Cell-free microRNAs as early predictors of graft viability during ex vivo normothermic machine perfusion of human donor livers

Background Cell-free microRNAs (miRs) have emerged as early and sensitive biomarkers for tissue injury and function. This study aimed to investigate whether the release of hepatocyte-derived microRNAs (HDmiRs) and cholangiocyte-derived miRs (CDmiRs) correlates with hepato-cholangiocellular injury and function during oxygenated, normothermic machine perfusion (NMP) of human liver grafts. Methods Donor livers (n = 12), declined for transplantation, were subjected to oxygenated NMP (6 hours) after a period of static cold storage (median 544 minutes (IQR 421-674)). Perfusate and bile samples were analyzed by qRT-PCR for HDmiR-122 and CDmiR-222. Spearman correlations were performed between miR levels and currently available indicators and classic markers. Results Both HDmiR-122 and CDmiR-222 levels in perfusate at 30 minutes of NMP strongly correlated with hepatocyte injury (peak perfusate AST) and cholangiocyte injury (peak biliary LDH). In bile, only CDmiR-222 correlated with these injury markers. For hepato-cholangiocellular function, both miRs in perfusate correlated with total bilirubin, while HDmiR-122 (in perfusate) and CDmiR-222 (in bile) correlated with bicarbonate secretion. Both the relative ratio of HDmiR-122/CDmiR-222 and AST in perfusate at 30 minutes significantly correlated with cumulative bile production, but only the relative ratio was predictive of histopathological injury after 6 hours NMP. Conclusion Early levels of HDmiR-122 and CDmiR-222, in perfusate and/or bile, are predictive of excretory functions and hepato-cholangiocellular injury after 6 hours NMP. These miRs may represent new biomarkers for graft viability and function during machine perfusion

Cell-free microRNAs as early predictors of graft viability during ex vivo normothermic machine perfusion of human donor livers

Background: Cell-free microRNAs (miRs) have emerged as early and sensitive biomarkers for tissue injury and function. This study aimed to investigate whether the release of hepatocyte-derived microRNAs (HDmiRs) and cholangiocyte-derived miRs (CDmiRs) correlates with hepato-cholangiocellular injury and function during oxygenated, normothermic machine perfusion (NMP) of human liver grafts. Methods: Donor livers (n = 12), declined for transplantation, were subjected to oxygenated NMP (6 hours) after a period of static cold storage (median 544 minutes (IQR 421-674)). Perfusate and bile samples were analyzed by qRT-PCR for HDmiR-122 and CDmiR-222. Spearman correlations were performed between miR levels and currently available indicators and classic markers. Results: Both HDmiR-122 and CDmiR-222 levels in perfusate at 30 minutes of NMP strongly correlated with hepatocyte injury (peak perfusate AST) and cholangiocyte injury (peak biliary LDH). In bile, only CDmiR-222 correlated with these injury markers. For hepato-cholangiocellular function, both miRs in perfusate correlated with total bilirubin, while HDmiR-122 (in perfusate) and CDmiR-222 (in bile) correlated with bicarbonate secretion. Both the relative ratio of HDmiR-122/CDmiR-222 and AST in perfusate at 30 minutes significantly correlated with cumulative bile production, but only the relative ratio was predictive of histopathological injury after 6 hours NMP. Conclusion: Early levels of HDmiR-122 and CDmiR-222, in perfusate and/or bile, are predictive of excretory functions and hepato-cholangiocellular injury after 6 hours NMP. These miRs may represent new biomarkers for graft viability and function during machine perfusion

Identification and Validation Model for Informative Liquid Biopsy-Based microRNA Biomarkers:Insights from Germ Cell Tumor In Vitro, In Vivo and Patient-Derived Data

Liquid biopsy-based biomarkers, such as microRNAs, represent valuable tools for patient management, but often do not make it to integration in the clinic. We aim to explore issues impeding this transition, in the setting of germ cell tumors, for which novel biomarkers are needed. We describe a model for identifying and validating clinically relevant microRNAs for germ cell tumor patients, using both in vitro, in vivo (mouse model) and patient-derived data. Initial wide screening of candidate microRNAs is performed, followed by targeted profiling of potentially relevant biomarkers. We demonstrate the relevance of appropriate (negative) controls, experimental conditions (proliferation), and issues related to sample origin (serum, plasma, cerebral spinal fluid) and pre-analytical variables (hemolysis, contaminants, temperature), all of which could interfere with liquid biopsy-based studies and their conclusions. Finally, we show the value of our identification model in a specific scenario, contradicting the presumed role of miR-375 as marker of teratoma histology in liquid biopsy setting. Our findings indicate other putative microRNAs (miR-885-5p, miR-448 and miR-197-3p) fulfilling this clinical need. The identification model is informative to identify the best candidate microRNAs to pursue in a clinical setting

