Exosome-derived miR-142-5p from liver stem cells improves the progression of liver fibrosis by regulating macrophage polarization through CTSB.

BACKGROUND: This study aims to explore the effect of liver stem cells (LSCs)-derived exosomes and the miR-142a-5p carried by them on the process of fibrosis by regulating macrophages polarization. METHODS: In this study, CCL4 was used to establish liver fibrosis model. The morphology and purity of exosomes (EVs) were verified by transmission electron microscopy, western blotting (WB) and nanoparticle tracing analysis (NTA). Real-time quantitative PCR (qRT-PCR), WB and enzyme-linked immunoadsorption (ELISA) were used to detect liver fibrosis markers, macrophage polarization markers and liver injury markers. Histopathological assays were used to verify the liver injury morphology in different groups. The cell co-culture model and liver fibrosis model were constructed to verify the expression of miR-142a-5p and ctsb. RESULTS: Immunofluorescence of LSCs markers CK-18, epithelial cell adhesion molecule (EpCam), and AFP showed that these markers were up-regulated in LSCs. In addition, we evaluated the ability of LSCs to excrete EVs by labeling LSCs-EVs with PKH67. We found that CCL4 and EVs were simultaneously treated at 50 and 100 µg doses, and both doses of EVs could reduce the degree of liver fibrosis in mice. We tested markers of M1 or M2 macrophage polarization and found that EVs reduced M1 marker expression and promoted M2 marker expression. Further, ELISA was used to detect the secreted factors related to M1 and M2 in tissue lysates, which also verified the above views. Further analysis showed that the expression of miR-142a-5p increased significantly with the increase of EVs treatment concentration and time. Further, in vitro and in vivo LSCs-EVs regulate macrophage polarization through miR-142a-5p/ctsb pathway and affect the process of liver fibrosis. CONCLUSION: Our data suggest that EVs-derived miR-142-5p from LSCs improves the progression of liver fibrosis by regulating macrophage polarization through ctsb.

HLSC-Derived Extracellular Vesicles Attenuate Liver Fibrosis and Inflammation in a Murine Model of Non-alcoholic Steatohepatitis.

Extracellular vesicles (EVs) are membrane vesicles released virtually by all cell types. Several studies have shown that stem cell-derived EVs may mimic both in vitro and in vivo the biological effects of the cells. We recently demonstrated that non-alcoholic steatohepatitis (NASH) is inhibited by treatment with human liver stem cells (HLSCs). The aim of the present study was to evaluate whether EVs released by HLSCs influence the progression of NASH, induced by a diet deprived of methionine and choline, in immunocompromised mice. EV treatment was initiated after 2 weeks of diet with a biweekly administration of three different doses. Bio-distribution evaluated by optical imaging showed a preferential accumulation in normal and, in particular, in fibrotic liver. EV treatment significantly improved liver function and reduced signs of liver fibrosis and inflammation at both morphological and molecular levels. In particular, we observed that, out of 29 fibrosis-associated genes upregulated in NASH liver, 28 were significantly downregulated by EV treatment. In conclusion, HLSC-derived EVs display anti-fibrotic and anti-inflammatory effects in a model of chronic liver disease, leading to an improvement of liver function.

Hepatitis C virus infection induces endoplasmic reticulum stress and apoptosis in human fetal liver stem cells.

The cellular mechanisms by which hepatitis C virus (HCV) replication might mediate cytopathic effects are controversial and not entirely clear. In this study, we found that blood-borne HCV (bbHCV) infection could lead to endoplasmic reticulum (ER)-stress and mitochondria-related/caspase-dependent apoptosis at the early stages of infection based on use of the highly efficient bbHCV cell culture model established previously. Sections of bbHCV-infected human fetal liver stem cells (hFLSCs) revealed convolution and nonlinear ER, cell vacuolization, swelling of mitochondria, and numerous double membrane vesicles (DMVs). The percentage of apoptotic hFLSCs infected by bbHCV reached 29.8% at 16 h postinfection, and the amount of cytochrome c increased remarkably in the cytosolic protein fraction. However, over time, apoptosis was inhibited due to the activation of NF-κB. The expression of NF-κB-p65, Bcl-xL, XIAP, and c-FLIPL in hFLSCs was increased significantly 24 h after in infection by bbHCV. The accelerated cell death cycles involving apoptosis, regeneration and repair by bbHCV infection might give rise to the development of cirrhosis, and ultimately to hepatocellular carcinogenesis. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

Dynamics of Proliferative and Quiescent Stem Cells in Liver Homeostasis and Injury.

