Histological Organization in Hepatocyte Organoid Cultures

Abstract

Hepatocytes and other cellular elements isolated by collagenase perfusion of the liver and maintained in defined culture conditions undergo a series of complex changes, including apoptosis and cell proliferation, to reconstruct tissue with specific architecture. Cultures in collagen-coated pleated surface roller bottles, with hepatocyte growth medium medium and in the presence of hepatocyte growth factor (HGF) and epidermal growth factor (EGF), form characteristic and reproducible tissue architecture composed of a superficial layer of biliary epithelial cells, an intermediate layer of connective tissue and hepatocytes, and a basal layer of endothelial cells. Dexamethasone, EGF, and HGF are required for the complete histological organization. Analysis of the structures formed demonstrates that the receptor tyrosine kinase ligands HGF and EGF are required for the presence, growth, and phenotypic maturation of the biliary epithelium on the surface of the cultures and for the formation of connective tissue in the cultures. Dexamethasone, in the presence of HGF and EGF, was required for the phenotypic maturation of hepatocytes. The results demonstrate the role of these molecules for the formation and phenotypic maturation of specific histological elements of the liver and suggest roles for these signaling molecules in the formation and structure of the in vivo hepatic architecture.

A mouse model for cystic biliary dysgenesis in autosomal recessive polycystic kidney disease (ARPKD)

“…Consistently, it has been shown that the contact to mesenchyme is essential for differentiation into biliary epithelium of isolated liver cells in vitro. 16 Analyzing Pkhd1ex40 mice showed that the failure of embryonic ductal plates to undergo architectural changes after successful delineation from the periportal hepatocytes retains the cholangiocytes in a proliferative and TGF-␤1-immunoreactive state. It appears that embryonic cholangioblasts in Pkhd1ex40 mice continuously stimulate mesenchymal cells to synthesize and accumulate excessive matrix.…”

“…For reverse transcription, 1 g total RNA was primed with 1 g oligo d(T) [12][13][14][15][16][17][18] , and transcription was performed by Superscript II RT following the manufacturer's description (Invitrogen). PCR was run with 35 cycles (1 minute at 94°C, 45 seconds at 53°C to 55°C, and 1 to 2 minutes at 72°C).…”

Embryonic development of the liver

“…Advances in organ culture systems and the availability of cultured hepatic progenitor cells have also been invaluable. 72,[80][81][82][83][84] During the mid-gestation stages of development, the hepatoblast population must expand to define the total volume of the liver. Studies in rat embryos have shown that between E13.5 and E20.5, the volume of the liver expands 84-fold, during which time the hepatoblasts undergo 8-doublings.…”

“…21 Consistent with the proposal that extracellular matrix is a significant determinant of hepatoblast cell fate, it has been demonstrated that the profile of gene expression within primary hepatic cultures is significantly affected by the composition of extracellular matrix. 79,80,118,[120][121][122][123] For example, culture of bipotential mouse embryonic liver cells in matrigel, a soluble basement membrane preparation that includes laminin, collagen IV, heparan sulfate proteoglycans, and entactin, induced them to express biliary epithelial cell markers and form ductile like structures. 83,124 In addition, cells lacking ␤1-integrin, a subunit of a heterodimeric protein complex that contributes to cell-matrix interactions, are unable to colonize the liver, and inhibition of the ␣3-integrin subunit expression blocks extracellular matrixinduced differentiation of a hepatic cell line.…”

Present and Future Developments in Hepatic Tissue Engineering for Liver Support Systems

“…Hetero-spheroids of hepatocytes and non-parenchymal hepatic cells on a synthetic polymer were shown to further enhance long-term liver functions (Yamada et al 2001). Moreover, Michalopoulos et al have suggested using the hepatocyte organoid (composed of proliferating hepatocytes and non-parenchymal hepatic cells) culture technique that a combination of certain cytokines, such as hepatocyte growth factor (HGF) and epidermal growth factor (EGF), and dexamethasone plays a role in the formation and structure of the in vivo-like architecture of hepatocytes (Michalopoulos et al 2001). Although hepatocytes in spheroids possess improved liverspecific functions and prolonged differentiated cell state in vitro, a concern is to be expressed regarding the size of the spheroids (Glicklis et al 2004).…”