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

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 16590577
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Gastroenterology
• Research Institution: Chiba University
• Principal Investigator: TOMIZAWA Minoru Chiba University, Chiba University Hospital, Assistant, 医学部附属病院, 助手 (90334193)
• Co-Investigator(Kenkyū-buntansha): NAKAGAWARA Akira Chiba University, Chiba Cancer Center Research Institute, Head, 研究局, 研究局長 (50117181)
• Project Period (FY): 2004 – 2005
• Keywords: embryonic stem cells / liver failure / hepatoctyes / urea cycle / glyconeogenesis / indocyanin green / C / EBP α / C / EBP β

Research Abstract

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 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.