BACKGROUND & AIMS: Adult liver stem cells are usually maintained in a quiescent/slow-cycling state. However, a proliferative population, marked by leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), was recently identified as an important liver stem cell population. We aimed to investigate the dynamics and functions of proliferative and quiescent stem cells in healthy and injured livers. METHODS: We studied LGR5-positive stem cells using diphtheria toxin receptor and green fluorescent protein (GFP) knock-in mice. In these mice, LGR5-positive cells specifically coexpress diphtheria toxin receptor and the GFP reporter. Lineage-tracing experiments were performed in mice in which LGR5-positive stem cells and their daughter cells expressed a yellow fluorescent protein/mTmG reporter. Slow-cycling stem cells were investigated using GFP-based, Tet-on controlled transgenic mice. We studied the dynamics of both stem cell populations during liver homeostasis and injury induced by carbon tetrachloride. Stem cells were isolated from mouse liver and organoid formation assays were performed. We analyzed hepatocyte and cholangiocyte lineage differentiation in cultured organoids. RESULTS: We did not detect LGR5-expressing stem cells in livers of mice at any stage of a lifespan, but only following liver injury induced by carbon tetrachloride. In the liver stem cell niche, where the proliferating LGR5+ cells are located, we identified a quiescent/slow-cycling cell population, called label-retaining cells (LRCs). These cells were present in the homeostatic liver, capable of retaining the GFP label over 1 year, and expressed a panel of progenitor/stem cell markers. Isolated single LRCs were capable of forming organoids that could be carried in culture, expanded for months, and differentiated into hepatocyte and cholangiocyte lineages in vitro, demonstrating their bona fide stem cell properties. More interestingly, LRCs responded to liver injury and gave rise to LGR5-expressing stem cells, as well as other potential progenitor/stem cell populations, including SOX9- and CD44-positive cells. CONCLUSIONS: Proliferative LGR5 cells are an intermediate stem cell population in the liver that emerge only during tissue injury. In contrast, LRCs are quiescent stem cells that are present in homeostatic liver, respond to tissue injury, and can give rise to LGR5 stem cells, as well as SOX9- and CD44-positive cells.

Tumor-promoting cyanotoxin microcystin-LR does not induce procarcinogenic events in adult human liver stem cells.

HL1-hT1 cell line represents adult human liver stem cells (LSCs) immortalized with human telomerase reverse transcriptase. In this study, HL1-hT1 cells were found to express mesenchymal markers (vimentin, CD73, CD90/THY-1 and CD105) and an early hepatic endoderm marker FOXA2, while not expressing hepatic progenitor (HNF4A, LGR5, α-fetoprotein) or differentiated hepatocyte markers (albumin, transthyretin, connexin 32). In response to microcystin-LR (MC-LR), a time- and concentration-dependent formation of MC-positive protein bands in HL1-hT1 cells was observed. Cellular accumulation of MC-LR occurred most likely via mechanisms independent on organic anion transporting polypeptides (OATPs) or multidrug resistance (MDR) proteins, as indicated (a) by a gene expression analysis of 11 human OATP genes and 4 major MDR genes (MDR1/P-glycoprotein, MRP1, MRP2 and BCRP); (b) by non-significant effects of OATP or MDR1 inhibitors on MC-LR uptake. Accumulation of MC-positive protein bands in HL1-hT1 cells was associated neither with alterations of cell viability and growth, dysregulations of ERK1/2 and p38 kinases, reactive oxygen species formation, induction of double-stranded DNA breaks nor modulations of stress-inducible genes (ATF3, HSP5). It suggests that LSCs might have a selective, MDR1-independent, survival advantage and higher tolerance towards MC-induced cytotoxic, genotoxic or cancer-related events than differentiated adult hepatocytes, fetal hepatocyte or malignant liver cell lines. HL1-hT1 cells provide a valuable in vitro tool for studying effects of toxicants and pharmaceuticals on LSCs, whose important role in the development of chronic toxicities and liver diseases is being increasingly recognized.

