Liver Stem Cells Research Articles

Human liver stem cells attenuate concanavalin A-induced acute liver injury by modulating myeloid-derived suppressor cells and CD4+ T cells in mice

BackgroundAcute liver failure (ALF) is a serious threat to the life of people all over the world. Finding an effective way to manage ALF is important. Human liver stem cells (HLSCs) are early undifferentiated cells that have been implicated in the regeneration and functional reconstruction of the liver. In this study, we aimed to evaluate the protective effects of the HLSC line HYX1 against concanavalin A (ConA)-induced acute liver injury.MethodsHYX1 cells were characterized by microscopy, functional assays, gene expression, and western blot analyses. We showed that HYX1 cells can differentiate into hepatocytes. We intraperitoneally injected HYX1 cells in mice and administered ConA via caudal vein injection 3, 6, 12, 24, and 48 h later. The effects of HYX1 cell transplantation were evaluated through blood tests, histology, and flow cytometry.ResultsHYX1 cells reduced the levels of alanine transaminase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) in serum and dramatically decreased the severity of liver injuries. Mechanistically, HYX1 cells promoted myeloid-derived suppressor cell (MDSC) migration into the spleen and liver, while reducing CD4+ T cell levels in both tissues. In addition, HYX1 cells suppressed the secretion of proinflammatory cytokines, such as tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), but led to increased interleukin-10 (IL-10) production.ConclusionsThese results confirm the efficacy of HLSCs in the prevention of the ConA-induced acute liver injury through modulation of MDSCs and CD4+ T cell migration and cytokine secretion.

From deceased to bioengineered graft: New frontiers in liver transplantation

In the worldwide context of graft shortage, several strategies have been explored to increase the number of grafts available for liver transplantation (LT). These include the use of marginal and living donors, split livers, and the improvement of marginal donor grafts (machine perfusion). However, recent advances in the understanding of liver organogenesis, stem cells, and matrix biology provide novel insights in tissue engineering. Today, the newest technologies and discoveries open the door to the development of new methods for organ implementation such as the recellularization of natural scaffolds, liver organoids, bio-printing, and tissue or generation of chimeric organs. These approaches might potentially to generate an unlimited source of grafts (allogenic or chimeric) which will be used in the near future for LT or as a temporary bridge toward LT. This qualitative review focuses on all methods of organ implementation and highlights the newest developments in tissue engineering and regenerative medicine.

Analysis for Remodeling of Hepatic Tissue Structures in 3D During Regeneration.

It has been demonstrated that the liver remarkably alter its tissue structures during regeneration, and various types of liver stem or progenitor cells locating in specific areas in the liver tissue contribute to regeneration. Therefore, it is important to analyze the dynamic rearrangement of the liver tissue structures in 3D for better understanding the process and mechanism of liver regeneration. Here we describe a macroscopic analysis method to visualize the whole 3D structure of the vasculatures and the biliary tree, which are dynamically remodeled during regeneration, and a microscopic analysis method to visualize detailed structures at the cellular level, which can compensate what cannot be detected in the macroscopic analysis.

Specific Labeling and Lineage Tracing of Periportal Hepatocytes Using Two-Step Genetic Recombination.

The liver is unmatched in regenerative capacity. However, when exhausted, the liver is predisposed to various diseases based on injury types and causal agents. Although hepatocytes have been proposed to be the main source of new hepatocytes during regeneration, the existence of specialized liver stem cells has been long debated. In mice, oval cells or ductal cells have been postulated as such stem/progenitor pool. Exhaustive works from different laboratories have shown that in genetically unmodified mice, oval cells, or by extension ductal cells, only contribute marginally in producing new hepatocytes during liver regeneration, thus indicating that hepatocytes are the main regenerative cell source. In this debated context, we identified a new population of periportal hepatocytes in the normal mouse liver. These cells we termed hybrid hepatocytes (HybHP) express low levels of the transcription factor Sox9. Using complementary lineage tracing tools, we demonstrated that HybHP regenerate the liver after chronic hepatocyte depleting injuries. Here, we describe the two-step genetic recombination method that allowed us to study HybHP's lineage in two established models of liver injury.