The potential and limitations of intrahepatic cholangiocyte organoids to study inborn errors of metabolism

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process

A Proof of Concept Study on Real-Pime LiMAx CYP1A2 Liver Function Assessment of Donor Grafts During Normothermic Machine Perfusion

No single reliable parameter exists to assess liver graft function of extended criteria donors during ex-vivo normothermic machine perfusion (NMP). The liver maximum capacity (LiMAx) test is a clinically validated cytochromal breath test, measuring liver function based on 13CO2 production. As an innovative concept, we aimed to integrate the LiMAx breath test with NMP to assess organ function. Eleven human livers were perfused using NMP. After one hour of stabilization, LiMAx testing was performed. Injury markers (ALT, AST, miR-122, FMN, and Suzuki-score) and lactate clearance were measured and related to LiMAx values. LiMAx values ranged between 111 and 1838 µg/kg/h, and performing consecutive LiMAx tests during longer NMP was feasible. No correlation was found between LiMAx value and miR-122 and FMN levels in the perfusate. However, a significant inverse correlation was found between LiMAx value and histological injury (Suzuki-score, R = − 0.874, P < 0.001), AST (R = − 0.812, P = 0.004) and ALT (R = − 0.687, P = 0.028). Furthermore, a significant correlation was found with lactate clearance (R = 0.683, P = 0.043). We demonstrate, as proof of principle, that liver function during NMP can be quantified using the LiMAx test, illustrating a positive correlation with traditional injury markers. This new breath-test application separates livers with adequate cytochromal liver function from inadequate ones and may support decision-making in the safe utilization of extended criteria donor grafts

Characterization of Antigen-Presenting Cell Subsets in Human Liver-Draining Lymph Nodes

T-cell immunity in the liver is tightly regulated to prevent chronic liver inflammation in response to antigens and toxins derived from food and intestinal bacterial flora. Since the main sites of T cell activation in response to foreign components entering solid tissues are the draining lymph nodes (LN), we aimed to study whether Antigen-Presenting Cell (APC) subsets in human liver lymph-draining LN show features that may contribute to the immunologically tolerant liver environment. Healthy liver LN, iliac LN, spleen and liver perfusates were obtained from multi-organ donors, while diseased liver LN were collected from explanted patient livers. Inguinal LN were obtained from kidney transplant recipients. Mononuclear cells were isolated from fresh tissues, and immunophenotypic and functional characteristics of APC subsets were studied using flowcytometry and in ex vivo cultures. Healthy liver-draining LN contained significantly lower relative numbers of CD1c+ conventional dendritic cells (cDC2), plasmacytoid DC (PDC), and CD14+CD163+DC-SIGN+ macrophages (MF) compared to inguinal LN. Compared to spleen, both types of LN contained low relative numbers of CD141hi cDC1. Both cDC subsets in liver LN showed a more activated/mature immunophenotype than those in inguinal LN, iliacal LN, spleen and liver tissue. Despite their more mature status, cDC2 isolated from hepatic LN displayed similar cytokine production capacity (IL-10, IL-12, and IL-6) and allogeneic T cell stimulatory capacity as their counterparts from spleen. Liver LN from patients with inflammatory liver diseases showed a further reduction of cDC1, but had increased relative numbers of PDC and MF. In steady state conditions human liver LN contain relatively low numbers of cDC2, PDC, and macrophages, and relative numbers of cDC1 in liver LN decline during liver inflammation. The paucity of cDC in liver LN may contribute to immune tolerance in the liver environment

Human Bile Contains Cholangiocyte Organoid-Initiating Cells Which Expand as Functional Cholangiocytes in Non-canonical Wnt Stimulating Conditions

