Dynamin-1-Like Protein Inhibition Drives Megamitochondria Formation as an Adaptive Response in Alcohol-Induced Hepatotoxicity.

Despite the growing global burden of alcoholic liver diseases, therapeutic options are limited, and novel targets are urgently needed. Accumulating evidence suggests that mitochondria adapt in response to ethanol and formation of megamitochondria in the livers of patients is recognized as a hallmark of alcoholic liver diseases. The processes involved in ethanol-induced hepatic mitochondrial changes, the impact on mitochondria-shaping proteins, and the significance of megamitochondria formation remain unknown. In this study, we investigated the mitochondrial and cellular response to alcohol in hepatoma cell line VL-17A. The mitochondrial architecture rapidly changed after 3 or 14 days of ethanol exposure with double-pronged presentation of hyperfragmentation and megamitochondria, and cell growth was inhibited. Dynamin-1-like protein (Drp1) was identified as the main mediator driving these mitochondrial alterations, and its genetic inactivation was determined to foster megamitochondria development, preserving the capacity of the cells to grow despite alcohol toxicity. The role of Drp1 in mediating megamitochondria formation in mice with liver-specific inactivation of Drp1 was further confirmed. Finally, when these mice were fed with ethanol, the presentation of hepatic megamitochondria was exacerbated compared with wild type fed with the same diet. Ethanol-induced toxicity was also reduced. Our study demonstrates that megamitochondria formation is mediated by Drp1, and this phenomenon is a beneficial adaptive response during alcohol-induced hepatotoxicity.

Cryopreserved neonatal hepatocytes may be a source for transplantation: Evaluation of functionality toward clinical use.

Neonatal livers are a potential source of good-quality hepatocytes for clinical transplantation. We compared viability and function of neonatal hepatocytes (NHs) and adult hepatocytes (AHs) and report their clinical use both intraportally and in alginate microbeads. Following isolation from donor livers, hepatocyte function was assessed using albumin, alpha-1-antitrypsin, and factor VII. Metabolic function was investigated by measuring resorufin conjugation, ammonia metabolism, uridine diphosphate glucuronosyltransferase enzyme activity, and cytochrome P450 (CYP) function following induction. Activation of the instant blood-mediated inflammatory reaction by NHs and AHs was investigated using an in vitro blood perfusion model, and tissue factor expression was analyzed using real-time polymerase chain reaction (RT-PCR). Clinical hepatocyte transplantation (HT) was undertaken using standard protocols. Hepatocytes were isolated from 14 neonatal livers, with an average viability of 89.4% ± 1.8% (mean ± standard error of the mean) and average yield of 9.3 × 106 ± 2.0 × 106 cells/g. Hepatocytes were isolated from 14 adult livers with an average viability of 78.6% ± 2.4% and yield 2.2 × 106 ± 0.5 × 105 cells/g. NHs had significantly higher viability after cryopreservation than AHs, with better attachment efficiency and less plasma membrane leakage. There were no differences in albumin, alpha-1-antitrypsin, and factor VII synthesis between NHs and AHs (P > 0.05). Neonatal cells had inducible phase 1 enzymes as assessed by CYP function and functional phase 2 enzymes, in which activity was comparable to AHs. In an in vitro blood perfusion model, AHs elicited increased thrombus formation with a greater consumption of platelets and white cells compared with NHs (28.3 × 109 versus 118.7 × 109 and 3.3 × 109 versus 6.6 × 109 ; P < 0.01). Intraportal transplantation and intraperitoneal transplantation of alginate encapsulated hepatocytes was safe, and preliminary data suggest the cells may activate the immune response to a lesser degree than adult cells. In conclusion, we have shown NHs have excellent cell viability, function, and drug metabolism making them a suitable alternative source for clinical HT. Liver Transplantation 24 394-406 2018 AASLD.

Considerations in the Design of Non-Clinical Development Programmes to Support Non-Viral Genetically Modified Mesenchymal Stromal Cell Therapies.

