Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium.

Abstract

Abstract. Mature adult parenchymal hepatocytes, typically of restricted capacity to proliferate in culture, can now enter into clonal growth under the influence of hepatocyte growth factor (scatter factor) (HGF/SF), epidermal growth factor (EGF), and transforming growth factor et (TGFo 0 in the presence of a new chemically defined medium (HGM). The expanding populations of hepatocytes lose expression of hepatocyte specific genes (albumin, cytochrome P450 IIB1), acquire expression of markers expressed by bile duct epithelium (cytokeratin 19), produce TGFot and acidic FGF and assume a very simplified morphologic phenotype by electron microscopy. A major change associated with this transition is the decrease in ratio between transcription factors C/EBPo~ and C/EBP[3, as well as the emergence in the proliferating hepatocytes of transcription factors AP1, NFKB. The liver associated transcription factors HNF1, HNF3, and HNF4 are preserved throughout this process. After population expansion and clonal growth, the proliferating hepatocytes can return to mature hepatocyte phenotype in the presence of EHS gel (Matrigel). This includes complete restoration of electron microscopic structure and albumin expression. The hepatocyte cultures however can instead be induced to form acinar/ductular structures akin to bile ductules (in the presence of HGF/SF and type I collagen). These transformations affect the entire population of the hepatocytes and occur even when DNA synthesis is inhibited. Similar acinar/ductular structures are seen in embryonic liver when HGF/SF and its receptor are expressed at high levels. These findings strongly support the hypothesis that mature hepatocytes can function as or be a source of bipotential facultative hepatic stem cells (hepatoblasts). These studies also provide evidence for the growth factor and matrix signals that govern these complex phenotypic transitions of facultative stem cells which are crucial for recovery from acute and chronic liver injury.

Enrichment of a bipotent hepatic progenitor cell from naïve adult liver tissue

“…Interestingly there was a more than 2-fold increase in the expression of CK-19 during this same time period. These findings are intriguing and we postulate that the sca-HPCs may be dedifferentiating in culture; a similar phenomenon had been demonstrated with mature hepatocytes in culture [25] and with other HPC populations [12]. Using a culture system that supports differentiation of hepatocytic cells along a biliary lineage the sca-HPCs did not differentiate towards a specific phenotype and while there was a loss of AFP expression with persistent expression of CK-19 there was also a decrease in albumin expression.…”

“…Immediately after isolation the sca-HPCs express CK-19, AFP, and albumin which persists for two weeks of culture under our established growth conditions. [ Figure 4 and Supplemental Data] Numerous culture conditions have been published with respect to supporting differentiation of liver cells towards biliary or hepatic phenotypes [25]. Based on this work the sca-HPCs are cultured under two distinct conditions to support differentiation.…”

Recovery of Mature Hepatocytic Phenotype following Bile Ductular Transdifferentiation of Rat Hepatocytes in Vitro

“…[9][10][11] Recently, we found that tumor necrosis factor-α (TNF-α) significantly enhanced ductular transdifferentiation (Nishikawa et al, submitted for publication).…”

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology

“…The medium factors selected for testing were HSA, OSM, human FGF4, human HGF, human EGF, NicA, and DexM. This selection was based on previous experience in the laboratory and on published results showing a supporting effect of these factors on liver cell maintenance and growth (Gomez-Lechon et al 1995;Strain et al 1991;Zarling et al 1986;Linsley et al 1989;Block et al 1996). The lower and higher concentrations for the seven factors were typical levels as reported in the literature or as used in previous experiments (Table 1).…”

“…This situation is particularly remarkable since research on hepatocyte cell biology has over the years uncovered the biological role of several extracellular growth and differentiation factors (Okita 2002). Of particular interest and relevance among these are (1) the hepatocyte growth factor (HGF), due to its ability to promote DNA synthesis (Gomez-Lechon et al 1995;Strain et al 1991); (2) albumin, which is involved in controlling osmotic conditions; (3) oncostatin M (OSM), due to its stimulation of fibroblasts and Kaposi cells (Zarling et al 1986;Linsley et al 1989); (4) fibroblast growth factors (FGF), which activate tissue repair; and (5) epidermal growth factor (EGF), due to its ability to support DNA synthesis in combination with insulin and glucagon (Block et al 1996). In order to attain optimal effects during cellular and metabolic experimentation and processing, it is particularly important to target the intracellular concentration levels of these protein factors.…”