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.

Interleukin-6 secreted by bipotential murine oval liver stem cells induces apoptosis of activated hepatic stellate cells by activating NF-κB-inducible nitric oxide synthase signaling.

Liver fibrosis is now well recognized as the causative factor for increased mortality from complications associated with liver pathologies. Activated hepatic stellate cells (HSCs) play a critical role in the progression of liver fibrosis. Therefore, targeting these activated HSCs to prevent and (or) treat liver disease is a worthwhile approach to explore. In the present in vitro study, we investigated the use of bipotential murine oval liver cells (BMOL) in regulating the functions of activated HSCs to prevent progression of liver fibrosis. We used a conditioned medium-based approach to study the effect of BMOL cells on activated HSC survival and function. Our data showed that BMOL cells block the contraction of activated HSCs by inducing apoptosis of these cells. We demonstrated that BMOL cells secrete soluble factors, such as interleukin-6 (IL-6), which induced apoptosis of activated HSCs. Using both pharmacological and molecular inhibitor approaches, we further identified that IL-6-mediated activation of NF-κB-iNOS-NO-ROS signaling in activated HSCs plays a critical role in BMOL-cell-mediated apoptosis of activated HSCs. Thus, the present study provides an alternative cell-based therapeutic approach to treat liver fibrosis.

[Protective effect of intraperitoneal transplantation of human liver-derived stem cells at different times against concanavalin A-induced acute liver injury in mice].

Objective: To investigate the protective effect of intraperitoneal transplantation of human liver-derived stem cells at different times against concanavalin A (ConA)-induced acute liver injury in mice. Methods: A total of 88 male C57BL/6 mice were randomly divided into normal control group (group C), ConA model group (group M), and human liver-derived stem cells (HYX1)+ConA group (group E); according to the interval between phosphate buffer/HYX1 injection and ConA injection, Groups M and E were further divided into 3-hour groups (M1 and E1 groups), 6-hour groups (M2 and E2 groups), 12-hour groups (M3 and E3 groups), 24-hour groups (M4 and E4 groups), and 48-hour groups (M5 and E5 groups). The levels of alanine aminotransferase (ALT), aspartate transaminase (AST), and total bilirubin (TBil) in peripheral blood were measured, liver tissue sections were used to observe pathological changes, and the Ishak score for liver inflammation was determined. The independent samples t-test was used for comparison between groups, and P < 0.05 was considered statistically significant. Results: The levels of ALT, AST, and TBil in group C were (36.25±1.16) U/L, (120.20±5.77) U/L, and (2.20±0.23) µmol/L, respectively; the levels of ALT, AST, and TBil and Ishak score were (8 721.23±837.39) U/L, (8 110.31±290.10) U/L, (8.41±0.10) µmol/L, and (13.32±1.30), respectively, in group M1, (8 334.31±666.50) U/L, (7 560.20±760.34) U/L, (10.40±0.80) µmol/L, and (12.67±0.81), respectively, in group M2, (8 960.75±551.93) U/L, (8 535.62±675.14) U/L, (10.95±1.43) µmol/L, and (14.57±0.65), respectively, in group M3, (8 618.57±886.40) U/L, (11 440.54 ± 1 327.86) U/L, (13.30±1.86) µmol/L, and (13.21±1.06), respectively, in group M4, and (10 170.13±1 112.37) U/L, (11 470.56±1 108.40) U/L, (12.75±1.55) µmol/L, and (15.07±1.58), respectively, in group M5. The levels of ALT, AST, and TBil and Ishak score were (1 016.35±163.47) U/L, (952.30±103.91) U/L, (7.77±0.62) µmol/L, and (3.50±0.21), respectively, in group E1, (42.10±6.20) U/L, (126.72±13.33) U/L, (3.41±0.53) µmol/L, and (2.01±0.40), respectively, in group E2, (44.21±4.30) U/L, (216.71±35.88) U/L, (3.47±0.44) µmol/L, and (2.13±0.25), respectively, in group E3, (2 909.69±212.14) U/L, (2 988.43±333.70) U/L, (7.03±0.93) µmol/L, and (4.70±0.50), respectively, in group E4, and (7 874.26±799.60) U/L, (10 940.54±947.35) U/L, (10.53±1.09) µmol/L, and (8.60±0.83), respectively, in group E5. Groups M1-M5 had significantly higher levels of ALT, AST, and TBil than group C (all P < 0.01), and groups M1-M4 had significantly higher levels of AST and ALT than groups E1-E4 (all P < 0.01), while there were no significant differences in the levels of AST and ALT between groups M5 and E5 (both P > 0.05). The pathological sections of liver tissue showed that compared with group M, group E had significant reductions in the degree of necrosis and Ishak score (both P < 0.05). Conclusion: Intraperitoneal transplantation of human liver-derived stem cells has a protective effect against ConA-induced acute liver injury in mice, and the injection at 6 and 12 hours in advance has the best protective effect.

