Activation, isolation, identification and culture of hepatic stem cells from porcine liver tissues

Utility of hepatic stem cells could provide a novel solution to the severe shortage of human donor livers, for treatment of liver-related diseases, due to their ability to proliferate and differentiate into functional hepatocytes. Porcine liver tissues also offer an alternative source from human donor livers. However, morphology, phenotype, successful isolation and culture of porcine hepatic stem cells still require much investigation.

In the present study, we performed partial hepatectomy to activate hepatic oval cells and developed a procedure utilizing enzymatic digestion and density gradient centrifugation to isolate and purify oval cells derived from porcine livers. We identified ovoid cells by their morphological characteristics and phenotypic properties, thereby providing definitive evidence for the presence of hepatic stem cells in porcine livers. Moreover, we established a culture system, using various growth factors, to provide nourishment for these cells.

By transmission electron microscopy, oval-shaped cells with ovoid nuclei, a high nucleus/cytoplasm ratio and few organelles were demonstrated. Flow cytometry and immunocytochemistry showed that freshly isolated oval cells expressed albumin, cytokeratin 19, alpha fetoprotein (AFP) and OV6 at high levels. Immunofluorescence revealed that porcine hepatic oval cells after culture expressed stem-cell factor, c-kit, Thy-1, CK19, OV6, and AFP. Taken together, this study provides a novel insight into morphological and phenotypic characteristics of porcine hepatic stem cells. Our ability for isolation and culturing porcine hepatic stem cells offers an abundant source of cells for transplantation and tissue engineering to help alleviate liver disease.