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Establishment, characterization, and long‐term maintenance of cultures of human fetal hepatocytes
Author(s) -
Lázaro Catherine A.,
Croager Emma J.,
Mitchell Claudia,
Campbell Jean S.,
Yu Changpu,
Foraker Jessica,
Rhim Jonathan A.,
Yeoh George C. T.,
Fausto Nelson
Publication year - 2003
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1053/jhep.2003.50448
Subject(s) - cd90 , biology , stem cell , cd34 , cell culture , population , hepatocyte , microbiology and biotechnology , cytokeratin , lineage markers , immunology , progenitor cell , in vitro , biochemistry , immunohistochemistry , genetics , demography , sociology
Abstract Cultured human hepatocytes have broad research and clinical applications; however, the difficulties in culturing rodent and human hepatocytes are well known. These problems include the rapid loss of the hepatocytic phenotype in primary culture and the limited replicating capacity of the cultured cells. We describe the establishment of serum‐free primary cultures of human fetal hepatocytes (HFHs) that retain hepatocytic morphology and gene expression patterns for several months and maintain sufficient proliferative activity to permit subculturing for at least 2 passages. Initially, HFH cultures contained 2 main cell types that morphologically resembled large and small hepatocytes. The fetal hepatocytes expressed α‐fetoprotein (AFP), cytokeratin (CK) 19, albumin, and other hepatic proteins. Treatment of the cultures with oncostatin M (OSM) increased cell size and enhanced cell differentiation and formation of bile canaliculi, probably through an effect on hepatocyte nuclear factor (HNF) 4α. Approximately 1 month after plating, multiple clusters of very small cells became apparent in the cultures. These cells had very few organelles and are referred to as blast‐like cells. Flow cytometric analysis of these cells showed that they express oval cell/stem cell markers such as CD90 (Thy‐1), CD34, and OV‐6 but do not stain with antibodies to β 2 ‐microglobulin. HFH cultures maintained for 9 to 12 months produced grossly visible organoids containing ductular structures that stained for CK18, CK19, and AFP. In conclusion, HFH cultures, which might contain a population of hepatic stem cells, constitute an excellent tool for a variety of studies with human hepatocytes, including the mechanisms of viral infection.