Influence of inflammation on the immunological profile of adult-derived human liver mesenchymal stromal cells and stellate cells.

BACKGROUND AIMS: Stem cell therapy for liver diseases has recently emerged as a promising alternative to liver transplantation. Eligible cells should have an appropriate immunophenotype. The aim of the present study was to define the immunological profile of two human liver-derived mesenchymal stromal cell populations, namely, stem cells (ADHLSC) and hepatic stellate cells (HSC). METHODS: The study was conducted under normal and inflammatory conditions with the use of human bone marrow mesenchymal stromal cells (BM-MSC) as reference. RESULTS: Like BM-MSC and ADHLSC, HSC were negative for hematopoietic (CD45) and endothelial (CD34) markers but positive for stromal markers. All cell types were constitutively positive for HLA class I and negative for human leukocyte antigen (HLA) class II and co-stimulatory molecules (CD80, CD86, CD134 and CD252). Inflammation induced the expression of CD40 in all cell types, but the highest values were observed on HSCs; high CD252 expression was only observed on HSC as compared with ADHLSC and BM-MSC. The expression of various adhesion molecules (CD54, CD58, CD106 and CD166) was dissimilar in these three cell types and was differentially influenced by inflammation as well. ADHLSC and HSC constitutively expressed the immunosuppressive molecule HLA-G, whereas CD274 expression was induced by inflammation, as in the case of BM-MSC. Moreover, all cell types expressed the two major natural killer ligands CD112 and CD115. CONCLUSIONS: Toll-like receptors (TLR) 1, 3, 4 and 6 messenger RNA was expressed by both cell types, whereas TLR 2, 5, 7, 9 and 10 were only expressed by ADHLSC. Inflammation increased the expression of TLR 2 and 3 by ADHLSC and HSC. Finally, both liver-derived cell types were immunosuppressive because they inhibited the proliferation of mitogen-activated T cells.

Proper BMP Signaling Levels Are Essential for 3D Assembly of Hepatic Cords from Hepatoblasts and Mesenchymal Cells.

