Acute liver failure (ALF) causes severe liver injury and a systemic inflammatory response, leading to multiorgan failure with a high short-term mortality. Bioartificial liver (BAL) therapy is a promising approach that is hampered by the lack of appropriate bioreactors and carriers to retain hepatic cell function and poor understanding of BAL treatment mechanisms in ALF and extrahepatic organ injury. Recently, we used a fiber scaffold bioreactor (FSB) for the high-density, three-dimensional (3D) culture of primary porcine hepatocytes (PPHs) combined with an absorption component to construct a BAL and verified its function in a D-galactosamine (D-gal)-induced ALF porcine model to evaluate its protective effects on the liver and extrahepatic organs.
Abstract Background and Aim Bioartificial livers (BALs) are considered as a solution to bridge patients with acute liver failure to liver transplantation or to assist in spontaneous recovery for patients with end‐stage liver disease. Pig is the best donor of hepatocytes for BALs in clinical trials, because metabolic and detoxification function of its liver are close to human. However, using pig hepatocytes for BALs remains controversial for safety concern owing to nonhuman proteins secretion. Herein, we attempt to establish modified pigs expressing humanized liver proteins, blood‐coagulation factor VII (F7), and albumin (ALB). These pigs should also be porcine endogenous retrovirus subtype C (PERV‐C) free so that their ability of transmitting PERV to human could be diminished seriously. Methods We devised both homology‐dependent and independent knock‐in approaches to insert a fusion of hF7 and hALB gene downstream the site of pig endogenous F7 promoter in pig fetal fibroblasts negative for PERV‐C. The modified pigs were then generated through somatic cell nuclear transfer. Results We obtained 14 and 10 cloned pigs by homology‐dependent and independent approaches, respectively. Among them, 19 cloned pigs were with expected gene modification and 13 are alive to date. These modified pigs can successfully express hF7 and hALB in the liver and serum, and the expressed hF7 exhibits normal coagulation activity. Conclusions The gene‐edited pigs expressing hF7 and hALB in the liver were generated successfully. We anticipate that our pigs could provide an alternative cell source for BALs as a promising treatment for patients with acute liver failure.
Laparoscopic anterior right hepatectomy (LARH) has been used in some hospitals. However, data on the feasibility and safety of this procedure are still limited, due to the demanding technical requirements. The primary objective of this study was to compare the clinical outcomes of LARH with those of laparoscopic conventional right hepatectomy (LCRH) in patients with large right hepatocellular carcinoma, as well as to confirm the safety and feasibility of LARH. Furthermore, the article presents a step-by-step description of the surgical procedures for LARH to help perform this surgery in the clinic. The principle of LARH is to first prioritize the hepatic inlet duct separation while separating the right hepatic perihepatic ligament after transecting the liver. From December 2015 to June 2022, 82 patients with large right hepatocellular carcinoma (maximum tumor diameter ≥ 5 cm), were recruited for the study. In this cohort, 54 and 28 patients underwent LARH and LCRH, respectively. The perioperative clinical data and survival outcomes of the two groups were compared. Compared with LCRH, LARH exhibited the advantages of less contact and extrusion, thereby leading to the achievement of superior results. Thus, we propose that LARH is the optimal choice for patients with large right hepatocellular carcinoma.
Abstract Background Circulating tumour cells (CTCs), especially mesenchymal CTCs, are important determinants of metastasis, which leads to most recurrence and mortality in hepatocellular carcinoma (HCC). However, little is known about the underlying mechanisms of CTC colonisation in pre-metastatic niches. Methods Detection and classification of CTCs in patients were performed using the CanPatrol™ system. A lentiviral vector expressing Prrx1-targeting shRNA was constructed to generate a stable HCC cell line with low expression of Prrx1. The effect of Prrx1 knockdown on stemness, migration, and drug resistance of the cell line was assessed, including involvement of SDF-1/CXCR4 signalling. Promising clinical applications of an inhibitor of STAT3 tyrosine phosphorylation, C188–9, and specific blockade with CXCR4 antibody were explored. Results The number of mesenchymal CTCs in blood was closely associated with tumour recurrence or metastasis. Pre-metastatic niche-derived SDF-1 could downregulate Prrx1, which induced the stemness, drug resistance, and increased expression of CXCR4 in HCC cells through the STAT3 pathway in vitro. In vivo , mice bearing tumours of Prrx1 low-expressing cells had significantly shorter survival. In xenograft tumours and clinical samples, loss of Prrx1 was negatively correlated with increased expression of CXCR4 in lung metastatic sites compared with that in the primary foci. Conclusions These findings demonstrate that decreased expression of Prrx1 stimulates SDF-1/CXCR4 signalling and contributes to organ colonisation with blood CTCs in HCC. STAT3 inhibition and specific blockade of CXCR4 have clinical potential as therapeutics for eliminating organ metastasis in advanced HCC.
