Jeroen de Jonge

Long-term culture of genome-stable bipotent stem cells from adult human liver

Summary Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.

Hepatocyte‐derived microRNAs as serum biomarkers of hepatic injury and rejection after liver transplantation

Recent animal and human studies have highlighted the potential of hepatocyte‐derived microRNAs (HDmiRs) in serum as early, stable, sensitive, and specific biomarkers of liver injury. Their usefulness in human liver transplantation, however, has not been addressed. The aim of this study was to investigate serum HDmiRs as markers of hepatic injury and rejection in liver transplantation. Serum samples from healthy controls and liver transplant recipients (n = 107) and peritransplant liver allograft biopsy samples (n = 45) were analyzed via the real‐time polymerase chain reaction quantification of HDmiRs (miR‐122, miR‐148a, and miR‐194). The expression of miR‐122 and miR‐148a in liver tissue was significantly reduced with prolonged graft warm ischemia times. Conversely, the serum levels of these HDmiRs were elevated in patients with liver injury and positively correlated with aminotransferase levels. HDmiRs appear to be very sensitive because patients with normal aminotransferase values (<50 IU/L) had 6‐ to 17‐fold higher HDmiR levels in comparison with healthy controls (P < 0.005). During an episode of acute rejection, serum HDmiRs were elevated up to 20‐fold, and their levels appeared to rise earlier than aminotransferase levels. HDmiRs in serum were stable during repeated freezing and thawing. In conclusion, this study shows that liver injury is associated with the release of HDmiRs into the circulation. HDmiRs are promising candidates as early, stable, and sensitive biomarkers of rejection and hepatic injury after liver transplantation. Liver Transpl 18:290–297, 2012.

Tissue-specific mutation accumulation in human adult stem cells during life

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.

The comprehensive complication index: a novel and more sensitive endpoint for assessing outcome and reducing sample size in randomized controlled trials

Objective:To test whether the newly developed comprehensive complication index (CCI) is more sensitive than traditional endpoints for detecting between-group differences in randomized controlled trials (RCTs). Background:A major challenge in RCTs is the choice of optimal endpoints to detect treatment effects. Mortality is no longer a sufficient marker in studies, and morbidity is often poorly defined. The CCI, integrating all complications including their severity in a linear scale ranging from 0 (no complication) to 100 (death), is a new tool, which may be more sensitive than other traditional endpoints to detect treatment effects on postoperative morbidity. Methods:The CCI was tested in 3 published RCTs from European centers evaluating pancreas, esophageal and colon resections. To compare the sensitivity of the CCI with traditional morbidity endpoints, for example, presence of any (yes/no) or only the most severe complications, all postoperative events were assessed, and the CCI calculated. Treatment effects and sample size calculations were compared using the CCI and traditional endpoints. Results:Although RCTs failed to show between-group differences using any or most severe complications, the CCI revealed significant differences between treatment groups in 2 RCTs—after pancreas (P = 0.009) and esophageal surgery (P = 0.014). The CCI in the RCT on colon resections confirmed the absence of between-group differences (P = 0.39). The required sample sizes in trials are up to 9 times lower for the CCI than for traditional morbidity endpoints. Conclusions:This study demonstrates superiority of the CCI to traditional endpoints. The CCI may serve as an appealing endpoint for future RCTs and may reduce the sample size.

End-to-end versus end-to-side esophagogastrostomy after esophageal cancer resection: a prospective randomized study.

Objective: To compare single-layered hand-sewn cervical end-to-side (ETS) anastomosis with end-to-end (ETE) anastomosis in a prospective randomized fashion. Background: The preferred organ used for reconstruction after esophagectomy for cancer is the stomach. Previous studies attempted to define the optimal site of anastomosis and anastomotic techniques. However, anastomotic stricture formation and leakage still remain an important clinical problem. Methods: From May 2005 to September 2007, 128 patients (64 in each group) were randomized between ETE and ETS anastomosis after esophagectomy for cancer with gastric tube reconstruction. Routine contrast swallow studies and endoscopy were performed. Anastomotic stricture within 1 year, requiring dilatation, was the primary endpoint. Secondary endpoints were anastomotic leak rate and mortality. Results: Ninety-nine men and 29 women underwent esophagectomy and gastric tube reconstruction. Benign stenosis of the anastomosis, for which dilatation was required, occurred more often in the ETE group (40% vs. ETS 18%, P < 0.01) after 1 year of follow-up. The overall (clinical and radiological) anastomotic leak rate was lower in the ETE group (22% vs. ETS 41%, P = 0.04). Patients with an ETE anastomosis suffered less often from pneumonia; 17% versus ETS 44%, P = 0.002 and had subsequently significantly shorter in-hospital stay (15 days vs. 22 days, P = 0.02). In-hospital mortality did not differ between both groups. Conclusion: ETS anastomosis is associated with a lower anastomotic stricture rate, compared to ETE anastomosis. However, prevention of stricture formation was at high costs with increased anastomotic leakage and longer in-hospital stay. This study is registered with the Dutch Trial Registry and carries the ID number OND1317772.