Generation of Hepatocyte-Like Cells by Different Strategies for Liver Regeneration

Incidence and mortality of liver disease has increased globally in recent years. Orthotopic liver transplantation is a well-developed, effective therapy even for the end-stage liver diseases. However, the application of the technique is limited by the short supply of donors and the complexity of orthotopic liver transplantation. Therefore, hepatocyte transplantation as a new therapeutic option was developed. Unfortunately, the routine supply of high quality human hepatocytes was also restricted and the liver donor was lacking, too. Thus, exploration and study for available and renewable sources of nondonor hepatocytes are both necessary and important. Researches have proved that nondonor hepatocyte-like cells can be derived from various types of cells including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), liver stem cells (LSCs), fibroblasts by lineage reprogramming, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). In this review, we describe recent advances in the field of the cell therapy in liver regeneration. In addition, we also discuss the existing restrictions in these types of therapies and ongoing developments.

Human liver stem cell-derived extracellular vesicles enhance cancer stem cell sensitivity to tyrosine kinase inhibitors through Akt/mTOR/PTEN combined modulation.

It is well recognized that Cancer Stem Cells (CSCs) sustain the initiation, the maintenance and the recurrence of tumors. We previously reported that extracellular vesicles (EVs) derived from human liver stem cells (HLSCs) were able to limit tumor development. In this study, we evaluated whether EV derived from HLSCs could act in synergy with tyrosine kinase inhibitors (TKIs) on apoptosis of CSCs isolated from renal carcinomas. For this purpose, we administered to renal CSCs, HLSC-EVs and TKIs, as co-incubation or sequential administration. We found that HLSC-EVs in combination with Sunitinb or Sorafenib significantly increased renal CSCs apoptosis induced by low TKI dose. At variance, no synergistic effect was observed when bone marrow mesenchymal stem cell-derived EVs were used. In particular, renal CSCs chemosensitivity to TKIs was enhanced when HLSC-EVs were either co-administered with TKIs or added after, but not before. CSC apoptosis was also incremented at a percentage comparable to that of co-administration when TKIs were loaded in HLSC-EVs. By a mechanistic point of view, Akt/mTOR and Erk and Creb intracellular pathways, known to be pivotal in the induction of tumor growth and survival, appeared modulated as consequence of TKIs/HLSC-EVs co-administration. Together, our results indicate that the synergistic effect of HLSC-EVs with TKIs may increase the response to TKIs at low doses, providing a rational for their combined use in the treatment of renal carcinoma.

Extracellular vesicles from human liver stem cells inhibit tumor angiogenesis.

Human liver stem-like cells (HLSC) and derived extracellular vesicles (EVs) were previously shown to exhibit anti-tumor activity. In our study, we investigated whether HLSC-derived EVs (HLSC-EVs) were able to inhibit tumor angiogenesis in vitro and in vivo, in comparison with EVs derived from mesenchymal stem cells (MSC-EVs). The results obtained indicated that HLSC-EVs, but not MSC-EVs, inhibited the angiogenic properties of tumor-derived endothelial cells (TEC) both in vitro and in vivo in a model of subcutaneous implantation in Matrigel. Treatment of TEC with HLSC-EVs led to the down-regulation of pro-angiogenic genes. Since HLSC-EVs carry a specific set of microRNAs (miRNAs) that could target these genes, we investigated their potential role by transfecting TEC with HLSC-EV specific miRNAs. We observed that four miRNAs, namely miR-15a, miR-181b, miR-320c and miR-874, significantly inhibited the angiogenic properties of TEC in vitro, and decreased the expression of some predicted target genes (ITGB3, FGF1, EPHB4 and PLAU). In parallel, TEC treated with HLSC-EVs significantly enhanced expression of miR-15a, miR-181b, miR-320c and miR-874 associated with the down-regulation of FGF1 and PLAU. In summary, HLSC-EVs possess an anti-tumorigenic effect, based on their ability to inhibit tumor angiogenesis.