Abstract Background: Hepatocellular carcinoma (HCC) is one of the major cause of cancer related deaths worldwide, due to high 5 year postoperative recurrence rate and individual heterogeneity. Thus, prognostic model has dramatically urgently needed on HCC in recent years. Serval research have reported that copy number amplification of the 8q24 chromosomal region is associated with low survival in many cancers. The objective of this study was to construct a multi-gene model to predict the prognosis of HCC. Methods: RNAseq and copy number variant (CNV) data of tumor tissue samples of HCC from TCGA (N = 328) was used to identify differentially expressed mRNAs of genes located on chromosomal 8q24 regions by Wilcox test. Univariate Cox and Lasso Cox regression were performed to screen and construct prognostic multi-gene signature in TCGA cohort (N = 119). The multi-gene signature was validated in ICGC cohort (N = 240).A nomogram for prognosis prediction was built and Gene Set Enrichment Analysis (GSEA) was used to further study the underlying molecular mechanisms. Results: A 7-gene prognosis signature model was established for predicting HCC prognosis. Using the model, we divided individuals into high-risk and low-risk sets with significantly different overall survival in training dataset(HR = 0.17, 95% CI 0.1–0.28; P ＜ 0.001) and in testing dataset (HR = 0.42, 95% CI 0.23–0.74; P = 0.002). Multivariate Cox regression analysis indicated that this signature was an independent prognostic factor of HCC survival. Nomogram including the prognostic signature was constructed and showed better predictive performance in short year (1 and 3 year) than long year (5 year) survival. Furthermore, GSEA revealed several significantly pathways, which may help explain the underlying molecular mechanism. Conclusions: The 7-gene signature was a reliable prognostic marker in HCC, which may provide meaningful information to therapeutic customization and treatment decision making.
Abstract Background: Hepatocellular carcinoma (HCC) is one of the majorcause of cancer related deaths worldwide, due tohigh 5 yearpostoperativerecurrence rate and individualheterogeneity.Thus, prognostic modelhas dramaticallyurgently neededon HCCin recent years.Serval research have reported that copy number amplificationof the 8q24 chromosomalregion is associated with lowsurvival in many cancers. The objectiveof this study was to construct a multi-gene modelto predict the prognosis of HCC. Methods: RNAseq and copy number variant (CNV)data of tumor tissue samples of HCC from TCGA(N = 328)wasused to identify differentially expressed mRNAs of genes located on chromosomal 8q24 regionsby Wilcoxtest.Univariate Cox and Lasso Cox regressionwereperformed to screenand constructprognostic multi-gene signature in TCGA cohort (N = 119). The multi-gene signature was validated in ICGCcohort(N = 240).A nomogram for prognosis prediction was built and Gene Set Enrichment Analysis (GSEA) was usedto further studythe underlying molecular mechanisms. Results: A 7-gene prognosis signaturemodelwas established for predicting HCC prognosis. Using the model, we divided individualsinto high-risk and low-risk setswith significantly different overall survival in training dataset(HR = 0.17, 95% CI 0.1–0.28; P ＜0.001) and in testing dataset (HR = 0.42, 95% CI 0.23–0.74; P =0.002). Multivariate Cox regression analysis indicatedthat thissignature was an independentprognostic factor ofHCC survival. Nomogram including the prognostic signature was constructed and showed better predictive performance in short year (1and 3year) than long year (5 year) survival. Furthermore, GSEA revealed several significantly pathways, which may help explain the underlying molecular mechanism. Conclusions: The 7-gene signature was areliableprognosticmarkerinHCC, which may provide meaningful informationto therapeutic customization and treatment decision making.