Fibrinolysis during liver transplantation is enhanced by using solvent/detergent virus-inactivated plasma (ESDEP®)

After the introduction of solvent/detergent-treated plasma (ESDEP®) in our hospital, an increased incidence of hyperfibrinolysis was observed (75% vs 29%;P = 0.005) compared with the use of fresh frozen plasma for liver transplantation. To clarify this increased incidence, intraoperative plasma samples of patients treated with fresh frozen plasma or ESDEP were analyzed in a retrospective observational study. During the anhepatic phase, plasma levels of d-dimer (6.58 vs 1.53 &mgr;g/mL;P = 0.02) and fibrinogen degradation products (60 vs 23 mg/L;P = 0.018) were significantly higher in patients treated with ESDEP. After reperfusion, differences increased to 23.5 vs 4.7 &mgr;g/mL (d-dimer, P = 0.002) and 161 vs 57 mg/L (fibrinogen degradation products, P = 0.001). The amount of plasma received per packed red blood cell concentrate, clotting tests, and levels of individual clotting factors did not show significant differences between the groups. &agr;2-Antiplasmin levels, however, were significantly lower in patients receiving ESDEP during the anhepatic phase (0.37 vs 0.65 IU/mL;P < 0.001) and after reperfusion (0.27 vs 0.58 IU/mL;P = 0.001). Analysis of &agr;2-antiplasmin levels in ESDEP alone showed a reduction to 0.28 IU/mL (normal >0.95 IU/mL) because of the solvent/detergent process. Therapeutic consequences for the use of ESDEP in orthotopic liver transplantation are discussed in view of an increased incidence of hyperfibrinolysis caused by reduced levels of &agr;2-antiplasmin in the solvent/detergent-treated plasma.

Outcome of Esophagectomy for Cancer in Elderly Patients

BACKGROUND This study analyzes the outcome of esophageal resection in patients 70 or more years of age, compared with patients aged less than 70 years and identifies risk factors for worse outcome in the elderly. METHODS Comorbidity, postoperative morbidity, in-hospital mortality and survival rates were compared between 811 patients aged less than 70 years and 250 patients aged 70 years or more who underwent esophagectomy for esophageal cancer in a single high-volume center from 1985 to 2005. RESULTS Groups were similar regarding surgical approach, resectability, and tumor stage. More patients aged 70 years or more had cardiovascular and respiratory concomitant disease. Among patients aged 70 years or more, the prevalence of adenocarcinoma and Barretts transformation was higher (67% versus 53% for patients aged less than 70 years, and 22% versus 15%, respectively). There were no differences in surgical complications (20% versus 17%). Nonsurgical complications occurred more in patients aged 70 years or more (35% versus 27%) and operative mortality was higher among elderly patients (8.4 versus 3.8%), as was in-hospital mortality (11.6% versus 5.4%). The disease-specific 5-year survival was lower for patients aged 70 years or more (27% versus 34%). The 1-year survival, reflecting the impact of operative morbidity and mortality, was 58% for patients aged 70 years or more and 68% for the patients aged less than 70 years (p = 0.002). Among patients aged 70 years or more, respiratory comorbidity and thoracoabdominal resection were risk factors for the occurrence of nonsurgical complications and respiratory comorbidity for in-hospital mortality. CONCLUSIONS Older patients have increased operative and in-hospital mortality and decreased 5-year survival after esophageal resection for cancer. Our results indicate that especially thoracoabdominal resection for esophageal carcinoma should be carefully considered for patients older than 70 years who suffer from respiratory disease.

