Isolation and Purification Method of Mouse Fetal Hepatoblasts
During development, liver precursors constitute a valuable source of pluripotent stem cells that present the ability to differentiate into both a hepatic and biliary lineage. In the present chapter, we report an experimental procedure developed by our group to isolate mouse fetal hepatoblasts (MFHs) with high purity. The method is based on a selective harvesting of the hepatic parenchymal cells from fetuses (E 14.5), followed by the sorting of E-cadherin+ progenitors through the use of magnetic beads and specific antibodies. This protocol allows the isolation of bipotent liver stem cells expressing both hepatic and biliary markers. Primary cultures of purified MFHs can be maintained under proliferation until confluence, leading to promotion of the differentiation process in the presence of hepatotrophic factors. By using a quantitative real-time polymerase chain reaction approach, we show the hepatospecific phenotype and the progressive maturation of MFHs, delineating early (α-fetoprotein), mid (albumin), and late (glucose-6-phosphatase) hepatic markers. Consequently, the model appears to be a valuable cell system for the study of molecular and cellular aspects occurring in hepatic differentiation.
Key wordsMouse fetal hepatoblasts MFHs E-cadherin Cell sorting Bipotent stem cells Hepatic differentiation
The author thanks Ayako Inoue for her efficient technical assistance, Takahiro Ochiya for his constructive comments on the study, and Takahashi Ryou-u for his advice regarding the cell sorting procedure. This work was supported by a Grant-in-Aid from the Third-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour, Welfare of Japan (H21-001).