title: Analysis of soluble factors in conditioned media derived from primary cultures of cirrhotic liver of biliary atresia.
abstract: Biliary atresia (BA) is a rare and serious liver disease in newborn infants. Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of BA may contain hepatic stem/progenitor cells in primary culture of NPC fractions. In this study, NPC fractions were subjected to primary or passage culture and found that clusters of hepatocyte-like cells appear even without adding hepatocyte growth factor (HGF) to the culture medium, but not in their passage culture used as a control. Based on these findings, conditioned media (CMs) were collected and soluble factors in the CMs were analyzed in order to elucidate the mechanism of the appearance of hepatocyte-like cells or their clusters. A large amount of active HGF consisting of α and β chains was detected in CMs derived from primary culture, but not in CMs from passage culture, as determined by western blot analysis, bone morphogenetic protein (BMP)-4, oncostatin M (OSM), and transforming growth factor (TGF)-β1 were not detected in any of the CMs. The number of hepatocyte-like cells in primary culture tended to decrease following treatment with the HGF receptor c-Met inhibitor, SU11274 in a dose-dependent manner. Furthermore, the clusters of hepatocyte-like cells tended to increase in size and number when freshly isolated NPC fractions were cultured in the presence of 10% of CMs collected after 3-4 wk of primary culture. In conclusion, these findings indicate that CMs derived from primary culture of NPC fractions of BA liver contain a large amount of active HGF, which may activate hepatic stem/progenitor cells and promote the appearance of hepatocyte-like cells or their clusters through HGF/c-Met signaling. The present study would lead to cell therapy using the patient's own cells for the treatment of BA.
authors.affiliation: Department of Liver Cell Biology, Kohno Clinical Medicine Research Institute, Tokyo, Japan. t.yamazaki@kcmi.or.jp.
authors.forename: Taisuke
authors.identifier: http://orcid.org/0000-0001-7669-0341
authors.initials: T
authors.lastname: Yamazaki
authors.affiliation: Department of Liver Cell Biology, Kohno Clinical Medicine Research Institute, Tokyo, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Wakai
authors.affiliation: Division for Advanced Medical Sciences, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Enosawa
authors.affiliation: Department of Liver Cell Biology, Kohno Clinical Medicine Research Institute, Tokyo, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Tokiwa
mesh_terms: D001656:Biliary Atresia / Q000378:metabolism* / Q000473:pathology; D017077:Culture Media, Conditioned / Q000737:chemistry* / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000737:chemistry / Q000302:isolation & purification*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000254:growth & development; D008103:Liver Cirrhosis / Q000378:metabolism* / Q000473:pathology; D010879:Piperazines / Q000494:pharmacology; D019859:Proto-Oncogene Proteins c-met / Q000037:antagonists & inhibitors / Q000235:genetics*; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: C478479:((3Z)-N-(3-chlorophenyl)-3-((3,5-dimethyl-4-((4-methylpiperazin-1-yl)carbonyl)-1H-pyrrol-2-yl)methylene)-N-methyl-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide); D017077:Culture Media, Conditioned; D007211:Indoles; D010879:Piperazines; D013449:Sulfonamides; D017228:Hepatocyte Growth Factor; D019859:Proto-Oncogene Proteins c-met
keywords: Active HGF; Biliary atresia; Hepatocyte-like cells; Non-parenchymal cell fractions; Soluble factors