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

背景与目的：为了将胚胎干细胞应用于肝功能衰竭，我们试图促进胚胎干细胞向肝细胞分化。方法：只有肝细胞具有尿素循环和糖异生作用。如果ES细胞在含有尿素循环和糖异生产物的培养液中培养，并添加其底物，预计其他细胞死亡，而从ES细胞分化的肝细胞存活。因此，我们尝试了一种不含精氨酸和葡萄糖，添加鸟氨酸和半乳糖(HSM)的培养液。RT-PCR检测白蛋白和葡萄糖-6-磷酸二酯酶(G6PD)的表达。吲哚青绿(ICG)只被肝细胞摄取。ICG阳性细胞为肝细胞。然后将ICG应用于培养上清液以进一步确认肝细胞。CCCAT/增强子结合蛋白(C/eBP)α和C/eBPβ是肝脏富集型转录因子，具有促进肝细胞分化的作用。它们有望促进ES细胞…的分化更多地进入肝细胞。体外连接构建表达C/eBPα或C/eBPβ的腺病毒载体。Western印迹分析证实腺病毒载体真的表达了C/eBPα或C/eBPβ。结果：与对照组相比，HSM培养的细胞数量减少了3%。RT-PCR结果显示存活细胞表达白蛋白和G6PD。加入ICG后，存活细胞呈ICG阳性，且阳性细胞数明显多于对照组。这些数据表明，ES细胞在HSM中比在对照培养液中更有效地分化为肝细胞。Western印迹结果表明，腺病毒真的表达(C/eBP)α或C/eBPβ。结论：HSM可促进ES细胞向肝细胞分化。构建表达C/eBPα或C/eBPβ的重组腺病毒。我们的下一个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。较少

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