A small molecule Hedgehog agonist HhAg1.5 mediated reprogramming breaks the quiescence of noninjured liver stem cells for rescuing liver failure

Liver is the second most transplanted organ according to United network for organ sharing. Due to shortage of compatible donors, surgical difficulties, immunological hindrance, and high postoperative cost, stem cell therapy is an attractive substitute of liver transplant for millions of patients suffering from hepatic failure. Due to several technical limitations such as viral integration, inefficient differentiation, and adult phenotypes and epigenetic memory of fibroblasts, induced pluripotent stem cells, mesenchymal stem cells, or induced hepatocyte may not present a great clinical substitute for liver transplant. We pioneered a novel technology for robust expansion of quiescent liver stem cells (LSCs) from mice via utilizing of Hedgehog agonist HhAg1.5 for 3 weeks. These expanded LSCs retained stem-like properties after multiple passaging and differentiated to hepatocytes and cholangiocytes. Grafting of ex vivo expanded LSCs in Fah-/- Rag2-/-Il2rg-/- knockout mice, significantly increased life span compared to control group (P < 0.001). Thus in this study, we provide a promising viable substitute for primary hepatocytes for regenerative medicine and for life-threatening metabolic liver diseases.

Codelivery of doxorubicin and elacridar to target both liver cancer cells and stem cells by polylactide-co-glycolide/d-alpha-tocopherol polyethylene glycol 1000 succinate nanoparticles.

PurposeLiver cancer is the third leading cause of cancer-related deaths worldwide. Liver cancer stem cells (LCSCs) are a subpopulation of cancer cells that are responsible for the initiation, progression, drug resistance, recurrence, and metastasis of liver cancer. Recent studies have suggested that the eradication of both LCSCs and liver cancer cells is necessary because the conversion of cancer stem cells (CSCs) to cancer cells occasionally occurs. As ATP-binding cassette (ABC) transporters are overexpressed in both CSCs and cancer cells, combined therapies using ABC transporter inhibitors and chemotherapy drugs could show superior therapeutic efficacy in liver cancer. In this study, we developed poly(lactide-co-glycolide)/d-alpha-tocopherol polyethylene glycol 1000 succinate nanoparticles to accomplish the simultaneous delivery of an optimized ratio of doxorubicin (DOX) and elacridar (ELC) to target both LCSCs and liver cancer cells.MethodsMedian-effect analysis was used for screening of DOX and ELC for synergy in liver cancer cells (HepG2 cells) and LCSCs (HepG2 tumor sphere [HepG2-TS]). Then, nanoparticles loaded with DOX and ELC at the optimized ratio (NDEs) were prepared by nanoprecipitation method. The cytotoxicity and colony and tumor sphere formation ability of nanoparticles were investigated in vitro, and the tissue distribution and antitumor activity of nanoparticles were evaluated in vivo.ResultsWe demonstrated that a DOX/ELC molar ratio of 1:1 was synergistic in HepG2 cells and HepG2-TS. NDEs were shown to exhibit significantly increased cytotoxic effects against both HepG2 and HepG2-TS compared with DOX-loaded nanoparticles (NDs) or ELC-loaded nanoparticles (NEs) in vitro. In vivo studies demonstrated that the nanoparticles exhibited better tumor targeting, with NDE showing the strongest antitumor activity with lower systemic toxicity.ConclusionThese results suggested that NDE represented a promising combination therapy against liver cancer by targeting both liver cancer cells and CSCs.

