Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells

Abstract

Landry et al. 1 first showed the reconstruction of a threedimensional cyto-architecture consisting of differentiated hepatocytes, bile duct-like cells, and deposited extracellular matrix (ECM) by the use of spheroidal aggregate culture of hepatic cells isolated from newborn rats. Thereafter, some attempts have been made to grow a hepatic organoid by the coculture of hepatocytes and fibroblasts. 2,3 As neither fibroblasts derived from the liver nor hepatocytes could proliferate, the size of the cell aggregates was limited and the position of the cells was disorderly, although differentiated functions were well maintained for a long time. To develop a hepatic organoid, the interactions not only between hepatocytes and nonparenchymal cells (NPCs) but also between ECM and hepatocytes must be well coordinated as many differentiated functions of mature hepatocytes (MHs), through which the homeostasis of life is sustained, should be maintained.We have reported that the proliferation of adult rat hepatocytes is observed in serum-free medium supplemented with 10 mmol/L nicotinamide and epidermal growth factor (EGF). [4][5][6][7][8] Other laboratories also reported that supplementation of the medium with nicotinamide and EGF stimulates the growth of the primary rat hepatocytes. 9,10 Most hepatocytes cultured in nicotinamide-supplemented medium can divide, and small hepatocytes (SHs), which are less than half as large as but are morphologically similar to MHs, proliferate so fast that the cells form colonies after 4 to 5 days of culture. They have been shown immunocytochemically and ultrastructurally to possess hepatic characteristics. 6 Recently, it was shown by our 11 and other laboratories 12 that a single SH can clonally proliferate and form a large colony consisting of more than 30 cells at day 10. The cells can grow and survive for more than 5 months. Thus, we previously proposed that rat hepatocytes may be classified into three types of cells with respect to their ability to divide: (1) cells that have a high potential to proliferate and form colonies in primary culture (type I cells; SHs), (2) cells for which the number of possible cell divisions is limited (type II cells), and (3) cells that lose the ability to divide (type III cells). 7,8 In addition, type II and type III cells may possess fully differentiated functions. Therefore, in this report we call both type II and type III cells MHs. Furthermore, we call the cell islands, which consist of SHs themselves and of a mixture of SHs and MHs, colonies.In the present experiment we showed that SHs could differentiate to MHs that interacted with hepatic NPCs and

Loss of MMP 13 attenuates murine hepatic injury and fibrosis during cholestasis

“…It is recognized that all cellular elements and supporting structures must be reconstituted to maintain the differentiated function of the liver. 43 Therefore, it is likely that degradation of the ECM by MMPs alters cell-matrix and cell-cell interactions and enhances hepatocyte susceptibility to necrosis and/or apoptosis caused by cholestasis. Loss of MMP-13 possibly attenuated this process, which is fundamental for the initiation of fibrotic repair post-liver injury in the BDL model.…”

Selective proliferation of rat hepatocyte progenitor cells in serum-free culture

“…Although the cells are less than half the size of mature hepatocytes (MHs), they possess hepatic characteristics 1 . SHs can clonally proliferate to form a colony and can differentiate into MHs by interacting with hepatic non-parenchymal cells (NPCs) 2,3 or as a result of treatment with gel derived from Engelbreth-Holm-Swarm sarcoma 4 . Thus, we consider that SHs may be 'committed progenitor cells' that can further differentiate into MHs.…”

“…013-12061) (see REAGENT SETUP) . BSA 2 O into a 1,000-ml graduated cylinder; add 100 ml 10Â Ca 2+ , Mg 2+ -free HANKS, 190 mg EGTA, 1 ml 1,000Â insulin stock solution and stir and adjust pH to 7.5 with 7 ml 1 M NaHCO 3 . Adjust to 1,000 ml and filter with a 0.2-mm filter.…”

Thermoreversible gelation polymer induces the emergence of hepatic stem cells in the partially injured rat liver

“…8 The procedure used for transmission electron microscopy (TEM) was previously described. 9 Immunostaining. Antibodies to c-Kit (Santa Cruz, CA), Thy1 (Pharmingen, Hamburg, Germany), ␣-fetoprotein (AFP; Dako Cytomation, Kyoto, Japan), albumin (Dako Cytomation), cytokeratin 19 (CK19; Novocastra Laboratories, Newcastle, UK and a gift of Dr. A. Miyajima, Tokyo University, Japan), proliferating cell nuclear antigen (PCNA; Dako Cytomation), Ki67 (Pharmingen), CCAAT/enhancer binding protein ␣ (C/EBP ␣; Santa Cruz), and multidrug-resistance associated protein 2 (MRP2; Alexis Biochemichals, San Diego, CA) were used.…”

“…The methods used for immunohistochemistry and immunocytochemistry were previously described. 9 3, 3Ј-Diaminobenzidine and BCIP/NBT (Dako Cytomation) were used as a substrate for colorization. Alexa 488 -conjugated and Alexa 594 -conjugated antibodies (Molecular Probes, Eugene, OR) were used as secondary antibodies.…”