Activation, isolation, identification and in vitro proliferation of oval cells from adult rat livers

Abstract

Oval cells, putative hepatic stem cells, could potentially provide a novel solution to the severe shortage of donor livers, because of their ability to proliferate and differentiate into functional hepatocytes. We have previously demonstrated that oval cells can be induced to differentiate into cells with morphologic, phenotypic, and functional characteristics of mature hepatocytes. In this study, we have established a new model combining ethionine treatment with partial hepatectomy to activate oval cells, then developed a procedure utilizing selective enzymatic digestion and density gradient centrifugation to isolate and purify such cells from heterogeneous liver cell population. We identified oval cells by their morphological characteristics and phenotypic properties, thereby providing definitive evidence of the presence of hepatic stem-like cells in adult rat livers. Viewed by transmission electron microscopy, they were small cells with ovoid nuclei, a high nucleus/cytoplasm ratio and few organelles, including mitochondria and endoplasmic reticulum. Flow cytometric assay showed that these cells highly expressed OV-6, cytokeratin-19 (CK-19) and albumin. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis displayed that the freshly isolated cells co-expressed albumin, cytokeratin-7 (CK-7) and CK-19 mRNA, indicating that they were essentially bipotential hepatic stem-like cells. Furthermore, we set up a culture system containing growth factors and a fibroblast feeder layer, to provide nourishment to these cells. Thus, we were able to culture them in vitro for more than 3 months, with the number of cells doubling 100 times. Gene expressions of albumin, CK-7 and CK-19 in the cells derived from the expanding colonies at day 95 were confirmed by RT-PCR analysis. These data suggested that the hepatic oval cells derived from adult rat livers possess a high potential to proliferate in vitro with a large increase in number, while maintaining the bipotential nature of hepatic stem cells.

Characterization of two distinct liver progenitor cell subpopulations of hematopoietic and hepatic origins

“…Independent of their origin, hepatic progenitor cells have been reported to be isolated, propagated and differentiated into hepatic-and pancreatic-islet-like cells in culture [28][29][30][31], thus providing an attractive renewable source of stem cells for cellular therapy. The goal of the current study was to further characterize the potential origin of hepatic progenitor cells in 2-AAF/CCl 4 -treated rat livers and to subsequently isolate and establish a stable oval cell line.…”

Downregulation of Multiple Stress Defense Mechanisms During Differentiation of Human Embryonic Stem Cells

“…H1 and hES-NCL1 lines were cultured and differentiated for 4,8,12,16, and 20 days as described above. To prepare lysates of undifferentiated cells, hESC were detached with collagenase IV (1 mg/ml), collected in a 1.5-ml tube (Eppendorf AG, Hamburg, Germany, http://www.…”

“…There are low numbers of mitochondria in liver and hematopoietic stem cells, which also show low oxygen consumption [12,13]. Human ESC (hESC) also contain very few mitochondria [14].…”

Bone Marrow-Derived Mesenchymal Stem Cells Promote Hepatic Regeneration after Partial Hepatectomy in Rats

“…In these situations, liver regeneration is triggered rapidly to restore hepatic function, and hepatic stem cells play an important role in regeneration [1]. Hepatic stem cells in liver tissues have high proliferation potential in vitro and can be induced to differentiate into hepatocytes [2,3]. However, hepatocyte proliferation may be inhibited if the injury is too severe [4], and liver regeneration is often compromised after surgery for malignancies [5,6].…”