Purpose: Keratin 19 (K19) is a known marker of poor prognosis and invasion in human hepatocellular carcinoma (HCC). However, the relationship between K19 and cancer stem cells (CSCs) is unclear. Here, we determined whether K19 can be used as a new CSC marker and therapeutic target in HCC.Experimental
It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 +/- 12.2% (means +/- SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes.
Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive factors that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and growth factors on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs. The transgene vector that contained enhanced green fluorescent protein (EGFP) under the control of human alpha-fetoprotein (AFP) enhancer/promoter was transfected into hESC lines. The transgenic hESCs were cultured on extracellular matrixes (collagen type I, laminin, and Matrigel) in the presence or absence of growth factors including hepatocyte growth factor (HGF), bone morphogenetic protein 4, fibroblast growth factor 4, all-trans-retinoic acid, and activin A. The expression of AFP-EGFP was measured by flow cytometry. The culture on Matrigel-coated dishes with 100 ng/ml activin A showed 19.5% of EGFP-positive proportions. Moreover, the sequential addition of 100 ng/ml activin A and 20 ng/ml HGF resulted in 21.7% and produced a higher yield of EGFP-positive cells than the group stimulated by activin A alone. RT-PCR and immunocytochemical staining revealed these EGFP-positive cells to differentiate into mesendoderm-like cells by use of activin A and then into hepatic endoderm cells by use of HGF. Two other hESC lines also differentiated into endoderm on the hepatic lineage by our method. In conclusion, we therefore found this protocol to effectively differentiate multiple hESC lines to early hepatocytes using activin A and HGF on Matrigel.
Objective: To evaluate the long-term outcomes of surgery for recurrent hepatocellular carcinoma (HCC). Background: HCC recurs with high incidence after liver resection. Little is known about long-term outcomes of patients undergoing surgery for recurrent HCC. Methods: Among 989 patients who underwent R0/R1 liver resection for HCC between 1995 and 2014, 676 patients who exhibited recurrence were included. Repeat surgery was performed in 128 patients (RS group), and not in the remaining 548 patients (NS group). Prognostic value after repeat surgery was evaluated by comparing survival after recurrence (SAR) between the RS and NS groups. Subgroup analyses according to the 3 recurrence patterns [intrahepatic recurrence (IHR), extrahepatic recurrence (EHR), and intra plus extrahepatic recurrence (IHR + EHR)] were performed. Results: Seventy-three of 430 patients (17.0%) with IHR, 17 of 57 patients (29.8%) with EHR, and 38 of 189 patients (20.1%) with IH + EHR underwent repeat surgery. Compared with the NS group, the RS group had better liver function and their time to recurrence was significantly longer (16.5 vs 11.4 months; P < 0.001). In the overall and 3 recurrence patterns, the 5-year SAR rate was better in the RS group compared with the NS group (RS vs NS group; overall, 53.0% vs 25.7%; IHR, 73.8% vs 37.2%; EHR, 30.0% vs 0%; IHR + EHR, 34.1% vs 10.6%; all P < 0.001, respectively). On multivariate analysis, repeat surgery was identified as an independent factor for better SAR (P < 0.001). Conclusion: Surgery for recurrent HCC may yield long-term survival for not only IHR but also for EHR in selected patients.
A whole-organ regeneration approach, using a decellularised xenogeneic liver as a scaffold for the construction of a transplantable liver was recently reported. Deriving suitable scaffolds was the first step towards clinical application; however, effective recellularisation remains to be achieved. This report presents a strategy for the improvement of the recellularisation process, using novel cell-seeding technique and cell source. We evaluated recellularised liver grafts repopulated through the portal vein or the biliary duct with mice adult hepatocytes or E14.5 foetal hepatocytes. More than 80% of the cells seeded through the biliary tree entered the parenchyma beyond the ductule-lining matrix barrier and distributed throughout the liver lobule. In contrast, about 20% of the cells seeded through the portal tree entered the parenchyma. The gene expression levels of foetal hepatocyte albumin, glucose 6-phosphatase, transferrin, cytokeratin 19, and gamma-glutamyl transpeptidase were increased in three-dimensional cultures in the native liver-derived scaffolds, and the activation of liver detoxification enzymes and formation of biliary duct-like structures were supported. The metabolic functions of liver grafts recellularised with different cell types were similar. These results suggest that biliary tree cell-seeding approach is promising, and that liver progenitor cells represent a good cell source candidate.
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