Noguchi Hirofumi, Miyagi-Shiohira Chika, Nakashima Yoshiki, Kinjo Takao, Kobayashi Naoya, Saitoh Issei, Watanabe Masami, Shapiro A M James, Kin Tatsuya
Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
Department of Basic Laboratory Sciences, School of Health Sciences in Faculty of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
Mol Ther Methods Clin Dev. 2019 Jan 29;13:243-252. doi: 10.1016/j.omtm.2019.01.011. eCollection 2019 Jun 14.
We recently demonstrated the generation of mouse induced tissue-specific stem (iTS) cells through transient overexpression of reprogramming factors combined with tissue-specific selection. Here we induced expandable tissue-specific progenitor (iTP) cells from human pancreatic tissue through transient expression of genes encoding the reprogramming factors OCT4 (octamer-binding transcription factor 4), p53 small hairpin RNA (shRNA), SOX2 (sex-determining region Y-box 2), KLF4 (Kruppel-like factor 4), L-MYC, and LIN28. Transfection of episomal plasmid vectors into human pancreatic tissue efficiently generated iTP cells expressing genetic markers of endoderm and pancreatic progenitors. The iTP cells differentiated into insulin-producing cells more efficiently than human induced pluripotent stem cells (iPSCs). iTP cells continued to proliferate faster than pancreatic tissue cells until days 100-120 (passages 15-20). iTP cells subcutaneously inoculated into immunodeficient mice did not form teratomas. Genomic bisulfite nucleotide sequence analysis demonstrated that the and promoters remained partially methylated in iTP cells. We compared the global gene expression profiles of iPSCs, iTP cells, and pancreatic cells (islets >80%). Microarray analyses revealed that the gene expression profiles of iTP cells were similar, but not identical, to those of iPSCs but different from those of pancreatic cells. The generation of human iTP cells may have important implications for the clinical application of stem/progenitor cells.
我们最近通过重编程因子的瞬时过表达结合组织特异性选择，证明了小鼠诱导组织特异性干细胞（iTS）的产生。在此，我们通过瞬时表达编码重编程因子OCT4（八聚体结合转录因子4）、p53小发夹RNA（shRNA）、SOX2（性别决定区Y框2）、KLF4（Kruppel样因子4）、L-MYC和LIN28的基因，从人胰腺组织中诱导出可扩增的组织特异性祖细胞（iTP）。将游离质粒载体转染到人胰腺组织中可有效产生表达内胚层和胰腺祖细胞遗传标记的iTP细胞。iTP细胞比人诱导多能干细胞（iPSC）更有效地分化为胰岛素分泌细胞。直到第100 - 120天（第15 - 20代），iTP细胞的增殖速度持续快于胰腺组织细胞。皮下接种到免疫缺陷小鼠体内的iTP细胞未形成畸胎瘤。基因组亚硫酸氢盐核苷酸序列分析表明，iTP细胞中的 和 启动子仍部分甲基化。我们比较了iPSC、iTP细胞和胰腺细胞（胰岛>80%）的整体基因表达谱。微阵列分析显示，iTP细胞的基因表达谱与iPSC相似但不完全相同，与胰腺细胞不同。人iTP细胞的产生可能对干细胞/祖细胞的临床应用具有重要意义。