Hepatocellular carcinoma (HCC) is an aggressive form of cancer characterized by a high recurrence rate following resection. Studies have implicated stromal and immune cells, which form part of the tumor microenvironment, as significant contributors to the poor prognoses of HCC patients. In the present study, we first downloaded gene expression datasets for HCC patients from The Cancer Genome Atlas database and categorized the patients into low and high stromal or immune score groups. By comparing those groups, we identified differentially expressed genes significantly associated with HCC prognosis. The Gene Ontology database was then used to perform functional enrichment analysis, and the STRING network database was used to construct protein-protein interaction networks. Our results show that most of the differentially expressed genes were involved in immune processes and responses and the plasma membrane. Those results were then validated using another a dataset from a HCC cohort in the Gene Expression Omnibus database and in 10 pairs of HCC tumor tissue and adjacent nontumor tissue. These findings enabled us to identify several tumor microenvironment-related genes that associate with HCC prognosis, and some those appear to have the potential to serve as HCC biomarkers.
Objective: Hepatocyte growth factor is cardioprotective, anti-apoptotic, and pro-angiogenic; yet, is limited in clinical translation due to its high manufacturing cost and low stability. We encapsulated an engineered HGF fragment (HGFf) in an engineered shear-thinning, temperature-responsive, injectable hydrogel (SHIELD). We hypothesized SHIELD would facilitate targeted, sustained intramyocardial delivery of HGFf, thereby attenuating myocardial injury and post-infarction remodeling. Methods: HGFf elution was tracked for 2 weeks via fluorophore conjugation. Cytoprotective effects of HGFf on rat cardiomyocytes were assessed with a WST-1 cell viability assay after a 48hr serum starvation. Adult male Wistar rats (n=21) underwent sham surgery or permanent ligation of the LAD followed by borderzone injection of 60μL PBS, SHIELD alone, or SHIELD encapsulating 10μg HGFf. Ventricular geometry and function, infarct size, and angiogenic response were assessed 4-weeks post-infarction. Results: HGFf elution was sustained for 14 days in vitro (Fig. 1A), remained active, and improved cardiomyocyte viability by 22% during serum starvation (Fig. 1B). SHIELD remained localized within the heart at day 16 following injection. Treatment with SHIELD-HGFf reduced infarct size and significantly increased ejection fraction compared to that of the controls (Fig. 1C and D). The improvements in function in SHIELD-HGFf treated animals were coupled with a significant increase in arterial density compared to that of the other study groups (Fig. 1E and F). Conclusions: Sustained delivery of a stable, protein engineered HGFf limits post-infarction adverse ventricular remodeling by increasing angiogenesis and reducing fibrosis. Combining HGFf with SHIELD improves clinical translatability by enabling catheter-based delivery and subsequent retention of this novel, potent angiogenic protein analog.
Background/Aims: The use of antibiotics to eliminate Mycoplasma contamination has some serious limitations. Mycoplasma contamination can be eliminated by intraperitoneal injection of BALB/c mice with contaminated cells combined with screening monoclonal cells. However, in vivo passage in mice after injection with contaminated cells requires a long duration (20-54 days). Furthermore, it is important to monitor for cross-contamination of mouse and human cells, xenotropic murine leukemia virus-related virus (XMRV) infection, and altered cell function after the in vivo treatment. The present study aimed to validate a reliable and simplified method to eliminate mycoplasma contamination from human hepatocytes. BALB/c mice were injected with paraffin oil prior to injection with cells, in order to shorten duration of intraperitoneal passage. Cross-contamination of mouse and human cells, XMRV infection and cell function-related genes and proteins were also evaluated.Methods: PCR and DNA sequencing were used to confirm Mycoplasma hyorhinis (M. hyorhinis) contamination in human hepatocyte C3A cells. Five BALB/c mice were intraperitoneally injected with 0.5 ml paraffin oil one week before injection of the cells. The mice were then intraperitoneally injected with C3A hepatocytes (5.0×106/ml) contaminated with M. hyorhinis (6.2±2.2×108 CFU/ml). Ascites were collected for monoclonal cell screening on the 14th day after injection of contaminated cells. Elimination of mycoplasma from cells was determined by PCR and Transmission Electron Microscopy (TEM). Human–mouse cell and XMRV contamination were also detected by PCR. Quantitative reverse transcription PCR and western blotting were used to compare the expression of genes and proteins among treated cells, nontreated infected cells, and uninfected cells. Results: Fourteen days after injection with cells, 4 of the 5 mice had ascites. Hepatocyte colonies extracted from the ascites of 4 mice were all mycoplasma-free. There was no cell cross-contamination or XMRV infection in treated cell cultures. Elimination of Mycoplasma resulted in partial or complete recovery in the expression of ALB, TF, and CYP3A4 genes as well as proteins. Proliferation of the treated cells was not significantly affected by this management. Conclusion: The method of elimination of Mycoplasma contamination in this study was validated and reproducible. Success was achieved in four of five cases examined.
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