Isolation
and long-term maintenance of hepatic progenitor cells (HPCs) from healthy,
non-injured adult livers remains challenging due to the lack of specific
surface markers for selection and a limited understanding of the mechanisms for
maintaining self-renewal. Previously, we identified a Sca-1 positive, bipotent
HPC population in the peri-portal region of adult liver, and found MAPK/ERK and
Wnt/β-Catenin pathways to be
synergistically involved in their proliferation. In this study, we report the
long-term culture of Sca-1 positive HPCs with epidermal growth factor (EGF) and
CHIR99021, a small molecule inhibitor of glycogen synthase kinase 3 (GSK-3).
Sca-1+ HPCs remain non-tumorigenic when passaged 35 times in vitro over 1 year. Flow cytometric analysis indicates that HPCs
are positive for Sca-1 and putative liver progenitor cell markers, including
CD13, CD24 and Prominin-1, but negative for hematopoietic/endothelial cell markers
CD31, CD34, CD45, CD90 and CD117. Immunocyto-chemistry and RT-PCR indicate
Sca-1+ HPCs express albumin (ALB), α-fetoprotein
(AFP), cytokeratin19 (CK19), Sox9 and a panel of special hepatic progenitor transcriptional
factors. Moreover, Sca-1+ HPCs are able to differentiate into hepatocyte-like
and cholangiocyte-like cells under appropriate culture conditions in vitro and can take part in liver
repopulation in an acetaminophen (APAP) induced liver injury mouse model. This
study provides a paradigm to capture and maintain HPCs from naive liver tissue
and offers a valuable cell model for investigating the molecular mechanisms
underlying the cell lineage relationship in normal liver.