Secreted Factors of Human Liver-Derived Mesenchymal Stem Cells Promote Liver Regeneration Early After Partial Hepatectomy

Rapid liver regeneration is required after living-donor liver transplantation and oncologic liver resections to warrant sufficient liver function and prevent small-for-size syndrome. Recent evidence highlights the therapeutic potential of mesenchymal stem cells (MSC) for treatment of toxic liver injury, but whether MSC and their secreted factors stimulate liver regeneration after surgical injury remains unknown. Therefore, the aim of this study is to investigate the effect of human liver-derived MSC-secreted factors in an experimental liver resection model. C57BL/6 mice were subjected to a 70% partial hepatectomy and treated with either concentrated MSC-conditioned culture medium (MSC-CM) or vehicle control. Animals were analyzed for liver and body weight, hepatocyte proliferation, and hepatic gene expression. Effects of MSC-CM on gene expression in a human hepatocyte-like cell line (Huh7 cells) were analyzed using genome-wide gene expression arrays. Liver regeneration was significantly stimulated by MSC-CM as shown by an increase in liver to body weight ratio and hepatocyte proliferation. MSC-CM upregulated hepatic gene expression of cytokines and growth factors relevant for cell proliferation, angiogenesis, and anti-inflammatory responses. In vitro, treatment of Huh7 cells with MSC-CM significantly altered expression levels of ~3,000 genes. Functional analysis revealed strong effects on networks associated with protein synthesis, cell survival, and cell proliferation. This study shows that treatment with MSC-derived factors can promote hepatocyte proliferation and regenerative responses in the early phase after surgical resection. MSC-CM may represent a feasible new strategy to promote liver regeneration in patients undergoing extensive liver resection or after transplantation of small liver grafts.

Mycophenolic acid augments interferon‐stimulated gene expression and inhibits hepatitis C Virus infection in vitro and in vivo

Mycophenolic acid (MPA) is a highly effective immunosuppressant that has broad antiviral activity against different viruses and can act in synergy with interferon‐α (IFN‐α) on hepatitis C virus (HCV) replication. MPA is a potent inosine monophosphate dehydrogenase (IMPDH) inhibitor but the antiviral mechanisms are less understood. The aim of this study was to investigate the inhibition of HCV infection by MPA and the molecular basis for its synergy with IFN‐α. The role of IMPDH and interferon‐stimulated genes (ISGs) was investigated in two HCV models using gain‐ or loss‐of‐function approaches. The in vivo effect of MPA treatment was studied in NOD/SCID mice engrafted with HCV replicon cells. Potent antiviral effects of MPA at clinically relevant concentrations were observed with both the subgenomic and JFH1‐derived infectious HCV models. MPA treatment in mice resulted in a specific and robust inhibition of HCV replication. Ectopic expression of an MPA‐resistant IMPDH2 mutant in HCV host cells completely reversed the antiproliferative effect of MPA but only partially affected the antiviral potency. However, similar to ribavirin, MPA induced expression of multiple antiviral ISGs, including interferon regulatory factor 1 (IRF1). Cotreatment of MPA with IFN‐α resulted in additive effects on ISG expression and enhanced IFN‐induced luciferase reporter activity. Knockdown of IRF1, but not IFITM3, significantly attenuated the inhibition of HCV replication by MPA. Conclusion: MPA exerts a potent anti‐HCV effect in vitro and in mice and acts in synergy with IFN‐α. MPAs antiviral activity partially depends on IMPDH but also involves stimulation of ISGs, providing a molecular basis for its synergy with IFN‐α. (HEPATOLOGY 2012;55:1673–1683)

Modeling rotavirus infection and antiviral therapy using primary intestinal organoids.

Despite the introduction of oral vaccines, rotavirus still kills over 450,000 children under five years of age annually. The absence of specific treatment prompts research aiming at further understanding of pathogenesis and the development of effective antiviral therapy, which in turn requires advanced experimental models. Given the intrinsic limitations of the classical rotavirus models using immortalized cell lines infected with laboratory-adapted strains in two dimensional cultures, our study aimed to model infection and antiviral therapy of both experimental and patient-derived rotavirus strains using three dimensional cultures of primary intestinal organoids. Intestinal epithelial organoids were successfully cultured from mouse or human gut tissues. These organoids recapitulate essential features of the in vivo tissue architecture, and are susceptible to rotavirus. Human organoids are more permissive to rotavirus infection, displaying an over 10,000-fold increase in genomic RNA following 24h of viral replication. Furthermore, infected organoids are capable of producing infectious rotavirus particles. Treatment of interferon-alpha or ribavirin inhibited viral replication in organoids of both species. Importantly, human organoids efficiently support the infection of patient-derived rotavirus strains and can be potentially harnessed for personalized evaluation of the efficacy of antiviral medications. Therefore, organoids provide a robust model system for studying rotavirus-host interactions and assessing antiviral medications.