Mouse Hepatic Oval Cells

HOCs Morphological Features in the Original Generation and Differentiation into BDEs

Hepatic oval cells (HOCs) can differentiate into cholangiocytes during liver regeneration, but the epigenetic mechanisms governing this transition remain unclear. Jin's team investigated whether histone methyltransferase SETD2 and its product H3K36me3 regulate HOC-to-cholangiocyte differentiation following partial hepatectomy and 2-acetamidofluorene exposure in mice.

A modified two-step enzyme digestion method established hepatic oval cell (HOC) proliferation in a mouse 2-AAF/PH model. Isolated HOCs were small, oval cells with high nuclear/cytoplasmic ratios (Fig. 1A). After 3 days in cytokine-containing medium, first-generation colonies showed rapid growth with gradually enlarging nuclei (Fig. 1B, C). By passages 2-3, cells became irregular-shaped with large volume and prominent nuclei (Fig. 1D, E). Passages 4-5 showed bile duct epithelial (BDE) differentiation with enlarged cell mass and volume (Fig. 1F, G). Passages 6-8 exhibited bile duct epithelium changes with pseudopodia-like growth and three-dimensional structure without bottom layering (Fig. 1H-J).

They investigated SETD2/H3K36me3 expression changes during HOC differentiation into BDEs. Although SETD2/H3K36me3 roles in cell differentiation are established in other stem cells, their dynamics in this process were previously unexplored. HOCs were induced to differentiate into BDEs using EGF (20 µg/l) + SCF (10 µg/l) + LIF (10 µg/l). Passages 5 and 8, distinguishable by morphology, were selected for analysis. Immunofluorescence showed SETD2 expressed in both nucleus and cytoplasm with significant increase from primary generation onward, while H3K36me3 was mainly nuclear and gradually increased during differentiation (Fig. 2Aa-d, Ba-d). Confocal microscopy of passage 3 BDEs confirmed SETD2 in nucleus/cytoplasm and H3K36me3 predominantly nuclear (Fig. 2Ca, b). RT-qPCR revealed SETD2 mRNA gradually increased and stabilized during differentiation (Fig. 2D). Western blotting showed SETD2 and H3K36me3 protein levels increased ~35% in passage 3 BDEs versus primary HOCs, and ~75% in passages 5 and 8 mature BDEs, thereafter stabilizing (Fig. 2E, F).