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

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 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. The transcription factor 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 mucosa fibroblasts (HOFs) and episomal plasmid vectors expressing transcription factors including Glis1.Materials and Methods: HOFs were established from 3-mm in diameter oral mucosa tissue using the skin trephine of a 23-year-old Asian man. Human iPSCs were generated from the established HOFs using the following episomal plasmid vectors: pCXLE-hOCT3/4-shp53-F that expresses OCT3/4 and 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 following 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.Conclusions: Our method, combining the use of HOFs and episomal plasmid vectors expressing OCT3/4, SOX2, KLF4, L-MYC, shRNA against p53, 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.

2015

収集根拠 : 博士論文（自動収集）
資料形態 : テキストデータ
コレクション : 国立国会図書館デジタルコレクション > デジタル化資料 > 博士論文
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 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. The transcription factor 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 mucosa fibroblasts (HOFs) and episomal plasmid vectors expressing transcription factors including Glis1.Materials and Methods: HOFs were established from 3-mm in diameter oral mucosa tissue using the skin trephine of a 23-year-old Asian man. Human iPSCs were generated from the established HOFs using the following episomal plasmid vectors: pCXLE-hOCT3/4-shp53-F that expresses OCT3/4 and 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 following 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.Conclusions: Our method, combining the use of HOFs and episomal plasmid vectors expressing OCT3/4, SOX2, KLF4, L-MYC, shRNA against p53, 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.
2015