BACKGROUND: Because the molecular mechanisms of morphogenesis of the hepatic cord and sinus are unclear, we investigated the involvement of bone morphogenetic protein (BMP4) in hepatic sinusoid morphogenesis. METHODS: We used embryonic chicken livers, which develop rapidly, as our model, and investigated expression of BMP-related genes. BMP4 activity was manipulated by overexpressing BMP4 and its antagonist, noggin. RESULTS: During hepatic cord morphogenesis, BMP4 and its receptors are expressed in both peri-sinusoidal cells and hepatoblasts as the sinusoids form, whereas noggin is expressed transiently in peri-sinusoidal cells at early stages. Suppression of BMP activity with noggin overexpression disrupted normal hepatic sinusoid structure, leading to liver congestion, failure of fibronectin deposition, and markedly reduced numbers of peri-sinusoidal cells. However, overexpression of BMP did not change sinusoidal morphology but increased endothelial cell number. Noggin overexpression resulted in disrupted cord organization, and dilated sinusoidal space, eventually leading to increased apoptosis and failed hepatocyte differentiation. CONCLUSIONS: Our results show that proper BMP signaling mediates peri-sinusoidal cell-hepatoblast interactions during development; this is essential for hepatic cord organization among hepatoblasts, endothelium, and presumptive hepatic stellate cells.

VEGF-FGF Signaling Activates Quiescent CD63+ Liver Stem Cells to Proliferate and Differentiate.

Understanding the liver stem cells (LSCs) holds great promise for new insights into liver diseases and liver regeneration. However, the heterogenicity and plasticity of liver cells have made it controversial. Here, by employing single-cell RNA-sequencing technology, transcriptome features of Krt19+ bile duct lineage cells isolated from Krt19CreERT; Rosa26R-GFP reporter mouse livers are examined. Distinct biliary epithelial cells which include adult LSCs, as well as their downstream hepatocytes and cholangiocytes are identified. Importantly, a novel cell surface LSCs marker, CD63, as well as CD56, which distinguished active and quiescent LSCs are discovered. Cell expansion and bi-potential differentiation in culture demonstrate the stemness ability of CD63+ cells in vitro. Transplantation and lineage tracing of CD63+ cells confirm their contribution to liver cell mass in vivo upon injury. Moreover, CD63+CD56+ cells are proved to be activated LSCs with vigorous proliferation ability. Further studies confirm that CD63+CD56- quiescent LSCs express VEGFR2 and FGFR1, and they can be activated to proliferation and differentiation through combination of growth factors: VEGF-A and bFGF. These findings define an authentic adult liver stem cells compartment, make a further understanding of fate regulation on LSCs, and highlight its contribution to liver during pathophysiologic processes.

In vitro characterization of 3D culture-based differentiation of human liver stem cells.

Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system, guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study, we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR, immunofluorescence, and Western blot analysis. Additionally, we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes, including CYP450, urea cycle enzymes, and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes, evident as early as 1 day post-differentiation. Interestingly, HLSCs transiently upregulated stem cell markers during differentiation, followed by downregulation after 7 days. Furthermore, differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24 h, with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system, its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications.

Large-Scale Expansion of Human Liver Stem Cells Using Two Different Bioreactor Systems.

The assessment of human liver stem cells (HLSCs) as cell therapeutics requires scalable, controlled expansion processes. We first focused on defining appropriate process parameters for HLSC expansion such as seeding density, use of antibiotics, optimal cell age and critical metabolite concentrations in conventional 2D culture systems. For scale-up, we transferred HLSC expansion to multi-plate and stirred-tank bioreactor systems to determine their limitations. A seeding density of 4000 cells cm-2 was needed for efficient expansion. Although growth was not significantly affected by antibiotics, the concentrations of lactate and ammonia were important. A maximum expansion capacity of at least 20 cumulative population doublings (cPDs) was observed, confirming HLSC growth, identity and functionality. For the expansion of HLSCs in the multi-plate bioreactor system Xpansion (XPN), the oxygen supply strategy was optimized due to a low kLa of 0.076 h-1. The XPN bioreactor yielded a final mean cell density of 94 ± 8 × 103 cells cm-2, more than double that of the standard process in T-flasks. However, in the larger XPN50 device, HLSC density reached only 28 ± 0.9 × 103 cells cm-2, while the glucose consumption rate increased 8-fold. In a fully-controlled 2 L stirred-tank bioreactor (STR), HLSCs expanded at a comparable rate to the T-flask and XPN50 processes in a homogeneous microenvironment using advanced process analytical technology. Ultimately, the scale-up of HLSCs was successful using two different bioreactor systems, resulting in sufficient numbers of viable, functional and undifferentiated HLSCs for therapeutic applications.

