Tomizawa Minoru, Shinozaki Fuminobu, Motoyoshi Yasufumi, Sugiyama Takao, Yamamoto Shigenori, Ishige Naoki
Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284‑0003, Japan.
Department of Radiology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284‑0003, Japan.
Mol Med Rep. 2017 May;15(5):3088-3092. doi: 10.3892/mmr.2017.6406. Epub 2017 Mar 28.
William's E (WE) is a suitable medium for the differentiation of human induced pluripotent stem (iPS) cells to the hepatocyte lineage. The aim of the present study was to investigate various growth factors in their ability to promote hepatocyte differentiation of iPS cells in WE medium. Human iPS 201B7 cells were cultured in WE medium supplemented with growth factors, and mRNA expression levels and promoter activities of α‑fetoprotein (AFP) and albumin were examined by reverse transcription‑quantitative polymerase chain reaction and luciferase assay, respectively. In addition, time course analysis of AFP mRNA expression was performed in 201B7 cells cultured in WE medium supplemented with oncostatin M. The results demonstrated that mRNA expression levels of AFP were significantly elevated by most growth factors tested as supplements in WE medium, except all‑trans retinoic acid, compared with cells cultured in ReproFF (a medium that maintains pluripotency). The highest increase in AFP mRNA expression levels was observed by oncostatin M stimulation. Albumin mRNA expression levels were increased by all‑trans retinoic acid and insulin‑transferrin‑selenium supplementation in WE medium compared with cells cultured in ReproFF. Oncostatin M supplementation significantly stimulated the promoter activity of the AFP gene, but no growth factor tested significantly stimulated the promoter activity of the albumin gene. By time course analysis, significant increase of AFP mRNA expression was observed on the sixth day post‑stimulation, compared with cells cultured in WE medium alone. In conclusion, the present study demonstrated that oncostatin M supplementation in WE medium was sufficient to initiate hepatocyte differentiation in iPS cells.
威廉姆斯E(WE)培养基是一种适合将人诱导多能干细胞(iPS细胞)分化为肝细胞谱系的培养基。本研究的目的是探究各种生长因子在促进iPS细胞在WE培养基中向肝细胞分化方面的能力。将人iPS 201B7细胞培养于添加了生长因子的WE培养基中,分别通过逆转录定量聚合酶链反应和荧光素酶测定法检测甲胎蛋白(AFP)和白蛋白的mRNA表达水平及启动子活性。此外还对在添加了制瘤素M 的WE培养基中培养的201B7细胞进行了AFP mRNA表达的时间进程分析。结果表明,与在ReproFF(一种维持多能性的培养基)中培养的细胞相比,在WE培养基中作为补充剂测试的大多数生长因子(全反式维甲酸除外)均能显著提高AFP的mRNA表达水平。制瘤素M刺激导致AFP mRNA表达水平升高最为显著。与在ReproFF中培养的细胞相比,在WE培养基中添加全反式维甲酸和胰岛素-转铁蛋白-硒可提高白蛋白mRNA表达水平。添加制瘤素M能显著刺激AFP基因的启动子活性,但所测试的生长因子均未显著刺激白蛋白基因的启动子活性。通过时间进程分析,与仅在WE培养基中培养的细胞相比,刺激后第6天观察到AFP mRNA表达显著增加。总之,本研究表明在WE培养基中添加制瘤素M足以启动iPS细胞向肝细胞的分化。