Islet-Like Structures Generated In Vitro from Adult Human Liver Stem Cells Revert Hyperglycemia in Diabetic SCID Mice

A potential therapeutic strategy for diabetes is the transplantation of induced-insulin secreting cells. Based on the common embryonic origin of liver and pancreas, we studied the potential of adult human liver stem-like cells (HLSC) to generate in vitro insulin-producing 3D spheroid structures (HLSC-ILS). HLSC-ILS were generated by a one-step protocol based on charge dependent aggregation of HLSC induced by protamine. 3D aggregation promoted the spontaneous differentiation into cells expressing insulin and several key markers of pancreatic β cells. HLSC-ILS showed endocrine granules similar to those seen in human β cells. In static and dynamic in vitro conditions, such structures produced C-peptide after stimulation with high glucose. HLSC-ILS significantly reduced hyperglycemia and restored a normo-glycemic profile when implanted in streptozotocin-diabetic SCID mice. Diabetic mice expressed human C-peptide and very low or undetectable levels of murine C-peptide. Hyperglycemia and a diabetic profile were restored after HLSC-ISL explant. The gene expression profile of in vitro generated HLSC-ILS showed a differentiation from HLSC profile and an endocrine commitment with the enhanced expression of several markers of β cell differentiation. The comparative analysis of gene expression profiles after 2 and 4 weeks of in vivo implantation showed a further β-cell differentiation, with a genetic profile still immature but closer to that of human islets. In conclusion, protamine-induced spheroid aggregation of HLSC triggers a spontaneous differentiation to an endocrine phenotype. Although the in vitro differentiated HLSC-ILS were immature, they responded to high glucose with insulin secretion and in vivo reversed hyperglycemia in diabetic SCID mice.

Post hepatectomy liver failure - A comprehensive review of current concepts and controversies.

Post hepatectomy liver failure (PHLF) comprises of a conundrum of symptoms and signs following major hepatic resections. The pathophysiology essentially revolves around disruption of the normal hepatocyte regeneration and disturbed liver homeostasis. Prompt identification of the pre-operative predictors of PHLF in the form of biochemical parameters and imaging features are of paramount importance for any hepatic surgeon and forms the cornerstone of its management. Treatment revolves around a goal-directed resuscitation of the systemic organ failure. Auxiliary support systems such as liver dialysis devices and stem cell therapy are still under investigational trials for treatment of the same. Orthotopic liver transplantation (OLT) is the last resort in most cases not responding to other measures.

Assessment of Hepatotoxic Potential of Cyanobacterial Toxins Using 3D In Vitro Model of Adult Human Liver Stem Cells.

Cyanotoxins microcystin-LR (MC-LR) and cylindrospermopsin (CYN) represent hazardous waterborne contaminants and potent human hepatotoxins. However, in vitro monolayer cultures of hepatic cell lines were found to recapitulate, poorly, major hepatocyte-specific functions and inadequately predict hepatotoxic effects of MC-LR and CYN. We utilized 3-dimensional (3D), scaffold-free spheroid cultures of human telomerase-immortalized adult liver stem cells HL1-hT1 to evaluate hepatotoxic potential of MC-LR and CYN. In monolayer cultures of HL1-hT1 cells, MC-LR did not induce cytotoxic effects (EC50 > 10 micromol/L), while CYN inhibited cell growth and viability (48h-96h EC50 ≈ 5.5-0.6 micromol/L). Growth and viability of small growing spheroids were inhibited by both cyanotoxins (≥0.1 micromol/L) and were associated with blebbing and disintegration at the spheroid surface. Hepatospheroid damage and viability reduction were observed also in large mature spheroids, with viability 96h-EC50 values being 0.04 micromol/L for MC-LR and 0.1 micromol/L for CYN, and No Observed Effect Concentrations <0.01 micromol/L. Spheroid cultures of adult human liver stem cells HL1-hT1 exhibit sensitivity comparable to cultures of primary hepatocytes and provide a simple, practical, and cost-effective tool, which can be effectively used in environmental and toxicological research, including assessment of hepatotoxic potential and effect-based monitoring of various samples contaminated with toxic cyanobacteria.