Naïve or Engineered Extracellular Vesicles from Different Cell Sources: Therapeutic Tools for Kidney Diseases.

Renal pathophysiology is a multifactorial process involving different kidney structures. Acute kidney injury (AKI) is a clinical condition characterized by tubular necrosis and glomerular hyperfiltration. The maladaptive repair after AKI predisposes to the onset of chronic kidney diseases (CKD). CKD is a progressive and irreversible loss of kidney function, characterized by fibrosis that could lead to end stage renal disease. In this review we provide a comprehensive overview of the most recent scientific publications analyzing the therapeutic potential of Extracellular Vesicles (EV)-based treatments in different animal models of AKI and CKD. EVs from multiple sources act as paracrine effectors involved in cell-cell communication with pro-generative and low immunogenic properties. They represent innovative and promising natural drug delivery vehicles used to treat experimental acute and chronic kidney diseases. Differently from synthetic systems, EVs can cross biological barriers and deliver biomolecules to the recipient cells inducing a physiological response. Moreover, new methods for improving the EVs as carriers have been introduced, such as the engineering of the cargo, the modification of the proteins on the external membrane, or the pre-conditioning of the cell of origin. The new nano-medicine approaches based on bioengineered EVs are an attempt to enhance their drug delivery capacity for potential clinical applications.

Distinct hepatocyte identities in liver homeostasis and regeneration.

The process of metabolic liver zonation is spontaneously established by assigning distributed tasks to hepatocytes along the porto-central blood flow. Hepatocytes fulfil critical metabolic functions, while also maintaining hepatocyte mass by replication when needed. Recent technological advances have enabled us to fine-tune our understanding of hepatocyte identity during homeostasis and regeneration. Subsets of hepatocytes have been identified to be more regenerative and some have even been proposed to function like stem cells, challenging the long-standing view that all hepatocytes are similarly capable of regeneration. The latest data show that hepatocyte renewal during homeostasis and regeneration after liver injury is not limited to rare hepatocytes; however, hepatocytes are not exactly the same. Herein, we review the known differences that give individual hepatocytes distinct identities, recent findings demonstrating how these distinct identities correspond to differences in hepatocyte regenerative capacity, and how the plasticity of hepatocyte identity allows for division of labour among hepatocytes. We further discuss how these distinct hepatocyte identities may play a role during liver disease.

Controversies Surrounding the Origin of Hepatocytes in Adult Livers and the in Vitro Generation or Propagation of Hepatocytes.

Epithelial cells in the liver (known as hepatocytes) are high-performance engines of myriad metabolic functions and versatile responders to liver injury. As hepatocytes metabolize amino acids, alcohol, drugs, and other substrates, they produce and are exposed to a milieu of toxins and harmful byproducts that can damage themselves. In the healthy liver, hepatocytes generally divide slowly. However, after liver injury, hepatocytes can ramp up proliferation to regenerate the liver. Yet, on extensive injury, regeneration falters, and liver failure ensues. It is therefore critical to understand the mechanisms underlying liver regeneration and, in particular, which liver cells are mobilized during liver maintenance and repair. Controversies continue to surround the very existence of hepatic stem cells and, if they exist, their spatial location, multipotency, degree of contribution to regeneration, ploidy, and susceptibility to tumorigenesis. This review discusses these controversies. Finally, we highlight how insights into hepatocyte regeneration and biology in vivo can inform in vitro studies to propagate primary hepatocytes with liver regeneration-associated signals and to generate hepatocytes de novo from pluripotent stem cells.

A case of small hepatocellular carcinoma with malignant ductular reaction.