Diseases of the bile duct (cholangiopathies) remain a common indication for liver transplantation, while little progress has been made over the last decade in understanding the underlying pathophysiology. This is largely due to lack of proper in vitro model systems to study cholangiopathies. Recently, a culture method has been developed that allows for expansion of human bile duct epithelial cells grown as extrahepatic cholangiocyte organoids (ncECOs) in non-canonical Wnt-stimulating conditions. These ncECOs closely resemble cholangiocytes in culture and have shown to efficiently repopulate collagen scaffolds that could act as functional biliary tissue in mice. Thus far, initiation of ncECOs required tissue samples, thereby limiting broad patient-specific applications. Here, we report that bile fluid, which can be less invasively obtained and with low risk for the patients, is an alternative source for culturing ncECOs. Further characterization showed that bile-derived cholangiocyte organoids (ncBCOs) are highly similar to ncECOs obtained from bile duct tissue biopsies. Compared to the previously reported bile-cholangiocyte organoids cultured in canonical Wnt-stimulation conditions, ncBCOs have superior function of cholangiocyte ion channels and are able to respond to secretin and somatostatin. In conclusion, bile is a new, less invasive, source for patient-derived cholangiocyte organoids and makes their regenerative medicine applications more safe and feasible

Protocol for the STRONG trial: stereotactic body radiation therapy following chemotherapy for unresectable perihilar cholangiocarcinoma, a phase I feasibility study

INTRODUCTION: For patients with perihilar cholangiocarcinoma (CCA), surgery is the only treatment modality that can result in cure. Unfortunately, in the majority of these patients, the tumours are found to be unresectable at presentation due to either local invasive tumour growth or the presence of distant metastases. For patients with unresectable CCA, palliative chemotherapy is the standard treatment yielding an estimated median overall survival (OS) of 12-15.2 months. There is no evidence from randomised trials to support the use of stereotactic body radiation therapy (SBRT) for CCA. However, small and most often retrospective studies combining chemotherapy with SBRT have shown promising results with OS reaching up to 33-35 months.METHODS AND ANALYSIS: This study has been designed as a single-centre phase I feasibility trial and will investigate the addition of SBRT after standard chemotherapy in patients with unresectable perihilar CCA (T1-4 N0-1 M0). A total of six patients will be included. SBRT will be delivered in 15 fractions of 3-4.5 Gy (risk adapted). The primary objective of this study is to determine feasibility and toxicity. Secondary outcomes include local tumour control, progression-free survival (PFS), OS and quality of life. Length of follow-up will be 2 years. As an ancillary study, the personalised effects of radiotherapy will be measured in vitro, in patient-derived tumour and bile duct organoid cultures.ETHICS AND DISSEMINATION: Ethics approval for the STRONG trial has been granted by the Medical Ethics Committee of Erasmus MC Rotterdam, the Netherlands. It is estimated that all patients will be included between October 2017 and October 2018. The results of this study will be published in a peer-reviewed journal, and presented at national and international conferences.TRIAL REGISTRATION NUMBER: NCT03307538; Pre-results

Mesenchymal Stromal Cell-Derived Factors Promote Tissue Repair in a Small-for-Size Ischemic Liver Model but Do Not Protect against Early Effects of Ischemia and Reperfusion Injury

Loss of liver mass and ischemia/reperfusion injury (IRI) are major contributors to postresectional liver failure and small-for-size syndrome. Mesenchymal stromal cell-(MSC-) secreted factors are described to stimulate regeneration after partial hepatectomy. This study investigates if liver-derived MSC-secreted factors also promote liver regeneration after resection in the presence of IRI. C57BL/6 mice underwent IRI of 70% of their liver mass, alone or combined with 50% partial hepatectomy (PH). Mice were treated with MSC-conditioned medium (MSC-CM) or unconditioned medium (UM) and sacrificed after 6 or 24 hours (IRI group) or after 48 hours (IRI + PH group). Blood and liver tissue were analyzed for tissue injury, hepatocyte proliferation, and gene expression. In the IRI alone model, serum ALT and AST levels, hepatic tissue damage, and inflammatory cytokine gene expression showed no significant differences between both treatment groups. In the IRI + PH model, significant reduction in hepatic tissue damage as well as a significant increase in hepatocyte proliferation was observed after MSC-CM treatment. Conclusion. Mesenchymal stromal cell-derived factors promote tissue regeneration of small-for-size livers exposed to ischemic conditions but do not protect against early ischemia and reperfusion injury itself. MSC-derived factors therefore represent a promising treatment strategy for small-for-size syndrome and postresectional liver failure