Abstract 5459: Regulation of IGF2 by TGF-β signaling in liver cancers and stem cell homeostasis

Abstract Development of hepatocellular carcinoma (HCC), which remains lethal, is associated with alterations in multiple factors including the transforming growth factor beta (TGF-β) signaling pathway. Previously we have uncovered a unique role for TGF-β signaling molecules, Smad3 and its adaptor β2SP, in suppressing stem cell transformation into cancer. Yet, while TGF-β plays a pleiotropic role including regulating stem cell differentiation, proliferation, and inflammation, mechanistic insight into the dichotomy of TGF-β, and its role in stem cell transformation remains poorly understood for these cancers. Here, we took an integrated approach to identify and validate effects of changes in this pathway in HCC and identify potential therapeutic targets such as IGF2. We extended our mechanistic studies associated with the regulation of insulin-like growth factor 2 (IGF2) in the context of the TGF-β-pathway, by utilizing both human liver cancer cell lines and TGF-β signaling-deficient mice (β2SP+/- and β2SP+/-/Smad3+/-) that develop liver cancers. We found the following: 1) IGF2 promotes stemness, overexpression leading to increased expression of stem cell genes that include SOX2 and ALDH1A1, and sphere formation in liver cancer cells; 2) A marked increase in IGF2 mRNA and protein in HepG2 cells and Huh7 cells with β2SP stable knockdown; 3) TGF-β signaling through β2SP/Smad3 significantly inhibits IGF2 transcription; ChIP assays validated binding of Smad3 at the human IGF2 promoter region between -859bp to -688bp upstream of the IGF2 transcriptional start site; 4) TCGA based integrated analyses identify specific HCC patient groups with altered TGF-β members and high expression of IGF2 that could be potentially targetable for IGF2; 5) TCGA analyses of 33 cancers also revealed that potentially “switching off” TGF-β-pathway (through decreased expression levels) leads to increased stemness validating findings of our TGF-β deficient mouse models that phenocopy a human cancer stem cell syndrome. In summary, TGF-β negatively regulates IGF2 transcription: loss of TGF-β signaling through β2SP/Smad3 leads to IGF2 activation, contributing to tumor progression. TGF-β-β2SP/Smad3 are important for IGF2 repression and maintaining stem cell homeostasis, which may result from TGF-β/β2SP/Smad3 and/or CTCF-dependent regulation of IGF2. Our studies provide a framework for new animal models of liver and gastrointestinal cancers and future new therapeutics. Citation Format: Shuyun Rao, Sobia Zaidi, Kazufumi Ohshiro, Jian Chen, Shoujun Gu, Wilma Jogunoori, Jon White, Bibhuti Mishra, Shulin Li, Rehan Akbani, Lopa Mishra. Regulation of IGF2 by TGF-β signaling in liver cancers and stem cell homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5459.

Abstract 162: UBE2T: A molecular regulator for cancer stemness and drug resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the common cancers worldwide. Poor prognosis of HCC patients is attributed to high frequency of tumor relapse and chemoresistant nature of this disease. Increasing evidence showed the critical role of cancer stem cells (CSCs) on tumor relapse and therapeutic resistance. Since normal stem cells and CSCs share high similarity, we would like to identify the molecules/pathways crucial in liver CSCs by determining what molecules involved in normal liver stem cells during liver regeneration. Comparison of expression profiles between early regenerating liver and intact liver revealed the upregulation of DNA Damage Response pathways in the self-renewing liver, in which Ubiquitin Conjugating Enzyme E2 T (UBE2T) was the most significantly upregulated. This, together with the publicly available dataset (GSE5975) shows upregulation of UBE2T in EpCAM-enriched liver CSC populations, suggest the potential role of UBE2T on regulation of cancer stemness. By qPCR analysis UBE2T mRNA level was overexpressed in 91% of the clinical HCC specimens (n=56) and associated with aggressive tumor behavior and poorer patients' survival. The overexpression of UBE2T was further confirmed by western blot and immunohistochemical analysis. By lentiviral based knock-down approach, we demonstrated the role of UBE2T in regulation of liver CSC properties, including self-renewal, tumorigenicity, drug resistance and expression of liver CSC markers. In orthotopic HCC model, UBE2T was found to play pivotal role in lung metastasis in vivo. Mechanistically, UBE2T is found to interact directly with E3 ligase Mule and regulate its protein expression via ubiquitination. Since Mule is found to directly degrade β-catenin protein, we are examining whether UBE2T mediates liver T-IC function through direct regulation of Mule-mediated β-catenin degradation. In conclusion, we have uncovered the novel role of UBE2T signaling cascade in regulation liver CSCs, which provides attractive therapeutic target for potential treatment of HCC. Citation Format: Pui Yu Ho, Kin Wah Lee. UBE2T: A molecular regulator for cancer stemness and drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 162.

