Differentiation of embryonic stem cells into hepatocytes by transcription factors that promote hepatocyte differentiation

通过促进肝细胞分化的转录因子将胚胎干细胞分化为肝细胞

• 批准号: 16590577
• 负责人: TOMIZAWA Minoru
• 金额: $ 2.3万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590577/
• 关键词: embryonic stem cells; liver failure; hepatoctyes; urea cycle; glyconeogenesis; indocyanin green; C; EBP α; EBP β; embryonic stem cell; アルギニン; オルニチン; グリセロール

Background & Aims : To apply embryonic stem (ES) cells into liver failure, we tried to promote ES cells to differentiate into hepatoccytes. Methods : Only hepatocytes have urea cycle and gluconeogenesis. It was expected that other cells died while hepatocytes differentiated from ES cells survived if ES cells were cultured in the medium with the products of the urea cycle and gluconeogenesis, and added with their substrates. We, thus, tried to invest a medium without arginine and glucose, added with ornithine and galactose (HSM). RT-PCR was performed to detect the expression of albumin and glucose-6-phospodiesterase (G6PD). Indocyanin green (ICG) is taken up by exclusively hepatocytes. The cells positive for ICG are hepatocytes. ICG was, then, applied to the medium to further confirm hepatocytes. CCCAT/enhancer binding protein (C/EBP) α and C/EBP β are liver enriched transcription factors, and promote hepatocyte differentiation. They were expected to promote differentiation of ES cells … More into hepatocytes. Adenovirus vector expressing C/EBP α or C/EBP β was constructed with in vitro ligation. Western blot was performed to confirm that the adenovirus vectors really expressed C/EBP α or C/EBP β. Results : The number of cells cultivated in HSM decreased to 3% compared to that of cells in control medium. RT-PCR revealed that the surviving cells expressed albumin and G6PD. When added with ICG, the surviving cells were positive with ICG, and the number of positive cells was larger than that of cells in control medium. These data indicated that ES cells differentiated into hepatocytes in HSM more effectively than in control medium. Western blot revealed that the adenoviruses really expressed (C/EBP) α or C/EBP β. Conclusion : HSM promoted the differentiation of ES cells into hepatocytes. The adenoviruses were constructed to express C/EBP α or C/EBP β. Our next experiment was to infect ES cells with the adenoviruses to promote hepatocyte differentiation of ES cells more effectively. Less

背景与目的：为了将胚胎干细胞（ES细胞）应用于肝衰竭的治疗，我们尝试促进ES细胞向肝细胞分化。方法：只有肝细胞具有尿素循环和尿素合成功能。如果将ES细胞培养在含有尿素循环和胚胎异生产物的培养基中，并加入它们的底物，则预期其他细胞死亡，而从ES细胞分化的肝细胞存活。因此，我们试图投资一种培养基，没有精氨酸和葡萄糖，添加了鸟氨酸和半乳糖（HSM）。RT-PCR检测白蛋白和葡萄糖-6-磷酸二酯酶（G6 PD）的表达。吲哚菁绿色（ICG）仅由肝细胞摄取。ICG阳性细胞为肝细胞。然后，将ICG应用于培养基以进一步确认肝细胞。CCCAT/增强子结合蛋白（C/EBP）α和C/EBP β是肝脏富集的转录因子，促进肝细胞分化。预期它们能促进ES细胞的分化 ...更多信息 转化成肝细胞体外连接法构建C/EBP α和C/EBP β的腺病毒载体。Western blot检测腺病毒载体是否真的表达C/EBP α或C/EBP β。结果：与对照培养基相比，在HSM中培养的细胞数量减少至3%。RT-PCR显示存活细胞表达白蛋白和G6 PD。加入ICG后，存活细胞呈ICG阳性，阳性细胞数明显多于对照组。这些数据表明，ES细胞分化成肝细胞在HSM比在对照培养基中更有效。Western blot结果显示腺病毒真核表达（C/EBP）α或C/EBP β。结论：HSM可促进ES细胞向肝细胞分化。构建表达C/EBP α或C/EBP β的腺病毒。我们的下一步实验是用腺病毒感染ES细胞，以更有效地促进ES细胞的肝细胞分化。少