Induced maturation of hepatic progenitor cells in vitro

Hepatic progenitor cells (HPCs) are a potential cell source for liver cell transplantation but do not function like mature liver cells. We sought an effective and reliable method to induce HPC maturation. An immortalized HP14.5 albumin promoter-driven Gaussian luciferase (ALB-GLuc) cell line was established from HPCs isolated from fetal mouse liver of post coitus day 14.5 mice to investigate the effect of induction factors on ALB promoter. HP14.5 parental cells were cultured in DMEM with different combinations of 2% horse serum (HS), 0.1 µM dexamethasone (DEX), 10 ng/mL hepatic growth factor (HGF), and/or 20 ng/mL fibroblast growth factor 4 (FGF4). Trypan blue and crystal violet staining were used to assess cell proliferation with different induction conditions. Expression of hepatic markers was measured by semi-quantitative RT-PCR, Western blot, and immunofluorescence. Glycogen storage and metabolism were detected by periodic acid-Schiff and indocyanine green (ICG) staining. GLuc activity indicated ALB expression. The combination of 2% HS+0.1 µM Dex+10 ng/mL HGF+20 ng/mL FGF4 induced the highest ALB-GLuc activity. Cell proliferation decreased in 2% HS but increased by adding FGF4. Upon induction, and consistent with hepatocyte development, DLK, AFP, and CK19 expression decreased, while ALB, CK18, and UGT1A expression increased. The maturity markers tyrosine aminotransferase and apolipoprotein B were detected at days 3 and 6 post-induction, respectively. ICG uptake and glycogen synthesis were detectable at day 6 and increased over time. Therefore, we demonstrated that HPCs were induced to differentiate into functional mature hepatocytes in vitro, suggesting that factor-treated HPCs may be further explored as a means of liver cell transplantation.

Effect of transplanted mesenchymal stem cells transfected with adenoviral ventor mediated HGF gene on snrvival volume of grain fat grafts / 中华医学美学美容杂志

ObjectiveTo investigate the effect of mesenchymal stem cells (MSCs) transfected with hepatic growth factor (HGF) gene on the survival volume of free grain fat grafts.Methods MSCs of male Wistar rats were transfected with Ad-GFP or Ad-HGF.The transfection infectivity of Ad-GFP to MSCs,the expression of HGF were measured using ELISA assay.150 rats were randomly divided into 5 groups:control group (group A),MSCs group (group B),Ad HGF group (group C),MSCs transplanted with Ad-GFP group (group D),and MSCs transplanted with Ad-HGF (MSCsHGF) group (group E).Then,the same volume of frain fat graft,mixed with DMEM LG andMSCs,Ad HGF,MSCs-GFP,MSCs HGF respectively,were transplanted to rats back,but control group were only mixed with DMEM-LG.Fat graft was obtained on days 3,5,7,14,28,and 60 after implantation.The volume of fat graft was measured by messcylinder,and the expression of HGF and CD34 in transplanted fat tissue were detected by immunohistochemistry.ResultsThe transfection infectivity of Ad-GFP to MSCs was 89.6 ％ at 100 MOI,the expression of HGF in MSCs culture medium reached to the level after being transfected with Ad-HGF for 48 h.Compared with other 4 groups,at days 3,5,7,and 14 post-transplantation,the expression of HGF in E group transplanted fat of group E had statistics significance (P＜0.05).The persentage of survival volume of fat graft in group E was significantly higher than that of other group ( P＜0.05) at days 28,and 60 post transplantation.ConclusionsMSCs transplanted with Ad-HGF could secrete HGF and increase the survival volume of fat grafts.

Expression of hepatic growth factor and C-met in reserved liver tissue after partial hepatectomy of hepatic fibrosis / 解剖学报

Objective To study the expression of hepatic growth factor(HGF) and C-met in reserved liver tissue after partial hepatectomy of rats with hepatic fibrosis. Methods Totally 130 SD rats were randomly divided into four groups: normal group (n=7), group of normal rats with partial hepatectomy(n=50),hepatic fibrotic group(n=7), and group of hepatic fibrotic rats with partial hepatectomy(n=66). Rats were killed after operation 12 hours, 1 day, 3 days, 5 days, 7 days and 14 days separately, then HGF and C-met of reserved liver tissues were detected with immunohistochemistry staining and Western blotting. Results In the group of normal rats with partial hepatectomy, immunohistochemistry staining indicated that the expression of HGF and C-met increased to get the peak point after partialhepatectomy 12 hours and 3 days respectively, and HGF maintained at the high level to the 7th day, then decreased gradually, finaly approched to the level of pro-operation at 14th day, but C-met fell sharply,and declined to the the level of pro-operation at the 14th day. In the group of hepatic fibrotic rats with partial hepatectomy, the expression of HGF and C-met decreased sharply after operation 12 hours, next HGF increased to get the peak point at the 1st day, and then declined speedily, and decreased to the bottom at the 14th day, but C-met declined to the bottom at the 3rd day, then increased slightly till the 7th day, affter that increased sharply to the summit at the 14th day. Western blotting analysis showed the results of HGF and C-met coincided with that of immunohistochemistry. Conclusion The high isochronous expression of HGF and C-met in hepatic tissue is propitious to hepatocellular division, Which indicates that the expresson out of step of HGF and C-met might be the key reason of hard regeneration of fibrosis liver after operation.

Hepatocyte Growth Factor is the Key Cytokine in Stimulating Potential Stem Cells in the Cord Blood into Hepatic Lineage Cells

BACKGROUND: This study was designed to investigate the role of the hepatocyte growth factor (HGF) with regards to differentiation of somatic stem cells originating from the human umbilical cord blood (UCB) into hepatic lineage cells in vitro culture system. METHODS: Mononuclear cells from UCB were cultured with and without HGF based on the fibroblast growth factor (FGF)-1, FGF-2, and stem cell factor. The cultured cells were confirmed by immunofluorescent staining analysis with albumin (ALB), cytokeratin-19 (CK-19), and proliferating cell nuclear antigen (PCNA) MoAb. ALB and CK-18 mRNA were also evaluated by reverse transcription-polymerase chain reaction. In order to observe changes in proliferating capacity with respect to the cultured period, CFSE with affinity to proliferating cells were tagged and later underwent flow cytometry. RESULTS: In the HGF-treated group, cultured cells had a large oval shaped appearance with adherent, but easily detachable characteristics. In the HGF-non treated group, these cells were spindle-shaped with strong adherent characteristics. Expressions of ALB and CK-19 were evident in HGF-treated group compared to non-expression of those in to HGF-non treated group. Dual immunostaining analysis of the ALB producing cells showed presence of PCNA in their nuclei, and ALB and CK-18 mRNA were detected on the 21st day of cultured cells in the HGF-treated group. CONCLUSION: Our findings suggest that HGF has a pivotal role in differentiating somatic stem cells of human UCB into hepatic lineage cells in vitro.