 and KLF4 expression (Figs.3a&f). KLF4 was the onlygene to exhibit higher expression in HOF cells compared with both the KhES-1 cells and HOF-iPSCs (Fig. 3f). C h a r a c t e r i z a t i o n o f H O F - i P S C s HOF-iPSCs were selected for characterization from amongthe picked clones after20 passages Table 3 ESC-like colonies obtained from HOFs. The number of colonies per 5×104 cells after cell reprogramming with episomal vectors. These data were obtained from three independent induction experiments using HOFs from a donor. Fig. 1 Excision of oral mucosal tissue by punch biopsy. Table 3 Fig. 1 ES–like Non-ES–like HOFs 54 23 52 24 58 29 5423 5224 5829