Morphogenetic events in mixed cultures of rat hepatocytes and nonparenchymal cells maintained in biological matrices in the presence of hepatocyte growth factor and epidermal growth factor

被引:100
作者
Michalopoulos, GK [1 ]
Bowen, WC
Zajac, VF
Beer-Stolz, D
Watkins, S
Kostrubsky, V
Strom, SC
机构
[1] Univ Pittsburgh, Sch Med, Dept Pathol, Pittsburgh, PA 15261 USA
[2] Univ Pittsburgh, Sch Med, Dept Cell Biol, Pittsburgh, PA 15261 USA
关键词
D O I
10.1002/hep.510290149
中图分类号
R57 [消化系及腹部疾病];
学科分类号
摘要
Hepatocytes were grown in chemically defined hepatocyte growth medium (HGM) containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF) on collagen-coated polystyrene beads in roller bottle cultures, forming clusters of beads, and proliferating hepatocytes and nonparenchymal cells, including fenestrated endothelium-forming vascular structures. Desmin-positive cells surrounded hepatocytes, Collagen types I and III were deposited in a diffuse manner whereas collagen type IV surrounded the clusters of the epithelial cells, forming a basement membrane. When the mixed cell clusters were implanted in Matrigel (Collaborative Research, Bedford, MA), hepatocytes grew in three dimensions, forming plates and ducts. Many single, long plates of hepatocytes were seen, suggesting progressive linear assembly guided by hepatocyte specific structural parameters. HGF, EGF and transforming growth factor-alpha (TGF-alpha) enhance these phenomena. HGF plus EGF elicited maximal response. TGF-beta 1 suppressed formation of the ducts and plates. Within three months in Matrigel, the cultures established monolayers composed of plates, ducts, and a well-delineated canalicular network. The mixed cultures expressed albumin, A1AT, AFP, transferrin, and CYPIIB1. Following implantation of the cell clusters in Matrigel, there was decreased expression of c-met, urokinase, urokinase receptor, and TGF-beta 1, Electron microscopy showed differentiated hepatocytes with nearly normal ultrastructure, The proliferating cell nuclear antigen (PCNA) labeling index was high (more than 80%) whereas the Bromo-deoxyaridine labeling index of ongoing DNA synthesis varied from 10% to 15%, These results show that the mixed cultures of proliferating hepatocytes and nonparenchymal cells can reproduce the hallmark structures of hepatic histological architecture while maintaining differentiation and the capacity to proliferate.
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页码:90 / 100
页数:11
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