Dental News - Accelerated generation of human induced pluripotent stem cells from human oral mucosa using episomal plasmid vectors and maternal transcription factor Glis1

Dr. Takahiro Kashiwagi et al.

Thu. 25. August 2016

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Objective: Induced pluripotent stem cells (iPSCs) possess high pluripotency and differentiation potential and may constitute a possible source of autologous stem cells for clinical applications. However, the lengthy reprogramming process (up to one month) remains one of the 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 primary human oral mucosal fibroblasts (HOFs) and episomal plasmid vectors expressing transcription factors, including Glis1.

Materials and methods: HOFs were established from oral mucosal tissue 3 mm in diameter from a 23-year-old Asian male using a skin trephine. Human iPSCs were generated 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.

Results: 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, including morphology, pluripotency-associated gene and protein markers, karyotype analysis and the ability to differentiate in vivo into all three germ layers.

Conclusion: 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 human iPSCs and facilitates the application of iPSC technology to biomedical research.

Editorial note: The full article was published in the 2/2016 issue of the Journal of Oral Science and Rehabilitation. Access the full article at www.dtscience.com.

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