Due to their immune suppressive pharmacology, regenerative capacity, and immune privileged status, mesenchymal stromal cells (MSCs) are an attractive cell type to treat a variety of diseases. Genetically engineered MSCs are currently in non-clinical and clinical development for a wide range of applications including the delivery of pro-drugs and therapeutic proteins or modified to enhance their regenerative potential. Unmodified MSCs have been shown to have good safety profiles in clinical development. The introduction of exogenous transgenes introduces possible additional risks that need to be assessed in non-clinical studies prior to initiating clinical studies. The use of ex vivo non-viral genetic modification approaches potentially reduces the risks associated with viral vector transfection approaches, including the potential for cell transformation. This review provides an overview of the regulatory-compliant non-clinical proof-of-concept and safety studies required to take MSC-based gene therapy products from the bench to the clinic.

Clinical application of hepatocyte transplantation: current status, applicability, limitations, and future outlook.

Introduction: Hepatocyte transplantation (HT) is a promising alternative to liver transplantation for the treatment of liver-based metabolic diseases and acute liver failure (ALF). However, shortage of good-quality liver tissues, early cell loss post-infusion, reduced cell engraftment and function restricts clinical application.Areas covered: A comprehensive literature search was performed to cover pre-clinical and clinical HT studies. The review discusses the latest developments to address HT limitations: cell sources from marginal/suboptimal donors to neonatal livers, differentiating pluripotent stem cells into hepatocyte-like cells, in vitro expansion, prevention of immune response to transplanted cells by encapsulation or using innate immunity-inhibiting agents, and enhancing engraftment through partial hepatectomy or irradiation.Expert opinion: To date, published data are highly encouraging specially the alginate-encapsulated hepatocyte treatment of children with ALF. Hepatocyte functions can be further improved through co-culturing with mesenchymal stromal cells. Moreover, ex-vivo genetic correction will enable the use of autologous cells in future personalized medicine.

Improving engraftment of hepatocyte transplantation using alpha-1 antitrypsin as an immune modulator.

For patients with non-cirrhotic liver-based metabolic disorders, hepatocyte transplantation can be an effective treatment. However, long-term function of transplanted hepatocytes following infusion has not been achieved due to insufficient numbers of hepatocytes reaching the liver cell plates caused by activation of the instant blood-mediated inflammatory reaction (IBMIR). Our aim was to determine if the natural immune modulator, alpha-1 antitrypsin (AAT), could improve engraftment of transplanted hepatocytes and investigate its mechanism of action. A tubing loop model was used to analyse activation of the IBMIR when human hepatocytes were in contact with ABO-matched blood and 4 mg/ml AAT. Platelet and white cell counts, complement and cytokine expression were analysed. To determine if AAT could improve short-term engraftment, female rats underwent tail vein injection of AAT (120 mg/kg) or water (control) prior to the intrasplenic transplantation of 2 × 107 male hepatocytes. At 48 h and 1 week, livers were collected for analysis. In our loop model, human hepatocytes elicited a significant drop in platelet count with thrombus formation compared to controls. Loops containing AAT and hepatocytes showed no platelet consumption and no thrombus formation. Further, AAT treatment resulted in reduced IL-1ß, IL-6 and IFN-γ and increased IL-1RA compared to untreated loops. In vivo, AAT significantly improved engraftment of rat hepatocytes compared to untreated at 48 h. AAT infusion may inhibit the IBMIR, thus improving short-term engraftment of donor hepatocytes and potentially improve the outcomes for patients with liver-based metabolic disease. KEY MESSAGES: • Alpha-1 antitrypsin (AAT) acts as an immune modulator to improve the efficacy of hepatocyte transplantation. • Treatment with AAT decreased thrombus formation and pro-inflammatory cytokine expression in a tubing loop model. • AAT significantly improved engraftment of donor hepatocytes within the first 48 h post transplantation.

Cell-based liver therapies: past, present and future.

Liver transplantation represents the standard treatment for people with an end-stage liver disease and some liver-based metabolic disorders; however, shortage of liver donor tissues limits its availability. Furthermore, whole liver replacement eliminates the possibility of using native liver as a possible target for future gene therapy in case of liver-based metabolic defects. Cell therapy has emerged as a potential alternative, as cells can provide the hepatic functions and engraft in the liver parenchyma. Various options have been proposed, including human or other species hepatocytes, hepatocyte-like cells derived from stem cells or more futuristic alternatives, such as combination therapies with different cell types, organoids and cell-biomaterial combinations. In this review, we aim to give an overview of the cell therapies developed so far, highlighting preclinical and/or clinical achievements as well as the limitations that need to be overcome to make them fully effective and safe for clinical applications.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

A New High Throughput Screening Platform for Cell Encapsulation in Alginate Hydrogel Shows Improved Hepatocyte Functions by Mesenchymal Stromal Cells Co-encapsulation.