Bioengineered bile ducts recapitulate key cholangiocyte functions

Investigation of diseases of the bile duct system and identification of potential therapeutic targets are hampered by the lack of tractable in vitro systems to model cholangiocyte biology. Here, we show a step-wise method for the differentiation of murine Lgr5+ liver stem cells (organoids) into cholangiocyte-like cells (CLCs) using a combination of growth factors and extracellular matrix components. Organoid-derived CLCs display key properties of primary cholangiocytes, such as expressing cholangiocyte markers, forming primary cilia, transporting small molecules and responding to farnesoid X receptor agonist. Integration of organoid-derived cholangiocytes with collagen-coated polyethersulfone hollow fiber membranes yielded bioengineered bile ducts that morphologically resembled native bile ducts and possessed polarized bile acid transport activity. As such, we present a novel in vitro model for studying and therapeutically modulating cholangiocyte function.

Raman spectroscopic investigation of the human liver stem cell line HepaRG

AbstractIn this work, Raman spectroscopic cell imaging approaches and a discrimination between HepG2, nondifferentiated hepatic stem cell line HepaRG, and differentiated hepatocyte‐like HepaRG cells are presented. Raman spectroscopic imaging was used to visualize the cell nuclei by means of false color imaging using a marker band, and a cell segmentation was performed by means of clustering. Furthermore, a 3‐class‐classification model based on the mean Raman spectra of individual cells was established for a classification between different cell types. A high average sensitivity of 96% was achieved by the applied classification model. Based on the results of clustering and classification, the main spectral contributions to different cell types and cell segments were analyzed in detail. Thereby, HepG2, nondifferentiated hepatic stem cell line HepaRG, and differentiated hepatocyte‐like HepaRG cells were Raman spectroscopically characterized and proven to be significantly different.

Hepatic stem cells and their enzymatic markers- A review

Stem cells are those cells which show capacity for self-renewal and ability to give rise to multiple differentiated cellular populations. Enzymatic activity, as a marker for cell proliferation and cell viability, is used by metabolic activity assays. Liver stem cells/progenitor cells can be a useful source of liver treatment. They can repopulate and restore injured liver. Fetal liver stem/ progenitor cells have been found to be more capable in this, but are subjected to ethical issues. Adult liver stem cells and stem cells from animals can be used. Alkaline phosphatase and lactate dehydrogenase are enzymatic markers of in vitro hepatocyte culture. During in vitro cell culture, in the culture medium, secreted alkaline phosphatase activity increases during exponential growth of cells, whereas low extracellular lactate dehydrogenase activity indicates increased number of viable cells. Alkaline phosphatase and lactate dehydrogenase activities can be used to assess hepatocytes proliferation in vitro.