Herein reported is the unique case of a small hepatocellular carcinoma (HCC) with several foci of a minor (10% in area) component of "malignant ductular reactions". The patient was 51-year-old man who was a drinker. HBV/HCV were negative. The tumor was small (12×10×11 mm), solid, expansile and reddish-brown, and contained fibrous septa. The background was cirrhotic without alcoholic features. Histologically, the tumor was well differentiated HCC, and, besides the HCC, it contained several small foci consisting of the following four biliary epithelial elements: clusters of small cells (CSC), ductules (D), ductular hepatocytes (DH), and bile ducts (BD). The proportion of area was as follows: HCC 90%, CSC 3%, D 3%, DH 2%, and BD 2%. These non-HCC elements were intimately admixed and formed several foci that were characteristically located in the fibrous septa (FS), except for CSC which were situated among HCC cells close to FS. There were gradual merges between HCC and CSC, CSC and D, D and DH, and D and BD, respectively. Cells of CSC and D resembled rat oval cells. Cells of these four elements had atypical features regarded as malignant. Immunohistochemically (IHC), HCC were positive for arginase, HepPar1, and less frequently CK7. CSC were positive for CK7. D were positive for arginase, HepPar1, CK7, CK19, EMA, and EpCAM. DH were positive for arginase, HepPar1, and CK7. BD were positive for CK7, CK19, EMA, EpCAM and mucin. Although such tumors as this have been termed stem cell-related cancers, our case lacked definite evidence for stem cell origin in histology as well as in the IHC that showed negativity for KIT, CD34, and OCT3/4. The above findings suggest that CSC, D, DH and BD are analogous to the ductular reaction seen in hepatic inflammation. Therefore, we termed the phenomenon "malignant ductular reaction". It is suggested in the present tumor that at first only HCC developed, and then HCC cells in the interface with FS transformed to CSC, like a fetal ductal plate. Then, the CSC gave rise to D, which in turn led to DH and BD in FS, all findings of which are most likely sequential considering embryonic biliary development. The idea that the present tumor was at first D carcinoma and then D developed on one hand into CSC and HCC, and on the other into DH and BD seems possible, but its probability appears low because the vast majority of the present tumor had the phenotype of HCC.

MiR-126 Regulates Properties of SOX9+ Liver Progenitor Cells during Liver Repair by Targeting Hoxb6.

Liver progenitor cells (LPCs) have a remarkable contribution to the hepatocytes and ductal cells when normal hepatocyte proliferation is severely impaired. As a biomarker for LPCs, Sry-box 9 (Sox9) plays critical roles in liver homeostasis and repair in response to injury. However, the regulation mechanism of Sox9 in liver physiological and pathological state remains unknown. In this study, we found that miR-126 positively regulated the expression of Sox9, the proliferation and differentiation of SOX9+ LPCs by suppressing the translation of homeobox b6 (Hoxb6). As a transcription factor, HOXB6 directly binds to the promoter of Sox9 to inhibit Sox9 expression, resulting in the destruction of the properties of SOX9+ LPCs in CCl4-induced liver injury. These findings revealed the role of miR-126 in regulating SOX9+ LPCs fate by targeting Hoxb6 in liver injury repair. Our findings suggest the potential role of miR-126 as a nucleic acid therapy drug target for liver failure.

Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration.

Hepatocyte proliferation is the principal mechanism for generating new hepatocytes in liver homeostasis and regeneration. Recent studies have suggested that this ability is not equally distributed among hepatocytes but concentrated in a small subset of hepatocytes acting like stem cells, located around the central vein or distributed throughout the liver lobule and exhibiting active WNT signaling or high telomerase activity, respectively. These findings were obtained by utilizing components of these growth regulators as markers for genetic lineage tracing. Here, we used random lineage tracing to localize and quantify clonal expansion of hepatocytes in normal and injured liver. We found that modest proliferation of hepatocytes distributed throughout the lobule maintains the hepatocyte mass and that most hepatocytes proliferate to regenerate it, with diploidy providing a growth advantage over polyploidy. These results show that the ability to proliferate is broadly distributed among hepatocytes rather than limited to a rare stem cell-like population.