Hepatocyte transplantation has emerged as an alternative to liver transplant for liver disease. Hepatocytes encapsulated in alginate microbeads have been proposed for the treatment of acute liver failure, as they are able to provide hepatic functions while the liver regenerates. Furthermore, they do not require immunosuppression, as the alginate protects the hepatocytes from the recipient's immune cells. Mesenchymal stromal cells are very attractive candidates for regenerative medicine, being able to differentiate into cells of the mesenchymal lineages and having extensive proliferative ability. When co-cultured with hepatocytes in two-dimensional cultures, they exert a trophic role, drastically improving hepatocytes survival and functions. In this study we aimed to (i) devise a high throughput system (HTS) to allow testing of a variety of different parameters for cell encapsulation and (ii) using this HTS, investigate whether mesenchymal stromal cells could have beneficial effects on the hepatocytes when co-encapsulated in alginate microbeads. Using our HTS platform, we observed some improvement of hepatocyte behavior with MSCs, subsequently confirmed in the low throughput analysis of cell function in alginate microbeads. Therefore, our study shows that mesenchymal stromal cells may be a good option to improve the function of hepatocytes microbeads. Furthermore, the platform developed may be used for HTS studies on cell encapsulation, in which several conditions (e.g., number of cells, combinations of cells, alginate modifications) could be easily compared at the same time.

Alginate Encapsulation of Human Hepatocytes and Assessment of Microbeads.

Alginate encapsulation of cells is an attractive technique in which alginate becomes polymerized entrapping the cells. The structure of formed microbeads/microcapsules is semipermeable as it allows oxygen and nutrients to go in, and waste products and other materials produced by the cells to go out. Here, we describe basic protocols for alginate encapsulation of human hepatocytes and methods for assessing the microbeads produced.

PARP14 promotes the Warburg effect in hepatocellular carcinoma by inhibiting JNK1-dependent PKM2 phosphorylation and activation.

Most tumour cells use aerobic glycolysis (the Warburg effect) to support anabolic growth and evade apoptosis. Intriguingly, the molecular mechanisms that link the Warburg effect with the suppression of apoptosis are not well understood. In this study, using loss-of-function studies in vitro and in vivo, we show that the anti-apoptotic protein poly(ADP-ribose) polymerase (PARP)14 promotes aerobic glycolysis in human hepatocellular carcinoma (HCC) by maintaining low activity of the pyruvate kinase M2 isoform (PKM2), a key regulator of the Warburg effect. Notably, PARP14 is highly expressed in HCC primary tumours and associated with poor patient prognosis. Mechanistically, PARP14 inhibits the pro-apoptotic kinase JNK1, which results in the activation of PKM2 through phosphorylation of Thr365. Moreover, targeting PARP14 enhances the sensitization of HCC cells to anti-HCC agents. Our findings indicate that the PARP14-JNK1-PKM2 regulatory axis is an important determinant for the Warburg effect in tumour cells and provide a mechanistic link between apoptosis and metabolism.

The glycosaminoglycan-binding domain of PRELP acts as a cell type-specific NF-kappaB inhibitor that impairs osteoclastogenesis.

Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a glycosaminoglycan (GAG)- and collagen-binding anchor protein highly expressed in cartilage, basement membranes, and developing bone. We observed that PRELP inhibited in vitro and in vivo mouse osteoclastogenesis through its GAG-binding domain ((hbd)PRELP), involving (a) cell internalization through a chondroitin sulfate- and annexin II-dependent mechanism, (b) nuclear translocation, (c) interaction with p65 nuclear factor kappaB (NF-kappaB) and inhibition of its DNA binding, and (d) impairment of NF-kappaB transcriptional activity and reduction of osteoclast-specific gene expression. (hbd)PRELP does not disrupt the mitogen-activated protein kinase signaling nor does it impair cell survival. (hbd)PRELP activity is cell type specific, given that it is internalized by the RAW264.7 osteoclast-like cell line but fails to affect calvarial osteoblasts, bone marrow macrophages, and epithelial cell lines. In vivo, (hbd)PRELP reduces osteoclast number and activity in ovariectomized mice, underlying its physiological and/or pathological importance in skeletal remodeling.