Human Hepatic Stellate Cells And Hepatocyte Co-Cultures Maintain Differentiation In Vitro And In Vivo

Lack of long-term cultures of mature human hepatocytes (Hep) that maintain liver-specific functions have hampered investigations of the effects of chronic drug toxicity and liver pathogens. We investigated if matrix-producing hepatic stellate cells (HSC) sustained Hep survival and differentiation in vitro and in vivo. Co-cultures (CC) survived 2 months in vitro, maintained expression of albumin, CYP2E1, CD81, and CK19 mRNAs, began to express α-fetoprotein and secreted α1-antitrypsin and fibrinogen. CC proliferated and required passaging every 3–4 weeks. CC prepared with Hep from newborn subjects expressed and sustained the hepatoblast markers Thy1 and Sca 1 in vitro. Transplanted Hep survived in CC for at least 8 weeks when engrafted in vascularized protein gels on the abdominal wall of SCID/bg mice. EM revealed that Hep ultrastructure and polarization was preserved and groups of epithelial cells formed bile duct-like structures in vivo. Our findings suggest that Hep-HSC CC sustain normal Hep morphology and differentiation. Accordingly, these CC could be used in studies of liver repopulation, hepatitis infections and long-term toxicity of drugs and alcohol. Supported by AGA RSA (MJH), NIH-R01HL085416 (JSP), USUHS MDA905-03-1-0006 (MHS) and NIH-RO1AA10541(MR) grants.