Proliferation and differentiation potential of mouse adult hepatic progenitor cells cultured <italic>in vitro</italic>

Abstract

This study aimed to isolate the stem cells or progenitors, if exist, from normal adult mouse liver and investigate their potential of proliferation and differentiation. Hepatocytes were isolated by modified two-step liver perfusion method and centrifugation, and then cultured in modified serumcontaining DMEM for observation more than 60 days.Immunofluorescence technique was applied to check the hepatocytes and to examine the formation of colonies with albumin, a-fetoprotein (AFP) and cytokeratin 19 (CK19). Results showed that some hepatocytes that were strongly positive for hepatocyte specific markers albumin on Day 1 in culture, could be activated at Days 2-3, followed by rapid proliferation and formation of colonies. The colonies could expand continually for more than 60 days. On Day 5, all the cells in the colony expressed hepatic stem cell (HSC) markers AFP. With the time of culture, some cells in colonies lost ability to divide at Days 13-15, and differentiated into cells which had a large cytoplasm and some two nuclei, similar to the appearance of mature hepatocytes morphologically. These differentiated cells demonstrated strong expression of albumin. Around Day 30, some big cells appeared in colonies and expressed bile duct cell marker CK19. Therefore, this subpopulation of mouse hepatocytes could acquire some characteristics of immature hepatocytes and showed the profile of hepatic progenitor cells with a high proliferating ability and bi-potential of differentiation. They were isolated from normal adult mouse, hence, named adult hepatic progenitor cells (AHPCs). Mouse AHPCs may be used as an HSC model for hepatocytes transplantation and hepatopathy study.

Early activated hepatic stellate cell-derived paracrine molecules modulate acute liver injury and regeneration

“…AHPCs were isolated by the two-step liver perfusion method by Seglen, with some modifications. 15 The basic culture medium consisted of modified low-glucose DMEM containing 10% fetal bovine serum, 10 mM nicotinamide, 1 mM Asc2P, 10 ng/ml epidermal growth factor, insulin-transferring selenium, dexamethasone, streptomycin, and penicillin and was plated on 35-mm culture dishes (BD Falcon, Bedford, MA, USA) coated with rat tail collagen (50 mg of dried tendon/0.1% acetic acid) at a low density of 1 × 10 4 cells/cm 2 . The culture conditions were basic medium (vehicle), basic medium mixed at a 20:1 ratio with the 25-fold concentrated HSC-CM (5% HSC-CM), and at a 5:1 ratio for 20% HSC-CM.…”