Surgical and comprehensive treatment for intrahepatic cholangiocarcinoma patients from the perspective of tumor heterogeneity

Intrahepatic cholangiocarcinoma(ICC) is generally found late along with extremely poor prognosis and lack of effective and standardized treatment. Tremendous heterogeneities exist in onset factors, geographic distribution, clinical manifestation, and biological characteristics of ICC patients. However, the relevant causes and mechanisms have not been clearly illustrated. Recent studies indicate that the diverse celluar origins of ICC, including liver cells, bile duct epithelial cells, glandular cells around the duct, and liver stem cells, etc. Besides, the tumorigenesis, progression and metastasis of ICC are closed related to metabolism such as glucose, lipid metabolism. It can be inferred that the varieties of biological characteristics and clinical manifestation of ICC patients are possibly closely linked with heterogeneous cellular origins, multiple metabolic mechanisms or pathways. Therefore, it's necessary to conduct research with regard to the connection between cellular origins, cell metabolism and clinical manifestation, biological characteristics, and make further classifications. Based on the accurate classification, surgeons can exert corresponding surgical and comprehensive treatment strategies on ICC patients, attaining the goal of individualized and standardized treatment, ultimately, improving prognosis and prolonging survival time.

Participation of liver stem cells in cholangiocarcinogenesis after aflatoxin B1 exposure of glutathione S-transferase A3 knockout mice.

Aflatoxin B1, arguably the most potent human carcinogen, induces liver cancer in humans, rats, trout, ducks, and so on, but adult mice are totally resistant. This resistance is because of a detoxifying enzyme, mouse glutathione S-transferase A3, which binds to and inactivates aflatoxin B1 epoxide, preventing the epoxide from binding to DNA and causing mutations. Glutathione S-transferase A3 or its analog has not been detected in any of the sensitive species, including humans. The generation of a glutathione S-transferase A3 knockout (represented as KO or -/-) mice has allowed us to study the induction of liver cancer in mice by aflatoxin B1. In contrast to the induction of hepatocellular carcinomas in other species, aflatoxin B1 induces cholangiocarcinomas in GSTA3-/- mice. In other species and in knockout mice, the induction of liver cancer is preceded by extensive proliferation of small oval cells, providing additional evidence that oval cells are bipolar stem cells and may give rise to either hepatocellular carcinoma or cholangiocarcinoma depending on the nature of the hepatocarcinogen and the species of animal. The recent development of mouse oval cell lines in our laboratory from aflatoxin B1-treated GSTA3-/- mice should provide a new venue for study of the properties and potential of putative mouse liver stem cells.

Extracellular Vesicles from Human Liver Stem Cells Reduce Injury in an Ex Vivo Normothermic Hypoxic Rat Liver Perfusion Model.

The gold standard for organ preservation before transplantation is static cold storage, which is unable to fully protect suboptimal livers from ischemia/reperfusion injury. An emerging alternative is normothermic machine perfusion (NMP), which permits organ reconditioning. Here, we aimed to explore the feasibility of a pharmacological intervention on isolated rat livers by using a combination of NMP and human liver stem cells-derived extracellular vesicles (HLSC-EV). We established an ex vivo murine model of NMP capable to maintain liver function despite an ongoing hypoxic injury induced by hemodilution. Livers were perfused for 4 hours without (control group, n = 10) or with HLSC-EV (treated group, n = 9). Bile production was quantified; perfusate samples were collected hourly to measure metabolic (pH, pO2, pCO2) and cytolysis parameters (AST, alanine aminotransferase, lactate dehydrogenase). At the end of perfusion, we assessed HLSC-EV engraftment by immunofluorescence, tissue injury by histology, apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, tissue hypoxia-inducible factor 1-α, and transforming growth factor-beta 1 RNA expression by quantitative reverse transcription-polymerase chain reaction. During hypoxic NMP, livers were able to maintain homeostasis and produce bile. In the treated group, AST (P = 0.018) and lactate dehydrogenase (P = 0.032) levels were significantly lower than those of the control group at 3 hours of perfusion, and AST levels persisted lower at 4 hours (P = 0.003). By the end of NMP, HLSC-EV had been uptaken by hepatocytes, and EV treatment significantly reduced histological damage (P = 0.030), apoptosis (P = 0.049), and RNA overexpression of hypoxia-inducible factor 1-α (P < 0.0001) and transforming growth factor-beta 1 (P = 0.014). HLSC-EV treatment, even in a short-duration model, was feasible and effectively reduced liver injury during hypoxic NMP.